Improvement of Avian Influenza DNA vaccine candidates:

The impact of antigen-targeting sequences

Margarida Susana Barradas dos Santos Azevedo

Thesis to obtain the Master of Science Degree in

Biotechnology

Examination Committe

Chairperson: Professor Doctor Arsénio do Carmo Sales Mendes Fialho Supervisors: Professor Doctor Gabriel António Amaro Monteiro

Doctor Miguel Agostinho Sousa Pinto de Torres Fevereiro Member of Committe: Doctor Ana Margarida Ferreira Henriques de Oliveira Mourão

November 2013

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Acknowledgments

I would like to express my gratitude to my supervisor, Professor Gabriel Monteiro, for accepting me in

this thesis, giving me the opportunity to work in such an interesting topic, and for the availability to receive me

at all times and to answer all my questions with helpful advice.

I would also like to thank my co-supervisor Doutor Miguel Fevereiro for receiving me at the virology

laboratory in INIAV and for the given support and the valuable suggestions and advice during the course of this

work.

My most important acknowledgement and sincere gratitude go to Doutora Ana Margarida Mourão, for

the unlimited patience to teach me and the encouragement, kind words and optimism even when faced with

disappointing results. I am certain that I would have not been successful in completing this work without your

help, perseverance and endless ideas to overcome difficulties.

To the group of people at INIAV that I had the pleasure to meet, thank you all for receiving me and

making me feel welcome. A special thanks to Arminda Batista and Tiago Luís for the help with the chickens, an

interesting and sometimes scary (but also funny) experience.

I’m also truly grateful to my lab colleagues and staff members at IST for all the help, support and

friendship during this year. I am especially grateful to Sofia Duarte and Jorge Paulo for all the patience and help

with the molecular cloning and RT-PCR, to Jonathan de La Vega, Ricardo Pereira and especially Salomé

Magalhães for being available and helpful with the flow cytometry analysis at Tagus Park and to Marina

Monteiro for all the help especially in my first months in the lab. To all my master’s colleagues I thank for the

friendship and support through these intense two years we shared, I am very thankful to have met you. To

Elisabete Freitas, a big word of gratitude for all the help in initiating my work and for always being available to

answer all my questions throughout this year.

Finally I must thank my friends and my family that understood my busy and stressful year and one way

or another showed support, patience and gave me great joy. To David, thank you for making me laugh in the

most stressful times and for being by my side.

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Abstract

DNA vaccination constitutes a safer and flexible alternative to conventional vaccines for delivery of

specific antigens to the immune system. pDNA vectors harboring a pathogen gene elicit both cellular and

humoral immune response, constituting the ideal vaccine platform for the elusive and fast evolving Avian

Influenza Virus. Intracellular antigen targeting can overcome setbacks of DNA vaccination, such as low antigen

expression and vaccine immunogenicity, by routing the synthesized antigen to different cell compartments and

major presentation pathways MHC I and MHC II and therefore modulating the immune response. In this work,

the targeting sequences E1A (adenovirus early region 1A) and LAMP (lysosomal-associated membrane protein-

1) were incorporated into DNA vaccine candidates encoding the AIV neuraminidase 3. Plasmid and mRNA

content and stability and protein expression were assessed by in vitro testing in Chinese hamster ovary (CHO)

cells. Results suggest the correct protein distribution, since the addition of LAMP and E1A-LAMP led to

decreased fluorescence levels associated with lysosome protein degradation, which was not observed in ER

targeting through the addition of E1A alone. Targeting sequences had no apparent influence on plasmid copy

number or mRNA transcript content. Groups of chickens were intramuscularly immunized with a DNA

vaccine/lipofectamine formulation, through a DNA prime-protein boost strategy. In vivo results showed that

plasmids N3, N3-LAMP and E1A-N3-LAMP triggered an anti-N3 antibody response. Furthermore, the N3-LAMP

plasmid showed enhanced humoral response, when compared to the N3, confirming the potential of the

antigen-targeting strategy for improvement of DNA vaccine effectiveness. The addition of E1A alone did not

elicit a humoral response, which can be attributed to the sorting of antigen to the ER and consequent loading

of peptides to a MHC I-restricted pathway, leading to a predominant cellular response, not studied in this work.

Keywords: DNA vaccine, Avian Influenza Virus, antigen-targeting sequences, adenovirus early region 1A (E1A),

lysosomal-associated membrane protein-1 (LAMP), MHC I and MHC II presentation pathways.

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Resumo

As vacinas de DNA constituem uma alternativa segura e flexível à vacinação convencional na

apresentação de antigénios específicos ao sistema imunitário. Apresentam a capacidade de gerar um resposta

imunitária tanto celular como humoral, constituindo a plataforma ideal no combate ao vírus da gripe aviária,

um vírus de rápida e constante evolução. A inclusão de sequências de direcionamento intracelular pode

superar alguns obstáculos das vacinas de DNA, tais como uma limitada expressão do antigénio e baixa

imunogenicidade, através do direcionamento do antigénio para diferentes compartimentos celulares e vias de

apresentação, modulando a resposta imunitária.

Neste trabalho, as sequências de direcionamento E1A, que dirige a proteína para o retículo

endoplasmático (RE) e para a via de apresentação MHC I, e LAMP, que dirige a proteína para o lisossoma e via

MHC II, foram incorporadas em vacinas de DNA que codifiam para o antigénio neuraminidase 3 do vírus da

gripe aviária. O número de cópias de plasmídeo e estabilidade do RNA mensageiro, bem como o nível de

proteína expressa foram avaliados através de ensaios in vitro em células CHO. Os resultados obtidos sugerem

um correcto direcionamento da proteína, uma vez que a adição das sequências LAMP e E1A-LAMP levou à

diminuição dos níveis de fluorescência, associados a degradação proteica nos lisossomas, o que não foi

observado no caso do direcionamento para o RE pela sequência E1A. Estas sequências parecem não ter tido

influência no número de cópias de plasmídeo ou na estabilidade das cópias de mRNA. Grupos de galinhas

foram imunizados pela via intramuscular com a formulação vacina de DNA/lipossomas, seguido de uma

inoculação com o antigénio N3. Os resultados do ensaio in vivo revelaram uma resposta humoral anti-N3

desencadeada pelos plasmídeos N3, N3-LAMP e E1A-N3-LAMP. N3-LAMP foi responsável por uma produção de

anticorpos mais elevada que o plasmído N3, confirmando o potencial desta estratégia para a melhoria da

potência das vacinas de DNA. A adição da sequência E1A resultou numa resposta humoral nula, o que pode ser

atribuído ao direcionamento da proteína para o RE e consequentemente para a via de apresentação MHC I, o

que provavelmente levou a uma resposta celular, não estudada neste trabalho.

Palavras-chave: vacinas de DNA, Virus da gripe aviária, sequências de direcionamento, E1A, LAMP, vias de

apresentação MHC I e MHC II.

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List of contents

Acknowledgments ................................................................................................................................................... iii

Abstract .................................................................................................................................................................... v

Resumo .................................................................................................................................................................. vii

List of contents ........................................................................................................................................................ ix

List of figures ........................................................................................................................................................... xi

List of tables ........................................................................................................................................................... xv

List of abbreviations ............................................................................................................................................. xvii

I. Literature Review ........................................................................................................................................... 1

I.1. Avian Influenza Virus .................................................................................................................................... 1

I.1.1. Influenza A Virion structure................................................................................................................... 3

I.1.2. Viral core/ genome structure ................................................................................................................ 4

I.1.3. The influenza virus replication cycle ..................................................................................................... 5

I.1.4. Host immune response ......................................................................................................................... 8

I.2. DNA vaccines .............................................................................................................................................. 12

I.2.1. Structure and mechanism of action .................................................................................................... 13

I.2.2. Advantages and drawbacks ................................................................................................................. 15

I.2.3. Improvement of DNA vaccine performance ........................................................................................ 16

I.2.4. Antigen-targeting sequences .............................................................................................................. 19

I.2.5. Production of DNA vaccines ................................................................................................................ 22

I.2.6. Plasmid DNA delivery .......................................................................................................................... 25

II. Background and objectives .......................................................................................................................... 29

III. Materials and methods ............................................................................................................................ 31

III.1. Design and production of DNA vaccines ................................................................................................... 31

III.1.1. Molecular cloning .............................................................................................................................. 32

III.1.2. Confirmation of clones by enzymatic digestion ................................................................................ 35

III.1.3. Confirmation of clones by automated sequencing ........................................................................... 35

III.1.4. Preparation of E. coli DH5 α cell banks for pVAX-E1A-N3-GFP and pVAX-E1A-N3-GFP–LAMP ......... 36

III.2. Plasmid Production and purification ........................................................................................................ 37

III.2.1. Alkaline lysis and hydrophobic interaction and size exclusion chromatographies (HIC-SEC) ........... 37

III.2.3. HiSpeed Plasmid Midi and Maxi Kit (Qiagen) .................................................................................... 39

III.3. In vitro assays in CHO cells........................................................................................................................ 39

III.3.1. Culture and transfection of CHO cells ............................................................................................... 39

III.3.2. Quantitative Real-time PCR for determination of plasmid copy number in transfected CHO cells .. 40

III.3.3. Flow cytometry analysis .................................................................................................................... 41

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III.3.4. Real-time reverse transcription PCR (RRT-PCR) ................................................................................ 42

III.3.5. Data and statistical analysis............................................................................................................... 42

III.4. In vivo assays in chickens .......................................................................................................................... 43

III.4.1. DNA vaccination of chickens ............................................................................................................. 43

III.4.2. N3 protein production and purification ............................................................................................ 43

III.4.3. Enzyme Linked Immunosorbent Assay (ELISA) .................................................................................. 43

III.4.4. Data and statistical analysis............................................................................................................... 44

I.V. Results and discussion .................................................................................................................................... 45

IV.1. DNA vaccine construction ........................................................................................................................ 45

IV.1.1. Construction of pVAX-E1A-N3-GFP and pVAX-E1A-N3-GFP-LAMP ................................................... 47

IV.2. Production of pDNA vaccines ................................................................................................................... 49

IV.3. In vitro assays ........................................................................................................................................... 52

IV.3.1. Flow cytometry analysis - Transfection efficiency ............................................................................ 52

IV.3.2. Flow cytometry analysis- Mean fluorescence of transfected cells ................................................... 53

IV.3.3. Analysis of plasmid copy number and mRNA content ...................................................................... 55

IV.3.4. Effect of gene and target sequence addition to plasmid stability, transcription and GFP expression

...................................................................................................................................................................... 59

IV.4. In vivo DNA vaccination ............................................................................................................................ 60

IV.4.1. Effect of Intracellular targeting sequences on the immune response .............................................. 62

IV. Final remarks and future work ................................................................................................................. 67

V. Bibliographic references ............................................................................................................................... 69

VI. Appendix .................................................................................................................................................. 77

Appendix I- plasmid pVAX1/lacZ features ........................................................................................................ 77

Appendix II- Growth curves of vectors used in molecular cloning pVAX-N3-GFP and pVAX-N3-GFP-LAMP .... 78

Appendix III- Calibration curves used for pDNA copy number quantification in transfected CHO cells, by RT-

PCR. ................................................................................................................................................................... 79

Appendix IV- Calibration curves used for mRNA copy number quantification in transfected CHO cells, by RRT-

PCR .................................................................................................................................................................... 80

Appendix V- N3 antigen gene and protein sequences and characteristics ....................................................... 81

Appendix VI- Molecular Weight Markers and Agarose gel electrophoresis analysis ........................................ 82

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List of figures

Figure 1- The processes of evasion from the immune system after influenza virus infection /vaccination. Due to

selective pressure, Antigenic drift and antigenic shift lead to antigenic changes in the circulating virus. Source:

WHO (World Health Organization) Collaborating centre for Reference and Research on Influenza. .................... 3

Figure 2 - Influenza A virion structure. The three envelope proteins: Glycoproteins NA (neuraminidase) and HA

(hemagglutinin) and ion-channel M2 protein. M1 protein; NEP/NS2 (nuclear export protein); The minimal unit

of replication, the RNP (ribonucleoprotein), is composed by RNA segments coated with the nucleoprotein (NP)

and the RNA polymerase (PB1, PB2 and PA). Horimoto et al. (2005). .................................................................... 4

Figure 3- Representation of Influenza A virus replication cycle. A) Virion structure and binding of HA to the sialic

acid residues in the host cell receptors; B) Virus is transported in the cell in a endocytic vesicle. The low pH in

the endossome triggers important mechanisms that lead to the release of the vRNPs into the cytoplasm and

their subsequent import into the cell nucleus; C) Once in the nucleus, the synthesis of viral mRNA is initiated by

the viral RNA polymerase by a process (“cap snatching”) in which 5’- capped RNA fragments are cleved from

host pre-mRNAs; D) Viral mRNAs are transported to the cytoplasm, where they are translated into viral

proteins. HA, NA and M2 are processed in the ER and glycosilated in the Golgi apparatus. These proteins are

finnaly transported to the cell membrane F) The new synthesised vRNPs are transported to the cytoplasm; G)

Once in the cell menbrane, vRNPs are incorporated in the newly formed virions, with the enveloped proteins,

The virions are released from the host cell, after the NA protein cleaves the ligation with the sialic acid

residues. E) NS1 protein role in blocking production of host-mRNAs. Adapted from Das et al. (2010). ................ 7

Figure 4- Dendritic cells (DCs) functional MHC I and MHC II presentation pathways (Villadangos & Schnorrer

2007). DCs have a dominant role in the presentation of antigens to naïve T lymphocytes and, therefore, priming

the immune response. MHC I molecules mainly present endogenous antigen via dependent or independent

proteasome and TAP pathway. MHC II molecules mainly acquire peptides processed in endosomal

compartments but also endogenous components of the endocytic pathway or that enter the endosome by

autophagy. Cross-presentation is a phenomenon that consists on the ability of DCs to deliver exogenous

antigens to the MHC I pathway. ........................................................................................................................... 10

Figure 5- Illustrative diagram of induction of cellular and humoral immunity upon influenza A viral infection.

Solid arrows correspond to induction of immune responses following primary IA viral infection. Dotted arrows

relate to immune memory mechanisms activated upon secondary infection with the virus, in which a more

rapid response is generated by virus-specific cells. (van de Sandt et al. 2012). ................................................... 11

Figure 6- Antibody immune response to avian influenza viruses. Antibodies against HA prevent virus

attachment. This is the only immune response that prevents infection. Antibodies against NA prevent release

of virions, binding the virus to the infected cell. Antibodies against M2 prevent the release of viral particles to

the extracellular space (Subbarao et al. 2007). .................................................................................................... 12

Figure 7- DNA vaccine clinical trials by vaccine target (2011) adapted from (Ferraro et al. 2011). Data obtained

from the clinicaltrials.gov database indicates that, currently, 130 trials with pDNA vaccines are listed as on-

going studies, as of August 2013. .......................................................................................................................... 13

Figure 8 - Schematic diagram of the essential elements in a DNA vaccine-vector. A basic vector includes an

antibiotic selection marker (KanR), a bacterial origin of replication (pUC ori), an eukaryotic promoter (pCMV), a

multiple cloning site to insert antigen sequences (MCS) and a polyadenylation signal sequence (polyA) (Ertl

2003). .................................................................................................................................................................... 14

Figure 9- The direct and indirect routes of antigen presentation, following the most common delivery methods

through injection to skin or muscle. DNA vector-encoded antigen is then expressed in myocytes or

keratinocytes. Antigen must be transferred to APCs for activation of T cells, usually a dendritic cell (DC). This

indirect process of transfer of antigenic material, possibly as apoptotic vesicles, is termed cross-presentation. A

small proportion of DNA is also taken up directly by DCs and the encoded antigen can then be processed and

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presented endogenously. Furthermore, antigen may be taken up by DCs through shed exogenous antigens.

(Rice et al. 2008) ................................................................................................................................................... 15

Figure 10- Example of several DNA vaccine targeting-strategies for modulation of the immune response

towards the MHC I or MHC II presentation pathways. After entering the nucleus of APCs (1), the plasmid DNA,

through the host cellular machinery, and the gene transcription is initiated, followed by protein production in

the citoplasm and processsing according to the respective modification. A) Signal seuences such as the human

tissue plasminogen activator leader peptide (tPA) and the secretory signal (Sc) lead the vaccine-encoded

protein to the extracellular space of transfected cells via the golgi apparatus; B) Unmodified antigen sequences,

lacking any targeting sequence, are expressed as cytoplasmic proteins and usually presented in the MHC I

molecules by default, but can also be presented in MHC II molecules through transport of cytosolic material via

autophagy; C) ubiquitin attachment leads the translated protein into the polyubiquitination pathway and

subsequently to the MHC I pathway; D) Signal sequences such as lysosomal integral membrane protein-II (LIMP

II) and lysosomal-associated membrane protein (LAMP) promote antigen transport to the lysosomes that

facilitate peptide presentation on MHC II molecules. Besides direct transfection of resident APCs as well as

somatic cells (2), APCs mediate the display of peptides on MHC II molecules after secreted vaccine-derived

antigens have been shed from transfected cells, captured and processed within the endocytic pathway (2) and

MHC I cross-presentation of exogenous antigen for example by engulfment of transfected and apoptotic cells.

Adapted from (Weinberger et al. 2013)................................................................................................................ 21

Figure 11- Schematic representation of the essential steps in production of plasmid DNA for therapeutic use

and vaccination. Supercoiled plasmid purification is achieved by a series of downstream processing unit

operations. Following cell lysis, the product can proceed directly to a single or dual mode chromatography or a

previous step of clarification and concentration can be performed. (Prather et al. 2003) .................................. 23

Figure 12- Schematic representation of plasmid DNA vectors and restriction sites used in molecular cloning for

the construction of pVAX-E1A-N3-GFP and pVAX-E1A-N3-GFP-LAMP and tested in in vitro and in vivo assays,

along with pVAX-GFP, used as a control in the experiments. Prokaryotic elements in the vaccine vectors for

plasmid replication, maintenance and selection in the bacterial host include a strong bacterial origin of

replication ColE1, a selectable marker for Kanamycin and an inducer of high level of transcription , the T7

promoter. Eukaryotic elements for expression in the mammalian host include the eukaryotic promoter pCMV, a

transcription polyadenylation/termination signal BGH polyA and the pathogen gene of interest (N3). Green

fluorescent protein is the reporter gene for assessment of protein localization and expression. Images obtained

with ApE © software. ............................................................................................................................................ 31

Figure 13 - Schematic illustration of the two designed DNA vaccine vectors, pVAX-E1A-N3-GFP and pVAX-E1A-

N3-GFP-LAMP, constructed by insertion of the adenovirus e1a endoplasmic reticulum sequence in the

previously constructed plasmids pVAX-N3-GFP and pVAX-N3-GFP-LAMP.Images obtained with ApE © software.

.............................................................................................................................................................................. 36

Figure 14 - Agarose gel electrophoresis, 4% (A) and 2% (B), for the confirmation of annealing procedure. A:

forward ss oligo (lane 1), reverse ss oligo (lane 2), annealed oligos (lane 3). B: forward ss oligo 10 µM (lane 1),

reverse ss oligo 10 µM (lane 2), Annealed sequence 2 µM (lane 3), 10 µM (lane 4) and 20 µM (lane5), annealed

sequence 10 µM after purification (lane 6). M- DNA molecular weight marker- GeneRuler 50bp DNA Ladder

(Bioline). ................................................................................................................................................................ 46

Figure 15 - Melt peak analysis of single-stranded forward (A) and reverse (B) oligonucleotides and double-

stranded annealed oligonucleotide (C). The Tm obtained for the ds oligo was 88,77oC. ..................................... 47

Figure 16 - Confirmation in 1% agarose gel electrophoresis of constructed plasmids pVAX- E1A-N3-GFP-(A) and

pVAX-E1A-N3-GFP -LAMP (B). Lanes 1A and 1B correspond to control plasmids pVAX-N3-GFP and pVAX-N3-

GFP-LAMP, respectively. Lanes 2A and 3B correspond to clones further sequenced and confirmed to be the

correct constructions. M- NYZDNA ladder III (NYZTech). ...................................................................................... 49

Figure 17- Analysis of purity and quality of pVAX-N3-GFP by 1% agarose gel electrophoresis. Plasmid samples

were obtained after neutralization of the lysate (lane 1 and 2), precipitation with isopropanol and clarification

with ammonium sulphate (lanes 3 and 4) and purification through HIC-SEC (lanes 5, 6 and 7). Lane M:

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Molecular-weight marker (NYZ DNA Ladder III,NYZTech). Purified pDNA samples in lanes 5, 6 and 7 correspond

to 20.25 µg, 110.5 µg and 65 µg per total sample (500 µL), respectively. ............................................................ 50

Figure 18 – Agarose gel electrophoresis (1%): (A): Assessment of quality of plasmids purified by High Speed

Maxi Kit (Qiagen), where is possible to detect a high amount of sc pDNA, with traces of linear and open circular

isoforms. (B): Confirmation of plasmid constructions by enzymatic digestion with restriction endonucleases

XbaI and KpnI. pVAX-GFP (1A, 1B); pVAX-N3-GFP (2A, 2B); pVAX-E1A-N3-GFP (3A, 3B); pVAX-N3-GFP-LAMP (4A,

4B); pVAX-E1A-N3-GFP -LAMP (5A, 5B). M- Molecular-weight marker (NYZ DNA Ladder III,NYZTech). .............. 51

Figure 19 - Evaluation of transfection efficiency assessed by flow cytometry 48h post-transfection. Results from

one experiement with triplicates for each plasmid are displayed. Differences between transfection efficy of

different plasmids were compared by performing the one way anlysis of variance test ANOVA (P < 0.05). Error

bars determine standard deviation between triplicates. ..................................................................................... 53

Figure 20- Mean fluorescence assessed by flow cytometry analysis, 48h post-transfection. Results from one

experiement with triplicates for each plasmid are displayed. Differences between mean levels of fluorescence

of cells transfected with different plasmids were compared by performing the the one way anlysis of variance

test ANOVA (P < 0.05). PVAX-GFP, used as a positive of control on the trasfection experiments had a mean

fluorescence value of 1081 ± 350 (MF ± SD). AU- arbitrary units. Error bars determine standard deviation

between triplicates. .............................................................................................................................................. 54

Figure 21- Plasmid copy number per cell determined 48h post-transfection, by quantitative Real-Time PCR.

Data displayed represents results from two independent assays, with three replicates for each plasmid

construct. In order to compare pDNA copies in cells transfected with the different plasmids, one way ANOVA

was performed, with a level of confidence of P < 0.05. ........................................................................................ 56

Figure 22- Plasmid DNA quality assessment by 1% agarose gel electrophoresis: pVAX-GFP (lane 1), pVAX-N3-

GFP (lane 2), pVAX-E1A-N3-GFP (lane 3) and pVAX-E1A-N3-GFP-LAMP (lane 4). M- DNA Molecular weight

Marker Hypperladder I (Bioline). .......................................................................................................................... 57

Figure 23 - Antibody production, measured by absorbance at 450 nm, against N3 antigen in chickens

immunized intramuscularly with N3, E1A-N3, N3-LAMP and E1A-N3-LAMP constructs (groups of 3 chickens). A

non-immunized chicken (CN) and two chickens immunized with pVAX-GFP vector (pVAX) represent the

negative controls. Immunizations with 100 µg of the DNA vaccines were carried out at days 0, 14 and 28 ( ).

Protein boost with 100 µL of purified N3 protein was administered at day 52 ( ). Data is representative of five

independent ELISA assays. Vertical bars represent standard deviation between measurements of each data

point. Antibody titers of each DNA vaccine were compared with the negative control and between each other.

Significant differences were analyzed by the unpaired two-tailed Student’s t-test. P < 0.05 was considered

statistically significant. .......................................................................................................................................... 61

Figure 24- Schematic diagram of pVAX1/LacZ. Source:

http://www.lifetechnologies.com/order/catalog/product/V26020 ..................................................................... 77

Figure 25- Growth curves for E.coli DH5α cells harboring pVAX-N3-GFP-LAMP and pVAX-N3-GFP. Cell culture

in Erlenmeyer flasks, under orbital agitation of 250 rpm and 37oC. ..................................................................... 78

Figure 26- Standard curves for the absolute quantification of plasmid copy number in transfected CHO cells,

obtained using pDNA masses ranging from 5 pg to 50000 pg per reaction, spiked with 12500 non-transfected

cells. Regression analysis is presented by r2 values. ............................................................................................. 79

Figure 27- Standard curves for the absolute quantification of mRNA copy number in transfected CHO cells,

obtained using pDNA masses ranging from 5 pg to 50000 pg per reaction. Regression analysis is presented by r2

values. ................................................................................................................................................................... 80

Figure 28- Analysis of functional domains of the N3 protein structure. Intracellular, extracellular and

transmembranar domains are discriminated. Source: http://www.cbs.dtu.dk/services/TMHMM/ .................... 81

Figure 29- DNA molecular weight marker HypperLadder TM

50 bp (Bioline®). Source and further product details:

http://www.bioline.com/h_prod_detail_ld.asp?itemid=152 ............................................................................... 82

Figure 30- DNA molecular weight marker NZYDNA Ladder III (NZYTech®). Source and further product details:

https://www.nzytech.com/site/vmchk/DNA-Markers/NZYDNA-Ladder-III .......................................................... 82

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List of tables

Table 1 - Adapted from (Bouvier & Palese 2008); The genome, the encoded proteins, and respective functions,

of avian influenza (A/Puerto Rico/1938/ H1N1). The PB2, PA, HA, NP and NA proteins are each encoded on a

separate RNA segment. The M2 and NEP are both expressed from spliced mRNAs, while PB1-F2 is encoded in

an alternate reading frame. .................................................................................................................................... 5

Table 2- The safety concerns regarding DNA vaccines and possible solutions. Adapted from (Glenting & Wessels

2005). .................................................................................................................................................................... 25

Table 3- Primers used in automated sequencing of plasmid DNA constructs ...................................................... 36

Table 4 - Real-time PCR forward and reverse primer sequences for assessment of eGFP gene presence. ......... 41

Table 5- Sequences of forward and reverse oligonucleotides used for the construction of the E1A insert. ....... 45

Table 6 - Expected fragment sizes of correct plasmid constructions, obtained by ligation of E1A sequence and

base vectors, after digestion with restriction enzymes MluI and NheI. ............................................................... 48

Table 7 - Expected fragment sizes, obtained with ApE© software, after double enzymatic digestion with

restriction enzymes KpnI and XbaI, for confirmation of correct pDNA vaccine vectors. ...................................... 51

Table 8- Determination of mRNA content per ng of total RNA, 48h post-transfection of CHO cells. Results

presented are the mean of three replicates per plasmid in one quantitative reverse transcription RT-PCR assay.

SD- Standard Deviation between triplicates. ........................................................................................................ 58

Table 9 – Comparison of parameters obtained by in vitro experiments for the five studied plasmids:

Transfection efficiency and mean fluorescence, obtained by flow cytometry analysis; Plasmid copy number and

mRNA content, assessed by RT-PCR. .................................................................................................................... 59

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List of abbreviations

AI- Avian influenza

AIV - Avian influenza virus

APC - Antigen presenting cell

BGH - Bovine growth hormone

bp - Base pair

CCL2 - Chemokine (C-C motif) ligand 2

CCR2 - C-C chemokine receptor type 2

cDNA - complementary deoxyribonucleic acid

cRNA – complementary ribonucleic acid

CHO - Chinese hamster ovary

CpG - Cytidine poly-guanine nucleotides

Ct - Threshold cycle

CTL – cytotoxic T lymphocyte

DC- denditric cells

DCW- dry cell weight

DMSO - Dimethyl sulfoxide

EDTA - Ethylenediamine tetraacetic acid

eGFP - enhanced GFP

ER - Endoplasmic reticulum

E1A - Adenovirus early region 1A

FBS - Foetal bovine serum

FDA - Food and Drug Administration

FSC - Forward scatter

GFP - Green Fluorescent Protein

HA – Hemagglutinin

HIC - Hydrophobic Interaction Chromatography

HPAI - Highly pathogenic avian influenza

IFN - interferon

IL - Interleukin

IPTG - Isopropylthio-β-galactoside

ISGF3- Interferon-stimulated gene factor 3

Kan – Kanamycin

kb - Kilobase

LAMP - Lysosome associated membrane proteins

LB - Luria Broth

LPAI - Low pathogenic avian influenza

LP - Low pathogenic

MCS - Multiple cloning sites

MEM - Modified Eagle’s Medium

MHC - Major histocompatibility complex

mRNA - Messenger ribonucleic acid

M1 - Matrix protein

M2 - Integral membrane protein

NA - Neuraminidase

NEAA - Non-essential amino acids

NEP - Nuclear export protein

NK - Natural killer cells

NLRP3 - NOD-like receptors pyrin domain containing 3

NP - Nucleocapsid protein

NS1 - Non-structural protein 1

NS2 – Non-structural protein 2

Oc- open circular

OD - Optical density

ORF - Open reading frame

PA - Polymerase acidic protein

PAMP – pathogen-associated molecular pattern

PB1 - Polymerase basic protein 1

PB2 - Polymerase basic protein 2

PBS - Phosphate buffered saline

pCMV - Cytomegalovirus promoter

pDNA - Plasmid DNA

PFA - Paraformaldehyde

PLAP - Placental alkaline phosphatase

PolyA – Polyadenylation

RNA - Ribonucleic acid

PRR – pattern recognition receptor

RIG-I - retinoic acid inducible gene-I

RNP - Ribonucleoprotein

RRT-PCR - Real time reverse transcription -

polymerase chain reaction

RT-PCR - Real time - polymerase chain reaction

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SEC – Size exclusion chromatography

Sc - supercolied

SSC - Side scatter

TAE - Tris-acetate-EDTA

TAP - Transporter associated with antigen

presentation

TCR - T-cell receptor

Th - T helper

TLR - Toll-like receptor

TMB – Tetramethylbenzidine

TNF – tumor necrosis factor

TPA - Tissue plasminogen activator signal

tRNA – Transfer RNA

UV - Ultraviolet

1

I. Literature Review

I.1. Avian Influenza Virus

All avian influenza virus subtypes are classified as Influenza virus type A. Influenza virus type A belongs

to the Orthomyxoviridae family along with the other genera influenza viruses, type B and C, and thogotovirus

(Lee & Saif 2009). One of the most significant differences between the three types of influenza viruses is their

range of hosts: influenza viruses type B and C are predominantly human pathogens, despite their occasional

detection and isolation in other species, such as seals (type B) and pigs (type C). On the contrary, influenza type

A natural host reservoirs consist mainly of water birds (ducks and shorebirds) but has also been isolated in non-

natural host species such as other avian species (chickens and turkeys), humans, pigs, horses, mink and marine

mammals and species of domestic and wild birds (Lee & Saif 2009). Avian influenza viruses are typically

nonpathogenic in wild aquatic birds, but the morbidity and mortality caused by them can increase significantly

when transmission to other species, such as domestic birds and mammals, occurs (Fouchier et al. 2005).

The disease caused by avian influenza virus can result in dramatically different effects, ranging from

asymptomatic infection to severe and potential fatal disease (Lee & Saif 2009). Besides characterization by

genetic profile, avian influenza viruses are also characterized by their ability to cause disease in poultry

(domesticated chicken, turkeys, ducks and geese), which are an important link between wild birds and humans

and eventually lead to a pandemic (Boyce et al. 2009).

Pandemic influenza is defined as a global outbreak of respiratory disease in humans caused by the

introduction of a novel Influenza A genotype to which the human population have no pre-existing exposure

and immunity (Boyce et al. 2009). An example of this is the emergence of high pathogenic H5N1 human

infections, mostly acquired from poultry, a great threat to human health, with a fatality rate of 60%, since 2003

(R. Gao et al. 2013)(Xu et al. 2011). In 2006, this influenza strain caused 97 human deaths, in a total of 176

reported cases of illness, across seven countries (Poland 2006). More recently, infection of humans with the

novel avian-origin H7N9 virus (R. Gao et al. 2013)(Li et al. 2013), a product of reassortment of viruses of avian

origin only, was observed for the first time in China, since human infection with N9 virus subtypes was never

previously documented(R. Gao et al. 2013). Besides the potential for human pandemics and importance

regarding animal health, outbreaks of avian influenza in poultry industries and independent farming result in

severe economic losses for the sector and in the number of birds that died or were killed under control policies

(Lupiani & Reddy 2009)(Halvorson et al. 2003) (Kamps et al. 2006) (Alexander 2000).

Based on severity criteria, these viruses can be classified as either high pathogenic avian influenza

(HPAI) viruses or low pathogenic avian influenza (LPAI) viruses (Boyce et al. 2009) (Swayne 2009), the latter

accounting for the majority of AIV detected in the natural reservoirs (wild aquatic birds). One of the major

differences between these two classes is the ability to cause systemic versus local replication (Lee & Saif 2009).

Subtypes H5 and H7 are usually related to HPAI, although most viruses of these subtypes are considered to be

of low pathogenicity (Boyce et al. 2009) (Alexander 2000). The hemagglutinin (HA) glycoprotein cleavage,

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further discussed below, is a major determinant of viral pathogenicity in birds. In LPAI viruses, the cleavage of

HA occurs extracellularly and only in tissues where appropriate proteases are present, which restricts the

replication, infectivity and spread of the virus, resulting in localized and therefore less severe infections, usually

in gastrointestinal system of the wild birds. On the contrary, the HA of HPAI viruses have a multibasic cleavage

site and are cleaved intracellularly by intracellular subtilisin -like proteases, a ubiquitous protease (Lee & Saif

2009). This results in fatal systemic infection in poultry, since the virus can replicate in a broad range of host

cells, damaging vital organs and resulting in death (Elsevier 2006) (Alexander 2000).

Transmission of the AI virus usually occurs through fecal-oral transmission, through feces and

contaminated water (Boyce et al. 2009) (Kamps et al. 2006). HPAI are more commonly found in the respiratory

system of birds and transmission by inhalation in the wild bird populations has been suggested (Boyce et al.

2009)(Spekreijse et al. 2011).

The three influenza viruses’ classification is based on the antigenic differences among the matrix

proteins (M) and the nucleocapsid proteins (NP). Influenza viruses type A are further characterized by the

serological subtype and antigenic properties of the glycoproteins expressed in the surface of virus particles,

hemagglutinin (HA) and neuraminidase (NA). The extended surveillance conducted regarding several avian

species resulted in the identification of a total of 16 different HA subtypes (H1-H16) and 9 NA subtypes (N1-N9)

(Bouvier & Palese 2008) (Fouchier et al. 2005), numbers that are likely to increase, due to extreme antigenic

variation of these surface glycoproteins. Recently, an influenza virus subtype, H17N10, was identified (Zhu et al.

2012). The hemagglutinin and neuraminidase genes present high variability in their sequences, only a small

percentage (less than 30%) of the amino acids are conserved in all the virus subtypes (Lee & Saif 2009). The

major feature of protective immune response against influenza A is the antibodies produced against these two

surface proteins, although there has been detectable humoral responses against some internal proteins (Košík

et al. 2012)(Xu et al. 2011).

Type A influenza is characterized by a high mutation rate, a common feature in RNA viruses.

Reassortment, due to mixing of gene segments of different strains and lineages during simultaneous infection,

is another mechanism of generation of variability in the viral genome (figure 1). This high genetic variability

results in a high adaptability and ability to evade the immune system by antigenic drift and antigenic shift

(Spackman 2008). Antigenic drift refers to accumulation of mutations in the glycoproteins NA and HA, due to

minor changes, such as amino acid substitution in one of the glycoproteins. On the other hand, antigenic shift

represents a more drastic and profound evolvement, resulting in the formation of virus subtype, when HA

and/or NA from one subtype are mixed or switched with another subtype, within the same replication cycle (Xu

et al. 2011) (Kamps et al. 2006).

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I.1.1. Influenza A Virion structure

Shape and size of influenza A virions vary accordingly to virus strain and passage history (Lee & Saif

2009). They are usually filamentous or spherical in shape and their size can vary from 80 to 120 nm in diameter

for the spherical forms and about 300 nm for the filamentous forms (Bouvier & Palese 2008).

The most distinct feature of the virus is the two types of glycoprotein projections, about 500

approximately, that detach from the lipid external virion bilayer (figure 2). This surface spikes, rod-shape

trimmers of hemagglutinin and mushroom-shape tetramers neuraminidase, exist in a ratio of four to one.

During virus replication, the HA protein is cleaved, by serine proteases, into HA1 and HA2. This post-

translational modification is essential to virus infectivity. The function of HA2 portion is thought to be

mediation of the fusion of virus envelope with cell membranes, while the HA1 portion contains the receptor

binding and antigenic sites (Bouvier & Palese 2008). HA is the primary viral antigen to which cells of the

immune system, by antibody production, respond to. The virus infectivity is usually neutralized by antibodies to

HA, so virus strains in order to evade this specific host defense, present frequent minor amino acid changes in

the antigenic sites, a process of antigenic drift. The accumulation of these alterations in multiple antigenic sites

will eventually result in the ineffectiveness of host antibodies against that virus strain (Elsevier 2006). The NA

glycoprotein has enzymatic activity, cleaving sialic acid residues from glycoproteins or glycolipids on the host

cell surface. The cleavage of sialic acid receptors, performed by NA, mediates the release of the new viral

particles from the infected cell. The virus envelope also contains a small number of copies of a third membrane

protein, the M2 protein, a tetramer that transverses the lipid envelope and has ion channel activity. These

three integral membrane proteins coat a matrix of M1 protein, which envelops the virion core and is thought to

play a vital role in virus assembly. With approximately 3000 copies per virion, the M1 protein is the most

Figure 1- The processes of evasion from the immune system after influenza virus

infection /vaccination. Due to selective pressure, Antigenic drift and antigenic

shift lead to antigenic changes in the circulating virus. Source: WHO (World

Health Organization) Collaborating centre for Reference and Research on

Influenza.

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abundant structural protein of influenza virus. Inside this envelope of M1 protein are found the nuclear export

protein (NEP) which also goes by the name of nonstructural protein 2 (NS2) and the ribonucleoprotein (RNP)

complex. This complex, the minimal functional unit of replication, consists of the viral RNA segments coated

with nucleoprotein (NP) and the RNA polymerase, composed of two “polymerase basic” and one “polymerase

acidic” subunits: polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2) and polymerase acidic

protein (PA) (Bouvier & Palese 2008).

I.1.2. Viral core/ genome structure

The influenza A virus genome consists of eight segments of negative-sense single-stranded viral RNA

(vRNA) (table 1). Influenza A RNA segments 1 to 6, with the exception of segment 2, encode one protein each,

the PB2, , PA, HA, NP and NA, respectively. Segment 7 and 2 encode two proteins each: M1 and M2 (by RNA

splicing) with overlapping reading frames and PB1 and PB1-F2, respectively. Segment 8 encodes for the non-

structural proteins NS1 (absent in virions but abundant in infected cells) and by mRNA splicing the NEP/ NS2,

which is involved in the viral RNP export from the host cell nucleus. Of the 11 proteins encoded by the 8 viral

RNA segments, only the NS1 protein is truly non-structural and is not packaged into the virion, functioning as a

regulatory protein in processes such as mRNA splicing and translation and a competitor against target cell’s

antiviral defenses. This feature makes the NS1 protein directly related to the pathogenicity of the influenza

virus strain and a possible target for vaccine development (Lee & Saif 2009).

The segmented genome displayed by the avian influenza virus facilitates the occurrence of antigenic

shift. This phenomenon causes an influenza A virus strain to acquire the HA segment and possibly the NA

segment as well of a different influenza A virus strain, resulting in the reassortment of segments and the

encoding of novel antigenic proteins by the resulting virus to which the host has no preexisting immunity.

Pandemic influenza is the result of a virus generated by antigenic shift, to which humans are immunologically

Figure 2 - Influenza A virion structure. The three envelope

proteins: Glycoproteins NA (neuraminidase) and HA

(hemagglutinin) and ion-channel M2 protein. M1 protein;

NEP/NS2 (nuclear export protein); The minimal unit of

replication, the RNP (ribonucleoprotein), is composed by

RNA segments coated with the nucleoprotein (NP) and

the RNA polymerase (PB1, PB2 and PA). Horimoto et al.

(2005).

5

naïve. One example of the lethality of this phenomenon is the “Spanish flu” (1918-1919), caused by the

influenza A (H1N1) virus, which killed about 50 million people worldwide. This high pathogenicity and global

spread was probably caused by the acquisition of novel surface proteins with unique antigenic properties to

which the human immune system of population at the time was naïve and therefore unprepared and unable to

respond efficiently (Bouvier & Palese 2008).

I.1.3. The influenza virus replication cycle

Influenza viruses recognize sialic acid (nine-carbon acidic monosaccharides) on the target host cell

surface. Virus attachment begins when the viral HA binds to the N-acetylneuraminic (sialic) acid residues on

glycoproteins or glycolipids on the cell surface. Being commonly found at the termini of many glycoconjugates,

sialic acids are ubiquitous not only in several cell types but also in different animal species. The nature of the

glycosidic linkage between the carbon of the terminal sialic acid and the carbon of the galactose residue on the

receptor will influence the specificity of the HA receptor binding. There are two types of linkage that can be

formed, α-2, 3-linkage (the carbon-2 of the sialic acid binds to the carbon-3 of galactose) or α-2,6-linkage (the

carbon-2 of the sialic acid binds to the carbon-6 of galactose). When the HA spikes recognize and bind the sialic

acid there is a preferential specificity for one linkage over the other type. Since the α-2,6-linkage predominates

in human tracheal epithelial cells, human influenza viruses have a preference for sialic acids attached to

galactose in a α-2,6 configuration. The α-2, 3 linkage is more common in duck gut epithelium, so naturally avian

influenza viruses show preference for this configuration (Bouvier & Palese 2008). There are also α-2,3 linkages

in human cells, but its low predominance explains on one hand why humans and other primates can be

infected with avian influenza viruses but, on the other hand, the low efficiency of its infection. It is also

Table 1 - Adapted from (Bouvier & Palese 2008); The genome, the encoded proteins, and respective functions, of avian influenza

(A/Puerto Rico/1938/ H1N1). The PB2, PA, HA, NP and NA proteins are each encoded on a separate RNA segment. The M2 and NEP are

both expressed from spliced mRNAs, while PB1-F2 is encoded in an alternate reading frame.

RNA Segment Encoded protein(s) Protein function

1 PB2 Polymerase subunit: mRNA recognition

2 PB1 Polymerase subunit: RNA elongation, endonuclease activity

PB1-F2 Pro-apoptotic activity

3 PA Polymerase subunit: protease activity

4 HA Surface glycoprotein: major antigen, receptor binding and fusion activities

5 NP RNA binding protein: nuclear import regulation

6 NA Surface glycoprotein: sialidase activity, virus release

7 M1 Matrix protein: vRNP interaction, RNA nuclear export regulation, viral budding

M2 Ion channel: virus uncoating and assembly

8 NS1 Interferon antagonist protein: regulation of host gene expression

NEP/NS2 Nuclear export of RNA

6

important to mention that there is a differential distribution of sialic acids in the human respiratory tract. The

α-2,3 linked sialylated proteins are more prevalent in the lower respiratory tract, which can explain the high

pathogenicity of some avian strains in humans, in spite of its low infectivity. Although fairly rare, infection in

humans by avian influenza viruses is therefore severe and potentially fatal.

After the attachment of the viral HA to the sialic residues on the target cell, the virus is endocytosed

(figure 3). In this process of uptake of the virus by receptor-mediated endocytosis, one of the key factors is the

acidity of the endosomal compartment, maintained by proton pumps in the endosomal membrane. It is the

low pH inside the endosomes (pH 5-6) that allows the uncoating of the influenza virus and the fusion reaction

between the viral envelope and the endosomal membrane. At low pH, there is a major conformational change

that occurs in the HA spike. This change consists in the exposure of the fusion peptide sequences of HA2 that

mediates the merging of the viral envelope with the target membrane. The fusion of the two membranes

results in the formation of a fusion pore, the entry site of the viral RNPs into the host cell cytosol (Elsevier

2006). The acidification of the viral interior is also crucial to the release of the viral RNPs to the cell cytoplasm.

The internal acidification of the virion, mediated by the M2 ion channel, results in the disruption of internal

protein-protein interactions, allowing the release of viral RNPs from the viral matrix into the cellular cytoplasm.

Once in the host cell cytosol, the RNP complexes are transported to the cell nucleus by nuclear

localization signals (NLSs), amino acid sequences that direct cellular proteins to import the RNPs and other viral

proteins into the host cell nucleus. The nucleus is the location of all influenza virus RNA synthesis. The negative-

sense viral RNAs are templates for the synthesis of positive-sense mRNAs by the viral RNA polymerase (PB1,

PB2 and PA), carried with the RNP complex. In a process called “cap snatching”, the PB1 and PB2 proteins steal

short 5’ capped regions from cellular mRNAs, required for efficient binding of ribosomes to the RNA, that serve

as primers for initiation of viral mRNA synthesis. The mRNAs, capped and polyadenylated, are then transported

back to the cytoplasm, where translation to protein occurs like host cell RNA. This process of cap snatching is

essential for the production of viral components, inhibiting the synthesis of host cell proteins. The RNA

polymerase also uses vRNA as template for the production of another positive-sense RNA species,

complementary RNA intermediates (cRNA), which are transcribed into multiple new copies of negative-sense

viral RNAs. These segments are exported back to the cytosol for assembly of new virus particles, a process

regulated by the viral proteins M1 and NEP/NS2. The NS1 protein has an important role in the inhibition of

production of host-mRNAs. This suppression is achieved by the inhibition of 3’-end processing of host-pre-

mRNAs. This mechanism not only alters the trafficking of host-mRNAs to the cytoplasm, but also interferes with

the production of immune system proteins such as interferon-β. The viral mRNAs do not require this

processing, so they are successfully transported to the cytoplasm for translation into viral proteins (Das et

al.2010). The growing polypeptide chains of envelope proteins HA, NA and M2 are transported to the

endoplasmic reticulum for glycosylation and folding into trimmers and tetramers. The proteins are then

trafficked to the Golgi apparatus for post-translational modifications. The three proteins, containing apical

sorting signals, are directed to the cell membrane for virion assembly. The M1 protein is thought to play a

crucial role in the assembly of RNP-NEP complex with the three envelope-bound proteins (Lee & Saif 2009).

7

The infectivity of Influenza virus is dependent on the correct virion packaging of a full genome of eight

RNA segments, a selective process, in which packaging signals on all RNA segments insure full genome

packaging into the majority of virus particles. Accumulation of M1 matrix protein at the cytoplasmic side of the

lipid bilayer induces virus budding. After budding of viral particles is completed, the viral NA cleaves the sialic

acid residues that are still interacting with the HA spikes, thus holding the virions attached to the cell

membrane. Besides its crucial sialidase activity in infection of host cells, NA also removes sialic acid residues

from the virus envelope, avoiding viral particle aggregation and it also performs a theorized role in aiding virus

Figure 3- Representation of Influenza A virus replication cycle. A) Virion structure and binding of

HA to the sialic acid residues in the host cell receptors; B) Virus is transported in the cell in a

endocytic vesicle. The low pH in the endossome triggers important mechanisms that lead to the

release of the vRNPs into the cytoplasm and their subsequent import into the cell nucleus; C)

Once in the nucleus, the synthesis of viral mRNA is initiated by the viral RNA polymerase by a

process (“cap snatching”) in which 5’- capped RNA fragments are cleved from host pre-mRNAs; D)

Viral mRNAs are transported to the cytoplasm, where they are translated into viral proteins. HA,

NA and M2 are processed in the ER and glycosilated in the Golgi apparatus. These proteins are

finnaly transported to the cell membrane F) The new synthesised vRNPs are transported to the

cytoplasm; G) Once in the cell menbrane, vRNPs are incorporated in the newly formed virions,

with the enveloped proteins, The virions are released from the host cell, after the NA protein

cleaves the ligation with the sialic acid residues. E) NS1 protein role in blocking production of

host-mRNAs. Adapted from Das et al. (2010).

8

infectivity by breaking down and removing the mucins, cilia and cellular glycocalyx in the respiratory cells

secretions, thus allowing the virus particules to enter the respiratory ciliated epithelium. Host antibodies and

inhibitor drugs directed against NA do not neutralize virus infection, instead prevent virus release from host

cell membranes and spreading of new formed viral particles, thus aiding in the inhibition of viral replication

(Matrosovich et al. 2004). Once released, virions can spread further throughout the respiratory tract.

I.1.4. Host immune response

The major function of the immune system is to recognize and eliminate foreign and harmful

microorganisms. This elaborate defense is composed of two main functional elements, the innate and the

adaptive immune response, which contrast by their time of response and mechanisms of pathogen recognition

(Spackman 2008).

I.1.4.1. Innate immune response

Besides its role as first physical barrier of defense, constituted by mucus and collectins, in the war

against invader pathogens, the innate immune system, which lacks specificity and memory, also provides

important signals for the more specific and organized cellular and humoral responses, the adaptive immune

response (García-Sastre 2006). The innate immune response is therefore responsible for the definition of the

overall immune response (Spackman 2008).

Influenza viruses primarily target the epithelial cells that line the respiratory tract. The initial responses

of the innate immune system derive from germ-line encoded receptors, the pattern recognition receptors

(PRRs), that recognize evolutionary conserved molecular markers in infection microbes, the pathogen-

associated molecular patterns (PAMPs) (van de Sandt et al. 2012). This response is characterized by the

production of interferons (IFN), cytokines and chemokines. The three main types of PRR involved in the

recognition of influenza virus type A are Toll-like receptors (TLR), the first to recognize influenza virus infection,

retinoic acid inducible gene-I receptors (RIG-I) and NOD-like receptors pyrin domain containing 3 (NLRP3). The

signaling pathways associated with the activation of such receptors result in the transcription of pro-

inflammatory cytokines (such as tumor necrosis factor TNF), chemokines and IFNs that are essential to the

recruitment of neutrophils, activation of macrophages and maturation of dendritic cells (DCs) (García-Sastre

2006).

Type I interferon (IFN α/β) response is an important and almost immediate element of the innate

immunity of animal cells against viral pathogens and includes a complex regulation of positive and negative

feed-back mechanisms. These molecules can be produced by all nucleated cells in response to viral infection

(Koyama et al. 2008). The synthesis and secretion of these molecules results in the generation of robust cellular

and humoral immune responses and of transcription factor ISGF3 (van de Sandt et al. 2012), resulting in the

induction of transcription of several antiviral genes (PKR, p56, PML, ADAR, etc.) (García-Sastre 2006). Their

9

antiviral properties include translation inhibition, induced apoptosis, RNA degradation, among others (van de

Sandt et al. 2012).

Influenza viruses are poor inducers of the INF α/β response (García-Sastre 2006), due to mechanisms

of evasion. Such is the importance of type I interferon in establishing antiviral immunity, that avian influenza

virus has evolved to produce a potent antagonist, the NS1 protein (McGill et al. 2009)(García-Sastre 2006). The

NS1 protein gene of the influenza A virus encodes the antagonist function of this type of response, mainly to its

capability to prevent INF β synthesis, by suppressing host-mRNA production through inhibition of the 3’-end

processing of host pre-mRNAs (Das et al.2010), and also attenuates the activation of different transcription

factors during viral infection. This is just an example of acquisition of viral genes during evolution that

antagonize the sophisticated immune response, allowing viruses to evade such responses and continuously

infect and cause disease.

The permanent vigilant and ready to respond cell types of the innate immune response against

influenza A viruses are essentially the alveolar macrophages and dendritic cells, the two primary phagocyte

populations in the lung (McGill et al. 2009), derived from a myeloid progenitor, and natural killer (NK) cells,

differentiated from the lymphoid lineage precursor, that shape the adaptive immune responses toward Th1-

type immunity (Sirén et al. 2004).

Alveolar macrophages are thought to have a regulatory role, existing in a quiescent state during

homeostasis (van de Sandt et al. 2012). Production of CCL2 (Chemokine (C-C motif) ligand 2) by the infected

epithelial cells during the initial phase of the influenza virus infection, attracts alveolar macrophages and

monocytes, which differentiate into monocyte-derived DCs and macrophages, through the CCR2 receptor. The

activated macrophages are responsible for the robust production of inflammatory cytokines, such as IL-6 and

TNF-α (McGill et al. 2009). Alveolar macrophages, on one hand, have a decisive role in limiting viral spread and

regulating adaptive immunity, especially towards the production of CD8+ T cells, but on the other hand,

through the production of NOS2 and TNF-α, contribute to excessive inflammation and have a deleterious effect

on lung tissue (McGill et al. 2009).

Dendritic cells (DCs) are professional antigen presenting cells that have an essential role in innate and

adaptive immune responses. Virus-infected DCs, through the triggering of pattern recognition receptors (such

as TLRs), produce inflammatory cytokines and antiviral interferons in response to viral infection and activate NK

cells (Eisenächer et al. 2007). The antigen processing and presenting functions of DCs are one of the

foundations of an efficient adaptive immune response. The priming of naïve T cells in vivo is the sole

responsibility of dendritic cells, so it makes sense to focus great importance on the antigen-processing

capabilities and mechanism of this cell type (Howarth & Elliott 2004) (figure 4). DCs acquire antigen via two

possible mechanisms: through direct infection with the virus (endogenous origin) or through phagocytosis of

infected apoptotic epithelial cells or viral particles (exogenous origin) (McGill et al. 2009) (Kutzler & Weiner

2008). The first mechanism leads to the degradation of viral proteins into small peptides by the proteasome,

that are transported to the endoplasmic reticulum (ER) via TAP (transporter of antigen processing), where they

are loaded into MHC I molecules. These molecules are transported via the Golgi apparatus to the cell

membrane, where they are presented and recognized by the CD8+ T cells. The second mechanism results in the

10

degradation of viral proteins in endosomes or lysosomes and presentation of antigen in MHCII molecules in the

cell membrane to CD4+ helper cells. These cells interact with B cells and CD8

+ T cells. The acquisition of

exogenous antigen by APCs can also result in cross-presentation, in which DCs endocytise viral particles and

antigens, which undergo proteolytic degradation and bind to MHC I molecules (Zajac 2008). The resultant

epitopes are presented to CD8+ T cells. Migration of DCs from the lungs to the draining lymph nodes, for

presentation to virus-specific T-cells, is a key step in the initiation of adaptive immune responses.

I.1.4.2. Adaptive immune response

Adaptive immunity constitutes a slower but more specific and potent response to viral infection,

consisting of humoral and cellular immunity and resulting in the formation of immunological memory, which

protects the host from subsequent infections (Zajac 2008) (figure 5).

Humoral immunity results, primarily, in the production of antibodies against viral glycoproteins NA

and HA (van de Sandt et al. 2012). Antibodies directed to HA prevent virus attachment to host cells and block

receptor-mediated endocytosis, neutralizing virus infectivity (Knossow & Skehel 2006). Antibodies directed

against NA, as discussed before, prevent release and spread of newly formed viral particles, shortening the

severity and duration of the disease (Matrosovich et al. 2004) (van de Sandt et al. 2012). M2 transmembrane

protein also elicits the formation of specific antibodies, that may be effective against different subtypes, since

Figure 4- Dendritic cells (DCs) functional MHC I and MHC II presentation pathways

(Villadangos & Schnorrer 2007). DCs have a dominant role in the presentation of antigens to

naïve T lymphocytes and, therefore, priming the immune response. MHC I molecules mainly

present endogenous antigen via dependent or independent proteasome and TAP pathway.

MHC II molecules mainly acquire peptides processed in endosomal compartments but also

endogenous components of the endocytic pathway or that enter the endosome by

autophagy. Cross-presentation is a phenomenon that consists on the ability of DCs to deliver

exogenous antigens to the MHC I pathway.

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the protein is highly conserved between different influenza A subtypes, as occurs with the antibodies produced

after infection against NP (Elsevier 2006) (van de Sandt et al. 2012). Only antibodies directed against HA can

prevent infection (figure 6).

Cell-mediated immunity is an equal important feature of adaptive immunity in the combat against

viral infection and it is inducted by CD4+ T cells and CD8

+T cells. The main difference between these two cells

types, is that T-cell receptors on CD4+ T cells recognize MHC II molecule-peptide combination, present on

dendritic cells, macrophages and B cells, instead of peptides loaded into MHC I molecules, which are

ubiquitously expressed in all cells (Zajac 2008). CD4+ T cells, also called T helper cells, have the ability to help

both B cells and CD8+ T cells, resulting in antibody production, class switching, cytotoxic T cell activity and

immunological memory. They are also essential in the fight against viruses, in the sense that they produce IFN-

γ and perforins, inducing lysis of infected cells (Zajac 2008). Based on their cytokine production profile, CD4+ T

cells can be differentiated in Th1, which produce IFN- and IL-2, among others, and promote CTL responses and

induction of memory CD8+ T cells (van de Sandt et al. 2012), and Th2 helper cells, which produce for instance

IL-4, IL-5 and IL-13 and are involved in antibody production (Zajac 2008). The differentiation of naïve CD4+ T

cells into effector T helper cells is regulated by two factors, T cell receptor engagement and the presence of a

certain type of cytokines. IL-12 and IFN- γ lead to a differentiation in Th1 cells, and IL-4 triggers a Th2 response

Figure 5- Illustrative diagram of induction of cellular and humoral immunity upon influenza A viral

infection. Solid arrows correspond to induction of immune responses following primary IA viral

infection. Dotted arrows relate to immune memory mechanisms activated upon secondary infection

with the virus, in which a more rapid response is generated by virus-specific cells. (van de Sandt et

al. 2012).

12

(Korn et al. 2009) (figure 5). This profile of CD4 +

T cells also features other class of helper cells, the Th17 cells,

which produce IL-17 and in the case of infection by influenza type A, counteraction of secondary bacterial

infections (van de Sandt et al. 2012) (Korn et al. 2009) and regulatory CD4 T cells that prevent autoimmunity by

suppression of auto reactive T cells (Zajac 2008).

I.2. DNA vaccines

Vaccination is the primary method for control of the influenza disease (Košík et al. 2012). The lack of

proofreading activity by the RNA polymerase leads to the recurrent appearance of several mutations in the

progeny virus (Deng et al. 2012) (van de Sandt et al. 2012).The unpredictable phenomenons of antigenic drift

and antigenic shift lead to conformational changes in the epitopes recognized by the neutralizing antibodies,

which renders the existing vaccines inefficient and consequently influenza vaccines must be updated every year

(Košík et al. 2012). The majority of vaccines in use are directed against HA, since humoral immune responses

are directed primarily against HA and in a lesser level against NA and M2 (Swayne 2009).

DNA vaccines consist in the expression of exogenous genes in vivo through a delivery platform, such as

naked plasmid DNA (Sun et al. 2013). This new generation of vaccines has been applied to several conditions,

from cancer (Trimble et al. 2009) and autoimmune diseases to infectious diseases caused by pathogens of viral

(Lim et al. 2012) (Laddy et al. 2009),parasitic (Dobaño et al. 2007) and bacterial origin (Ingolotti et al. 2010). The

first licensed DNA vaccine was targeted to West Nile virus for immunization in horses (2005). Other licensures

include vaccine targets Infectious haematopoietic necrosis virus for salmon (2005), Growth hormone releasing

Figure 6- Antibody immune response to avian influenza viruses. Antibodies

against HA prevent virus attachment. This is the only immune response that

prevents infection. Antibodies against NA prevent release of virions, binding

the virus to the infected cell. Antibodies against M2 prevent the release of viral

particles to the extracellular space (Subbarao et al. 2007).

13

hormone (2007) for swine and other agricultural animals and melanoma for dogs (2007) (Kutzler & Weiner

2008).

This method of vaccination presents several advantages in comparison to more traditional methods

currently in use and its rapid development as resulted into a variety of human clinical trials (Trimble et al. 2009)

(Fioretti et al. 2010) (Ferraro et al. 2011) (MacGregor et al. 1998) (figure 7).

Whole inactivated virus vaccines, the most widely used influenza vaccines (Xu et al. 2011), only confer

optimized protection to the circulating strain of the virus (Kim et al. 2012) (Xu et al. 2011), therefore it would

be ideal to produce a vaccine that elicits antibody response against different influenza subtypes, providing

broad cross-strain protection. Other types of vaccines in use include Subunit vaccines of surface glycoproteins,

hemagglutinin (HA) but also neuraminidase (NA) and the M2 surface protein (Deng et al. 2012), and live

attenuated vaccines (Košík et al. 2012), which have the advantage of providing antibody response against

circulating influenza and cellular immunity against viral proteins which remain conserved among different

subtypes (Xu et al. 2011). The major drawback of this type of vaccine is the possibility of reversion back to the

natural virus.

I.2.1. Structure and mechanism of action

A DNA vaccine is a bacterial plasmid encoding the antigen of interest under the control of a

mammalian promoter. It constitutes a simple vehicle for in vivo transfection, presenting the ability of

generating an immune response once it is delivered to the host.

The required transcriptional elements essential for an optimal gene transcription and expression are

an eukaryotic promoter that drives the expression of the gene, a multiple cloning site and a polyadenylation

(polyA) signal for protein production in transfect mammalian cells, that mediates mRNA cleavage and efficient

export to the cytoplasm (Ertl 2003) (Williams 2013) (figure 8). The prokaryotic elements essential in the

9% 2%

22%

11%

11%

2%

9%

5%

29%

Hepatite B

Hepatite C

HIV- prevention

HIV-treatment

HPV

Influenza H1N1

Influenza H5N1

Malaria

Figure 7- DNA vaccine clinical trials by vaccine target (2011) adapted from (Ferraro et

al. 2011). Data obtained from the clinicaltrials.gov database indicates that, currently,

130 trials with pDNA vaccines are listed as on-going studies, as of August 2013.

14

plasmid backbone of a DNA vaccine are an origin of replication (ORF), a selection marker (usually an antibiotic

resistance gene), These elements and respective alternatives will be further discussed in the optimization

strategies chapter.

The mechanisms involved in the development of the immune responses following DNA immunization

are still not fully understood and are under intense investigation (Fioretti et al. 2010). Plasmid DNA vaccination

is believed to mimic the natural immune system mechanisms (discussed in detail in chapter I.1.4) and gene

expression pathways triggered by infection with viral or attenuated pathogens.

Firstly, the vaccine formulation with the gene sequence of interest is delivered to the skin

(intradermally), subcutaneum or muscle by a chosen delivery method that has significant effect in the types of

cells that are transfected (Fioretti et al. 2010). Immune response is associated with two models, direct and

indirect presentation of antigen to antigen presenting cells (APCs) (figure 9). Non-bone marrow derived cells

such as myocytes, keratinocytes and fibroblasts, non-professional cells, are one of the primary cells transfected

after plasmid delivery. Once in the host, the plasmid is taken up by such local transfected cells and the antigen

is produced and acquired by APCs (cross-presentation), a mechanism believed to be the main route of priming

(Fioretti et al. 2010). Resident antigen presenting cells (APCs), such as dendritic cells and macrophages, are also

transfected, a process called direct priming. The antigen, using the host cellular machinery, is expressed,

processed and presented by the two classes, I and II, of major histocompatibility (MHC) molecules, a usual

mechanism that occurs during natural infection (Babiuk et al. 2008). APCs, now loaded with host-synthesized

antigen, travel to the lymph nodes where they present antigenic peptide-MHC complexes to naïve T cells,

leading to the initiation of the immune response, by the activation and expansion of T cells or activation of B

cells and antibody production (Prather et al. 2003). It is important to note that the most effective vaccines are

those that elicit both humoral and cellular responses, thus stimulating both CD4+ and CD8

+ T cells.

Figure 8 - Schematic diagram of the essential elements in a DNA

vaccine-vector. A basic vector includes an antibiotic selection marker

(KanR), a bacterial origin of replication (pUC ori), an eukaryotic

promoter (pCMV), a multiple cloning site to insert antigen sequences

(MCS) and a polyadenylation signal sequence (polyA) (Ertl 2003).

15

I.2.2. Advantages and drawbacks

DNA vaccination presents several advantageous characteristics when compared to conventional

vaccination methods. Regarding production, most of the vaccine types currently in use are produced in

embryonated chicken eggs (Košík et al. 2012) (Laddy & Weiner 2007) (Kamps et al. 2006). The supply of such

biological material may impact negatively the need for sufficient amount of vaccine within a short period of

time. DNA vaccination presents rapid and flexible production without the need of developing virus in

embryonated chicken eggs and inactivating it (Košík et al. 2012) (Swayne 2009) or production and purification

of recombinant proteins (Li et al. 2012). DNA vaccines are easier and less expensive to manufacture, due to

their production in prokaryotic systems. The time frame for DNA vaccine production can be of only 1 month, a

period significantly smaller than the time needed for conventional vaccine production (from 4 to 9 months)

(Kutzler & Weiner 2008).

The platform of nucleic acid vaccination offers the possibility of expression of several genes in the

same plasmid, mixing of plasmids in the DNA vaccine cocktail and the use of several genetic adjuvants,

resulting in multiple engineering options and approaches for construction of the ideal vaccine candidate. DNA

vaccines are stable and offer reproducible and easy production (small-scale) and isolation and facilitated

storage and distribution (no need for refrigeration) (Kutzler & Weiner 2008) (Babiuk et al. 2008), a great

advantage in vaccinating populations where storage conditions are not ideal. Regarding safety, DNA vaccine

Figure 9- The direct and indirect routes of antigen presentation, following the most

common delivery methods through injection to skin or muscle. DNA vector-

encoded antigen is then expressed in myocytes or keratinocytes. Antigen must be

transferred to APCs for activation of T cells, usually a dendritic cell (DC). This

indirect process of transfer of antigenic material, possibly as apoptotic vesicles, is

termed cross-presentation. A small proportion of DNA is also taken up directly by

DCs and the encoded antigen can then be processed and presented endogenously.

Furthermore, antigen may be taken up by DCs through shed exogenous antigens.

(Rice et al. 2008)

16

plasmids do not induce anti-vector immunity (Williams 2013), are non-life and non-replicating, which

represents a no risk of reversion back to virulence and recovery of a disease-causing phenotype, unlike live

vaccines, or secondary infection (Košík et al. 2012) (Kutzler & Weiner 2008) (Babiuk et al. 2008). Viral mediated

gene transfer, such as retroviruses and adenoviruses have the high risk of toxicity and insertional mutagenesis

(Fioretti et al. 2010), as well as a high cost, despite their stability and high transfection rates (Josefsberg &

Buckland 2012). One of the major advantages of DNA vaccines is their ability to induce broad immunity, both

humoral and cellular with antigen presentation on both MHC I and MHC II pathways. Most conventional

vaccine types, especially subunit and inactivated vaccines, have a tendency to induce a strong Th2-type of

response (Babiuk et al. 2008), which lacks in the induction of cellular immunity, an extremely important feature

in the defense against viral pathogens. Several molecular and transcriptional elements as well as delivery of

plasmid can be modified in order to tune the type and magnitude of the desired immune response.

The advantages of DNA immunization remain unrealized especially due to lack of efficacy, when

translated to large animals and human trials. Despite all this potential, DNA vaccines also come with some

concerns and drawbacks. From a safety viewpoint, there are some potential issues related to the potential of

integration of plasmid DNA into cellular DNA (Kutzler & Weiner 2008), generation of anti-DNA antibodies

(Babiuk et al. 2008) and the possibility of antibiotic resistance transferred to the patient. Furthermore, there

are some concerns regarding the effect of plasmid DNA in the host’s own genetic material, namely the event of

insertional mutagenesis, leading to the activation or deactivation of oncogenes or tumor-suppressor genes

(Ferreira et al. 2000) (Kutzler & Weiner 2008), problems shared with viral vectors, and chromosomal instability,

but none of these events have been witnessed in several studies and trials of DNA vaccination (Kutzler &

Weiner 2008). The major issue regarding DNA vaccination is the low immunogenicity in larger species of

mammals and in humans, despite good results in mice (Kutzler & Weiner 2008). Ineffective immune responses

include low levels of memory T and B cells, a critical point for effective and successful vaccination. There have

been several developments and strategies to overcome these setbacks. These strategies are directed at

different stages of vaccine production, such as optimization of the plasmid backbone, optimization of vaccine

formulation and the use of novel delivery methods.

I.2.3. Improvement of DNA vaccine performance

One of the major set-backs regarding DNA vaccination is the low immunogenicity generated in larger

animals and in humans. Deliver of pDNA into the nucleus of the transfected cells and subsequent transgene

expression, can be improved by optimization of the eukaryotic and bacterial regions of the DNA vaccine vector.

Regarding DNA vaccination against Influenza A virus, research has mainly focused on the hemagglutinin and

neuramidase antigens. Other influenza viral proteins, such as internal PB1 protein have been studied and

results show fair antibody immunity in mice (Košík et al. 2012). But in order to achieve a more broad and

protective immune response, delivery of multiple antigens may be necessary (Ertl 2003). This reasoning was

approached for example, in a study conducted by Jimenez et al. 2007 (Jimenez et al. 2007) , in which the

17

combination of a pDNA vaccine encoding highly-conserved M2 and NP proteins formulated with the cationic

lipid formulation Vaxfectin TM

provided high level of protection against lethal challenge with H3N2 and H1N1. In

another approach, a dual-promoter bivalent DNA vaccine expressing HA and NP (nucleoprotein) proteins

elicited high levels of cellular and humoral immunity in mice against two strains of virus, H5N1 and H9N2 (Xu et

al. 2011). Such study suggests that the combination of internal and surface viral proteins in the same vaccine

might have an important role in the development of a broad-spectrum vaccine and of cross immunity against

different viral strains, one of the most important setbacks of conventional vaccines for influenza.

I.2.3.1. Plasmid design

Firstly, the molecular aspects of vaccine engineering are discussed, mainly the optimization of

transcriptional elements in the plasmid backbone that can lead to improved gene transcription and expression.

The backbone of these expression plasmids consist of three essential elements: an origin of replication and

selection marker, which aim to allow plasmid propagation and maintenance during large-scale production; an

expression cassette consisting of a promoter and a polyadenylation (polyA) signal, separated by multiple

cloning sites (MCS) and finally the coding sequence for the target antigen, which must be cloned between the

promoter and the polyA signal (Ertl 2003) (Garmory et al. 2003). There are several readily available plasmids in

the market that include all these required elements and that can be modified by the researcher in order to

better suit its requirements in terms of design.

Plasmid DNA is the ultimate product of interest for production of DNA vaccines and consequently it is

essential to achieve large quantities of this material. Maintenance of plasmid at high copy number in bacterial

cells makes this production easier and efficient, resulting in improved DNA yields per gram of cells. The E. coli

ColE1 origin of replication is a popular choice in the construction of a high-copy number vector (Garmory et al.

2003). A mutant ColE1 replicon, that lacks the RNA (Rom) protein and contain a point mutation in the RNA II

sequence, is currently the most used origin of replication and can be found in the pUC series of plasmids

(Bower & Prather 2012). These two mutations lead to a higher copy number (500-700) as the culture is

increased from 30oC to 42

oC. Most existing DNA vaccines used for therapeutics include the pMB1 based rop-

pBR322 origin, the rop- pUC origin or the ColE1 based pMM1 origin (Williams et al. 2009). Another alternative

to the pUC–vectors already available that has recently gained attention is the runaway R1 origin of replication.

R1-based vectors could be well suited for the production of plasmid DNA because, at temperatures above 37oC,

the plasmids lose control of the copy number, leading to about 2000 plasmid copies per chromosome (Williams

et al. 2009) (Nordström 2006). Due to one of the two mutations, its transcription is temperature dependent

and, as a result, R1-based plasmids have been successfully used in temperature-induced recombinant protein

production but not studied for the production of plasmid DNA for therapeutic purposes. Recently, Bower and

Prather (2012) (Bower & Prather 2012) constructed and characterized runaway R1-containing vectors,

pDMB02-GFP and pDMB-ATG. The plasmid DNA production yields were compared to the pUC-based DNA

vaccine, and RNA and protein expression of the plasmid replication initiator were also studied, confirming the

18

high yield and high quality of plasmid DNA, the dependence on temperature and protein RepA availability, the

mechanistic factors that may limitate the R1 replication and the need to further study such vectors, in order for

this approach to constitute a industry-relevant option for the DNA vaccine market.

Selection of plasmid-containing cells is commonly achieved by antibiotic resistance markers.

Resistance to kanamycin (kanR) is the most used antibiotic resistance markers. The use of ampicillin (β-lactam

antibiotic) or penicillin is not recommended, for example by FDA guidelines (Ertl 2003) (Vandermeulen et al.

2011), due to potential allergic reactions to more sensitive individuals caused by residual antibiotic in the final

product. Safety concerns regarding antibiotic selection, such as dissemination of antibiotic resistance genes to

the patient enteric bacteria, led to the design of alternative selection methods to increase the safety profile of

plasmid backbones. The World Health Organization and the European Agency for the Evaluation of Medicinal

Products (EMA) also have guidelines directed at the use of antibiotic selection markers, specifying the attention

needed regarding this issue especially in the matters of plasmid-based vaccination (Vandermeulen et al. 2011).

Besides safety, antibiotic markers such as kanamycin (1 kb) also increase plasmid size, which decreases

transfection efficiency (Williams et al. 2009) (Ribeiro et al. 2012). Antibiotic-free systems of plasmid selection

include operator-repressor titration, RNA based selector markers and complementation of auxotrophic

bacterial strains, among others (Williams et al. 2009) (Vandermeulen et al. 2011).

The promoter, a component of extreme importance, drives the expression of the gene of interest.

Different types of promoters have been used for optimal expression of mammalian genes, from the oncogenic

viruses such as Rous sarcoma virus or SV40 (Kutzler & Weiner 2008). Nowadays, most DNA vaccine studies

include the constitutive human cytomegalovirus (CMV) promoter since it drives high levels of constitutive

protein expression in a wide range of mammalian tissues, accomplishing greater immunogenicity and

enhancing antigen-induced immune responses at higher levels than, for example, host-specific promoters

(Kutzler & Weiner 2008) (Laddy et al. 2009) (Saade & Petrovsky 2012).

The optimization of the initiation start site for protein synthesis is one of the strategies used to

improve the expression of the transgene product, improving protein production. This can be achieved by the

insertion of the human T-cell leukemia virus type I R region (HTLV-I R) 5’ UTR downstream of the CMV

promoter (Williams 2013) (Saade & Petrovsky 2012) and the inclusion of the Kozak consensus sequence around

the initiation codon (Laddy & Weiner 2007) (Saade & Petrovsky 2012)(Ertl 2003), essential in the protein

translation and recognition by the eukaryotic ribosome (Garmory et al. 2003). Codon optimization to match

high use codons for the target species also correlates with higher translational efficiencies in mammalian cells

and increased immune responses may be obtained by DNA vaccination when this specific codon usage is

applied (Garmory et al. 2003) (Williams 2013). The use of double stop codons is important to achieve correct

termination, through the prevention of the formation of oversized or misfolded proteins (Kutzler & Weiner

2008).

19

I.2.3.2. Immunostimulatory sequences and Co-delivery of immune modulators

In addition to the several possible changes that can be applied to modify the behavior and consequent

outcome of the DNA plasmid construct, the immune response can be enhanced or adapted through the co-

delivery of immune modulators, such as cytokines, chemokines and co-stimulatory molecules (Kutzler &

Weiner 2008) (Laddy & Weiner 2007). The co-injection of such adjuvants has a substantial effect on the

immunogenicity of the vaccine (Kutzler & Weiner 2008) (Babiuk et al. 2008). Examples of these molecules are

the cytokine interleukin 2 (IL-2), a potent Th1-type cytokine, capable of inducing cellular and humoral immunity

and activating NK cells and the co-stimulatory molecules B 7.1 and B7.2, members of the Ig family, associated

with IL-2 secretion and cellular proliferation and CD40 (Laddy & Weiner 2007). Both the cellular immunity and

the humoral immunity can be enhanced by the use of genetic adjuvants, other examples being IL-15 and IL-18,

resulting in more effective vaccine candidates (Lim et al. 2012) and IL-12 (Naderi et al. 2013).

Regarding more novel adjuvants, Jalilian et al. (Jalilian et al. 2010) developed an avian influenza DNA

vaccine, encoding the H5 gene and the MDP-1 gene of Mycobacterium bovis as a potential genetic adjuvants.

Results showed that indeed the application of this genetic adjuvant resulted in higher antibody titers in

chickens, when compared to the DNA vaccine encoding the hemagglutinin gene alone.

The immune stimulation caused by bacterial DNA when administered to mammals is mediated

through cystidine-phosphate-guanosine (CpG) motifs, which interact with Toll-like receptor 9 (Koyama et al.

2008) and induce the production of several innate immune stimulatory molecules and activate B-cells,

macrophages, dendritic cells, among others (Ertl 2003). Encoding such unmethylated CpG motifs in the DNA

vaccine plasmid can indeed have an effect on growth, differentiation and survival of several cell types and

enhance antigen-specific immune responses (Ertl 2003) (Kutzler & Weiner 2008).

I.2.4. Antigen-targeting sequences

Several efforts have been made in order to enhance the immunogenicity of DNA vaccines and

targeting strategies represent a valuable mechanism for optimization of immune responses.

Stimulation of both CD4+ T cells and CD8

+ naïve T cells into T-helper cells and CTL, respectively, is

essential for the generation of an effective immune response against intracellular pathogens (Thomson et al.

1998) (Kim et al. 2003). A strategy for improved immunological responses is the targeting of the pDNA or the

encoded antigen to APCs (Williams et al. 2009), namely dendritic cells, the most potent antigen-presenting cells

(APC) in vivo that initiate the adaptive immunity. This has been achieved by attaching antigens to ligands or

antibodies that specifically bind to DC receptors (Cao et al. 2013) (Nchinda et al. 2008) (Deliyannis et al. 2000)

and specific modified non-viral vectors directed to DCs (Lu et al. 2007). This strategy has been successfully

applied to influenza, breast tumor and melanoma, resulting in much higher level of antibody and antigen-

specific T cells, and therefore improving efficacy of the DNA vaccines.

20

Targeting the DNA-plasmid vaccine encoded proteins to specific sub-cellular compartments within the

transfected cell is also a strategy that leads to improved immunological results. This can be achieved by the

addition of a targeting peptide that routes antigens intracellularly to protein-processing machineries

(proteasome, endosome/lysosome) or to cellular secretion for example, helping to improve and modulate the

processing and presentation of such DNA-encoded antigens via the class I and class II MHC molecules,

enhancing CD8 + T cell, CD4

+ T cell and antibody responses (figure 10).

Generation of epitopes capable of inducing CD8+ T cell response is associated with antigen

degradation in the proteasome of an APC (Rodriguez et al. 2001), so attachment to proteasomal-targeting N-

terminal ubiquitin tag increases the availability of antigenic peptides for binding to the MHC I molecules and

therefore increase cytotoxic T cell responses (Williams 2013) (Ullas 2012) (Howarth & Elliott 2004).

The production of antibodies and also the CTL response are both dependent on a efficient CD4+ T cell

activation, which is dependent on antigen degradation via the endosomal/lysosomal compartments in the cell

cytosol and delivery to the MHC II pathway (Thomson et al. 1998) (Carvalho et al. 2010) (Dobaño et al. 2007).

The lysosomal targeting sequences of LAMP (lysosomal-associated membrane protein) are the most commonly

used signal sequence for subcellular targeting (Li et al. 2012). LAMP molecules are localized in the outer

membrane of lysosomes with a trafficking pathway from the Golgi complex mediated by adaptor protein

binding to the carboxyl-terminal recognition sequence of an 11-amino acid cytoplasmic tail. This LAMP

trafficking pathway coincides with that of MHC II in specialized vesicular compartments of immature APCs, sites

associated with the formation of antigenic peptide-MHC II complexes (Marques et al. 2003). Therefore, this

sequence directs the protein to compartments that contain class II MHC molecules, such as the lysosome and

results in an enhanced MHC II-mediated response (Dobaño et al. 2007) in several systems, such as influenza

virus (Thomson et al. 1998), HIV (Rowell 1995) (Goldoni et al. 2011) and human papilloma virus (Kim et al.

2003). Its association in DNA vectors allows for the processing of antigens of endogenous origin through the

MHC II pathway, by directing the cytosolic protein to the lysosome.

Endoplasmic reticulum (ER) targeting enhances DNA vaccine immunogenicity. The early region 1A

(E1A) sequence, which is the first viral transcription unit expressed following infection by human adenovirus

(Loewenstein et al. 2006) and the signal ERTS (Xu et al. 2005), derived from the Adenovirus E3 leader sequence,

when attached to proteins facilitate entrance in endoplasmic reticulum and therefore loading to compartments

involved in the MHC I pathway (Carvalho et al. 2010) (Xu et al. 2005), a pathway essential for the development

of a CTL response. Targeting peptides into the ER increases such response, since there are two mechanisms by

which antigens are loaded into MHC I molecules, one involves the proteasome degradation and TAP

transportation and the other is proteasome and TAP independent: both involve the ER. The proteasome is the

major non-lysosomal pathway responsible for general protein degradation and is also involved in the

preparation of epitopes for MHC I presentation (Berhane et al. 2011). Once in the ER lumen, peptides

generated by the proteasome and transported by TAP bind to the MHC I molecules, which are subsequently

released from the ER and transported to the cell surface via the constitutive secretory pathway. On the other

hand, exogenous antigens may also enter the MHC I pathway, even after being degraded in the phagolysosome

(Grommé & Neefjes 2002).

21

Targeting of antigen to secretion, through the addition of a secretory signal (sc) (Henriques et al. 2007)

such as the human tissue plasminogen activator leader peptide (tPA) (Ertl 2003) (Weinberger et al. 2013)

(Williams et al. 2009), promotes the release of antigen to the extracellular space. The secreted antigen can be

taken up by APCs, and becomes available for presentation in MHC II molecules. It is important to mention that

part of the antigen can be presented in MHC I molecules, through the mechanism of cross-presentation, and

that the success of secretion promoted by these targeting sequences is also antigen-dependent, since some

proteins have specific sequences or motifs that can retain it in the membrane, for example (Ertl 2003).

Figure 10- Example of several DNA vaccine targeting-strategies for modulation of the immune response

towards the MHC I or MHC II presentation pathways. After entering the nucleus of APCs (1), the plasmid DNA,

through the host cellular machinery, and the gene transcription is initiated, followed by protein production in

the citoplasm and processsing according to the respective modification. A) Signal seuences such as the human

tissue plasminogen activator leader peptide (tPA), a secretory signal (Sc) that leads the vaccine-encoded

protein to the extracellular space of transfected cells via the golgi apparatus; B) Unmodified antigen sequences,

lacking any targeting sequence, are expressed as cytoplasmic proteins and usually presented in the MHC I

molecules by default, but can also be presented in MHC II molecules through transport of cytosolic material via

autophagy; C) ubiquitin attachment leads the translated protein into the polyubiquitination pathway and

subsequently to the MHC I pathway; D) Signal sequences such as lysosomal integral membrane protein-II (LIMP

II) and lysosomal-associated membrane protein (LAMP) promote antigen transport to the lysosomes that

facilitate peptide presentation on MHC II molecules. Besides direct transfection of resident APCs as well as

somatic cells (2), APCs mediate the display of peptides on MHC II molecules after secreted vaccine-derived

antigens have been shed from transfected cells, captured and processed within the endocytic pathway (2) and

MHC I cross-presentation of exogenous antigen for example by engulfment of transfected and apoptotic cells.

Adapted from (Weinberger et al. 2013).

22

Kim et al. (Kim et al. 2003), combined intracellular targeting with a strategy to prolong dendritic cell

life, which resulted in a synergetic effect on the enhancement of the potency of the immune response, namely

an increased CD8+ T cell response, stronger immune memory and enhanced antitumor effects. Their results

support the hypothesis that a DNA vaccination strategy combining intracellular targeting with the prolongation

of the DC life enhanced the antigen-specific immune response to a greater level than intracellular targeting

alone, reveling that this strategy of improvement of DNA vaccines can be further optimized. Recently,

Weinberger et al. (2013) (Weinberger et al. 2013) applied this strategy of sub-cellular antigen-targeting to the

development of DNA vaccines for counterbalancing and protecting from allergic sensitization to a specific

allergen, by modulating the immune response to a Th1-balanced immune response, aligning targeting

strategies to the proven potential of DNA vaccines to induce anti-inflammatory and immune regulation (Fissolo

et al. 2012). The constructed DNA vaccines targeted several subcellular compartments such as: the

extracellular space, by the human tissue plasminogen activator leader peptide (tPA) sequence; ubiquitin (Ubi)

that leads the antigen into the polyubiquitination pathway and the 20AA C-terminal tail of the lysosomal

integral membrane protein-II (LIMPII) sequence, which as the LAMP sequence described above, promotes

targeting to the lysosome. Targeting proved to be a valuable strategy in modulating the humoral immune

response and in the optimization of anti-allergic gene vaccines, rendering the vaccine hypoallergenic without

compromising its efficacy.

I.2.5. Production of DNA vaccines

The development and production of DNA vaccines starts with the design, selection of the expression

vectors and adequate microorganisms, usually Escherichia coli, transformation and clone selection and

cultivation medium formulation (upstream processing), cell growth (fermentation step) and the isolation and

purification of the supercoiled plasmid DNA (downstream processing) (Ferreira et al. 2000) (Ghanem et al.

2012) (figure 11). As previously mentioned, plasmid DNA vectors have lower transfection efficiencies, when

compared to viral vectors. Due to several physical and biochemical barriers, only a small percentage of

plasmids presented to the cells reach the nucleus and are expressed (estimations point to 1 in every 1000

plasmids (Ferreira et al. 2000). Therefore, in order to stimulate and enhance the immune system, pDNA

vaccination treatments require milligrams of plasmid DNA. Purification methods must result in large amounts

of pDNA production (Carvalho et al. 2009). At a small scale, plasmid DNA is relatively easy to produce and

purify for vaccination, but under non optimized laboratory conditions resulting volumetric yields tend to be

very low (5-40 mg/L) and the utilization of laboratory-scale purification methods at industrial scales also leads

to poor yields (Prather et al. 2003) (Ghanem et al. 2012).

23

Large-scale production of pharmaceutical

pDNA must be free of contaminant bacterial

components, have a competitive and cost-

effective yield per volume of broth and must

also follow regulatory guidelines. Efficient

purification processes of pDNA for

therapeutic or research purposes are

needed in order to achieve considerable

amounts of homogenous product, consisting

mainly of supercoiled (sc) pDNA (>90%),

according to regulatory guidelines, with high

degree of purity (>97%), free from host

genomic DNA (gDNA), host proteins, RNA

and endotoxins (Diogo et al. 2005). Plasmid

DNA in bacteria mainly exists in the

supercoiled form or as covalently closed

circular DNA, forms that are very tight and

enable the pDNA to fit inside the cell. The

presence and content of sc plasmid DNA is

an indicator of plasmid quality and sc pDNA

also promotes the unwinding and strand

separation during replication and

transcription (Sousa et al. 2008) and

originates more effective immunogenic

responses, namely CD8+

T cell responses

(Pillai et al. 2009) and produces higher transfection efficiencies (Laddy & Weiner 2007) (Li et al. 2011). Other

plasmid isoforms, such as open circular (oc) pDNA and linear pDNA, are products of the degradation of sc

pDNA, which can occur during even careful pDNA purification processes, and are less efficient in inducing gene

expression (Sousa et al. 2008). These unwanted conformations are caused by single strand nicking, which

results in the relaxed form of sc pDNA, the oc pDNA, and double–strand nicking, by restriction endonucleases,

which originates linear DNAs. The diversity of biomolecules present in Escherichia coli and plasmid DNA initial

extracts constitutes one of the main challenges in achieving high yields for supercoiled pDNA with

chromatographic purification methods. Furthermore, such impurities share many physical and chemical

properties with sc pDNA: negative charge (RNA, gDNA, endotoxins), hydrophobicity (endotoxins) and molecular

mass (gDNA, endotoxins).

Plasmid fermentation processes ideally maximize the volumetric yield (mg pDNA/L), which facilitates

fermentations at smaller and more economical scales, and the specific yield (mg/g DCW), which tends to

improve plasmid purity and yield in downstream processing (Williams et al. 2009). Other key parameters of

Figure 11- Schematic representation of the essential steps in production

of plasmid DNA for therapeutic use and vaccination. Supercoiled plasmid

purification is achieved by a series of downstream processing unit

operations. Following cell lysis, the product can proceed directly to a

single or dual mode chromatography or a previous step of clarification

and concentration can be performed. (Prather et al. 2003)

24

fermentation include cell density, plasmid copy number and homogeneity, which are strongly influenced by

host strain, fermentation mode (in general, plasmid quality and yield is higher from fed-batch rather than batch

fermentation (Williams 2013)), medium composition, metabolic burden and harvesting point (Urthaler et al.

2005). One of the most critical features of pDNA production with a high percentage of supercoiled isoform is

the separation from other isoforms in downstream purification processes. It is of high importance to optimize

fermentation process in order to maximize retention and harvest of sc pDNA. Following fermentation, a series

of purification steps take place: cell harvest, cell lysis, followed by optional clarification and concentration, and

a final purification step. Downstream processing starts with cell disintegration in order to release the plasmid.

Cell lysis through mechanical methods is not adequate for large-scale production due to the sensitivity of

polynucleotides, as is enzymatic disruption due to economical and regulatory drawbacks (Urthaler et al. 2005).

In light of this, alkaline-lysis has been the procedure of choice for this first step of pDNA recovery, first

described by Birnboim and Doly (1979) (Ferreira et al. 2000) (Urthaler et al. 2005). The purification processes

are extremely important, since many supercoiled pDNA contaminants share similar characteristics with our

desired product, such as negative charge, identical size and hydrophobicity (Ghanem et al. 2012). The

clarification and concentration, achieved for example with the use of lithium chloride and ammonium acetate

and PEG (polyethylene glycol) for concentration, steps aim to concentrate the pDNA, by eliminating cell

impurities, proteins, low molecular nucleic acids and high molecular RNA. Chromatography, both as a single or

combined mode, is considered the method with highest resolution (Urthaler et al. 2005) and is used in order to

remove the other forms of plasmid DNA present in the sample, linear and open circular, genomic DNA and

endotoxins (Ferreira et al. 2000). The most common used chromatographic techniques are anion-exchange

(AIEC), hydrophobic interaction (HIC) and size-exclusion (SEC) chromatographies

(Urthaler et al. 2005).

Hydrophobic-interaction chromatography (HIC) separates sc pDNA from the more hydrophobic single-stranded

nucleic acids, RNA, gDNA and endotoxins, exploiting the differences in their behavior once exposed to

hydrophobic ligands (sheparose and cellulose-based materials) in the chromatographic column. In the presence

of high salt concentrations (for example 1.5 M of ammonium sulphate), more hydrophobic solutes, such as the

aforementioned impurities, are retained in the column and pDNA is eluted first. This behavior is explained by

the fact that pDNA has a minimal interaction with the HIC support, due to the fact that the hydrophobic

aromatic bases are protected inside the packed double-helix. A decreasing ionic-strength gradient causes the

other more hydrophobic molecules to elute from the HIC column. This method has the ability to handle heavily

contaminated samples, without the need for pre-treatments such as digestion with RNase (Diogo et al. 2003).

Size-exclusion chromatography (SEC) explores the differences in sizes of small impurities such as RNA,

endotoxins and proteins, and large molecules such as pDNA, which are not able to get inside the agarose beads

(Ferreira et al. 2000). The efficiency of SEC is increased if low amounts of impurities are present in the injected

sample, therefore this methodology is ideal when it is performed as a final purification step (Ferreira et al.

2000).

All these processes must attend not only to a cost-effective necessity, but also to the strict guidelines

for plasmid DNA vaccine commercialization, presented by the U.S. Food and Drug Administration (Fda 2007),

which includes the requirement for a preclinical safety evaluation, focusing in the assessment of

25

immunogenicity, possible adverse effects caused by the use of cytokines and other immunomodulatory agents,

safety and tolerability of the dose and systemic toxicity. Table 2 discriminates the major concerns associated

with plasmid DNA vaccines and their production hosts. It is important to consider these concerns early in the

development of a DNA vaccine, in order to achieve efficient solutions. Regarding the most used production

host, E.coli, its greatest advantages are the efficiency, high yield of pDNA and well established downstream

processes. Disadvantages include the highly immunogenic endotoxin (lipopolysaccharides - LPS) in its outer

membrane (Glenting & Wessels 2005) and possible product contamination.

Table 2- The safety concerns regarding DNA vaccines and possible solutions. Adapted from (Glenting & Wessels 2005).

Safety Concern Possible Solution

Genetic elements Transfer of plasmid to host flora Narrow host-range replication region

Non antibiotic plasmid marker

Germline Integration Avoidance of mammalian replication region

Insertional mutagenesis and oncogenesis Artificial DNA for promoter, intron and signal

sequence Avoidance of human-homologous DNA

Adverse effects of encoded peptide(s) Artificial signal sequences

Avoidance of mammalian replication region Evaluation of vaccine peptide case-by-case

Induction of autoimmune reactions Minimized plasmids

Production host Endotoxins and biogenic amines Use of gram-positive organism (Eg.: Lactococcus

lactis)

Transferable antibiotic resistance genes Determination of minimal inhibitory

concentrations (MIC’S) Screening for transferability

Genetic instability Analysis of plasmid population by sequencing and mass spectrometry

Pathogenicity Use of food-grade organism

I.2.6. Plasmid DNA delivery

Plasmid administration route is directly correlated with the effectiveness and immunogenicity of DNA

vaccines (Bansal et al. 2008)(Laddy & Weiner 2007).

The most common method of immunization used in DNA vaccine studies is the intramuscular route

(Kutzler & Weiner 2008). Intramuscular injection delivers the DNA plasmid dissolved in PBS, water or other

formulation, into the muscle tissue of the host. There is a limited number of APCs in the muscle tissue, which

results in the predominant transfection of myocytes by pDNA (Fioretti et al. 2010) (Laddy & Weiner 2007). DNA

uptake and internalization by cells upon injection in the muscle is mostly inefficient (Kutzler & Weiner 2008)

26

(Sun et al. 2013). The performance of intramuscular delivery method can be improved by associating the

plasmid DNA with chemical adjuvants such as cationic polymers and liposomal formulations. When compared

with immunization of pDNA alone, liposomes and chitosan can increase plasmid DNA copy number at the site

of injection and also enhance tissue distribution and extend half-life of pDNA by protecting it against

intracellular degradation (Sun et al. 2013) (Henriques et al. 2009).

Intradermal delivery systems (both by needle/syringe) by injecting plasmid encoding antigen has also

been explored. After the stratum corneum layer of the skin, cutaneous dendritic cells (epidermal Langerhans

cells (LC) and dermal dendritic cells), which are potent APCs, can be reached and are able to induce immune

responses, both cellular and humoral by stimulation of naïve T lymphocytes in the lymph nodes (Larregina et al.

2001). This approach is also used in needle-free systems, such as Biojector© (Saade & Petrovsky 2012)

(Carvalho et al. 2009), where a plasmid DNA solution is sprayed through the skin with the goal of direct

transfection of LC cells (Saade & Petrovsky 2012). The skin is the most accessible organ of the human body, is

easily monitored as well as being a highly immunocompetent target organ. It is important to notice that pDNA-

immunization via the skin requires smaller amounts of DNA than the needed for intramuscular injection. One of

the challenges in DNA vaccine is the delivery of plasmid DNA to elicit similar responses in larger animals and

humans as in murine models. Novel DNA delivery systems, among other strategies, can help reaching this goal.

In order for the plasmid to express its antigen, the plasmid must reach the nucleus without being degradated

by nucleases in the cytosol and in the endosomal and lysosomal compartments. Injection of plasmid usually

results in a low number of plasmids that can overcome these obstacles and express the encoded antigen.

Particle-mediated epidermal delivery (PMED), often called gene-gun, is able to perform a direct

intracellular DNA directly to cultured cells and whole organisms for several purposes: plant genetic

engineering, gene therapy and DNA vaccine delivery, for example (Dean et al. 2005). This approach consists in

the acceleration of DNA-coated gold particles, whether by employing an electric discharge or compressed

helium, directly into the cytoplasm and nuclei of cells in the epidermis of the skin, so both keratinocytes and

epidermal antigen-presenting cells (Langerhans cells) are targeted and obstacles of gene delivery in vivo, such

as DNA degradation, are potentially overturned (Larregina et al. 2001). This mechanism will result in antigen-

presentation via direct transfection and cross-priming, leading to a more efficient DNA delivery and gene

expression. LCs are antigen-presenting cells that are able to migrate to draining lymph nodes and are

responsible for inducing primary cellular responses and immunological memory, while the non-migratory

keratinocytes produce antigen at their localization in the skin and are the major contributors to the magnitude

of the antibody response. One of the more important advantages of this method is the ability to achieve strong

immune responses with smaller amounts of pDNA when compared with intramuscular inoculation (Saade &

Petrovsky 2012). In human studies, results with intramuscular injection show a modest immune response with

doses of 5 mg of DNA administered, while PMED vaccines showed greater immunological success, both cellular

and humoral, with 10 µg or less DNA (Dean et al. 2005). Regardless, the gene gun approach has been limited by

its high cost and Th2-biased immune response (Kim et al. 2012).

The use of Microneedle patches for DNA vaccination is a valuable approach that can overcome the

difficulties and specificities regarding other methods of skin vaccination, as discussed previously. Microneedles

27

efficacy and simplicity has been demonstrated for influenza vaccination using whole-inactivated virus, virus-like

particle and protein subunit vaccines. Kim et al. (Kim et al. 2012) studied the use of Microneedles for DNA

vaccination against influenza for the first time, in order to obtain increased immunogenicity but also simplicity

of manufacture and administration. An H5 avian influenza DNA vaccine was administered to the skin using

Microneedles and intramuscular vaccination, with application of the same dose. The Microneedle approach

consists of a patch of solid metal Microneedles coated with pDNA that are then applied to the skin without the

need for medical expertise and then DNA coating dissolves off the Microneedle within minutes, being delivered

into antigen-presenting cells in epidermis and dermis (Josefsberg & Buckland 2012). Their results show that this

delivery method translates indeed, in the mouse model, in a higher production of virus-specific antibodies and

consequently a superior protection when compared to intramuscular immunization. Despite this, DNA

immunizations by Microneedles did not fully protect the mice from lethal viral challenge.

Electroporation (EP) is a technique aimed at the improvement of the entrance of macromolecules,

such as chemotherapy drugs to kill specific tumor cells (Kutzler & Weiner 2008) or nucleic acids, either in vivo

or in vitro. It was first an experimental technique that now is used in several clinical trials aimed at the delivery

of drugs and nucleic acids. It consists in the application of brief electric pulses, that cause temporary and

reversible permeabilization of the cell membrane (Laddy et al. 2009). During membrane destabilization,

macromolecules present in the extracellular space gain access to the intracellular environment. After cessation

of electric pulses, the membrane slowly regains its original form. Following injection of plasmid DNA, electric

needles or patches are inserted around the site of injection to deliver these electric pulses. EP mediated

delivery can be used both with intramuscular and intradermal delivery systems. This versatility allows the

delivery of plasmid to both muscle and skin cells. There have been several studies regarding vaccine candidates

and electroporation-based delivery for a wide range of infectious diseases and cancer (Xu et al. 2011) (Saade &

Petrovsky 2012). Comparison of delivery with or without EP showed a clear increase (10-100 fold) of immune

responses as well as immunological protection in various animal models of disease, including highly pathogenic

avian influenza (Laddy et al. 2009), proving the efficacy of in vivo electroporation as a means to DNA vaccine

delivery. It is important to notice that the results from the several studies with this delivery strategy are

variable, due in part to the fact that the efficacy also depends on a number of other factors such as pulse

patterns, voltage, electrode configuration (needle or patches) and DNA vaccine formulation. Concerns

regarding this approach include the risk of plasmid integration into the host genome (Wang et al. 2004) and

pain upon delivery, especially when associated with intramuscular delivery. Gene delivery via electroporation

in clinical studies suggest that DNA delivered using electroporation devices may elicit immune responses on par

with more traditional vaccines and that this vaccine platform may indeed have broad applicability.

28

29

II. Background and objectives

Avian influenza viruses represent important veterinary pathogens for animal health, due to their

ability to cause disease, either asymptomatic or highly pathogenic, in poultry. AIV has had both an effect on the

economy of poultry industries but is also involved in the emergence of viral strains capable of infection and

spread of disease in humans and, therefore, constitutes a major concern to public health. In the last century,

novel genetic elements with presumed avian origin have caused pandemics among humans, such as the

“Spanish flu” in 1918 (H1N1), the H5N1 highly pathogenic strain in 2006 and more recently, in 2013, the

outbreak and human infection of a novel avian-origin AIV H7N9.

Due to production, safety, logistical, surveillance and monetary constraints, current vaccination

approaches are not optimal. Furthermore, due to the antigenic variant characteristics of the virus, vaccines

may need to be updated with frequency to new circulating strains. Immunization with DNA vectors harboring

viral antigens, such as HA and NA, presents features that could overcome these difficulties, such as ease and

low cost of large-scale production and administration and the advantage of being able to provide cross-strain

protection by encoding several antigens, eliciting both humoral and cellular responses, essential in eliminating

viral pathogens. Influenza DNA vaccines have been safe and effective in providing protective immunity against

viral infection, but low transfection efficiency and low immunogenicity in large animals are still important

hurdles regarding DNA vaccination. Several strategies have been employed to overcome such setbacks, from

different administration routes and formulations, to prime boost strategies and novel vaccine design. Antigen-

targeting sequences have improved immunogenicity of DNA vaccines, by modulating the type of immune

response elicited.

In this work, DNA vaccine candidates harboring the sequence of the neuraminidase 3 antigen of AIV, a

transmembrane protein with 52 kDa and 470 amino acids (Appendix V), were constructed with antigen-

targeting sequences and tested in vitro and in vivo through mammalian cell transfection and immunization of

chickens, respectively. Neuraminidase is one of the major surface glycoproteins of the AI virions, being

responsible for cleavage of sialic acid residues on host cell surface, thus mediating the release of newly formed

virions. Humoral response is manly constituted of antibodies against the more present HA surface protein, but

also against NA and internal proteins M2 and NP. Antibodies against NA do not prevent infection, but are

essential in the prevention of viral spread. Targeting sequences E1A (adenovirus early region 1A), LAMP

(Lysosomal associated membrane protein) and their combination were added to the DNA vectors, in order to

potentiate immune response. The E1A targeting sequence directs the antigen to endoplasmic reticulum,

potentiating loading into MHC I molecules and thus enhancing a CD8+ T cell response. On the contrary, addition

of the LAMP sequence promotes targeting to the lysosome/endosome, loading into MHC II molecules and

therefore contributes to CD4+ T cell response. A DNA prime/protein boost strategy was also employed in vivo,

envisioning the enhancement of the humoral response and the eliciting of immunological memory.

30

31

III. Materials and methods

III.1. Design and production of DNA vaccines

The plasmid pVAX-GFP (figure 12), used as a control in all the experiments, was previously constructed

by modification of the commercial plasmid vector pVAX1LacZ (Invitrogen) (Appendix I), by replacement of the

lacZ reporter gene with the eGFP (Green Fluorescent Protein) gene, at the Nucleic Acid Engineering Group,

Institute for Biotechnology and Bioengineering, at Instituto Superior Técnico. Details of construction are

described elsewhere (Azzoni et al. 2007). pVAX-N3-GFP (figure 12), encoding for the N3 protein fused with

GFP, tested in this work for its potential as efficient DNA vaccine and as a control for the effect of targeting

sequences in DNA vaccine improvement, was previously produced and transformed in Escherichia coli DH5α

cells by Ana Margarida Mourão at LNIV. The plasmid pVAX-N3-GFP-LAMP, harboring the targeting sequence

LAMP, was constructed and meticulously studied along with the plasmids previously mentioned in another

master thesis work “DNA vaccines against avian influenza virus: Enhancing Immune response by protein

targeting” (Freitas 2012) (figure 12).

Figure 12- Schematic representation of plasmid DNA vectors and restriction sites used in molecular cloning for the construction of

pVAX-E1A-N3-GFP and pVAX-E1A-N3-GFP-LAMP and tested in in vitro and in vivo assays, along with pVAX-GFP, used as a control

in the experiments. Prokaryotic elements in the vaccine vectors for plasmid replication, maintenance and selection in the

bacterial host include a strong bacterial origin of replication ColE1, a selectable marker for Kanamycin and an inducer of high level

of transcription, the T7 promoter. Eukaryotic elements for expression in the mammalian host include the eukaryotic promoter

pCMV, a transcription polyadenylation/termination signal BGH polyA and the pathogen gene of interest (N3). Green fluorescent

protein (GFP) is the reporter gene for assessment of protein localization and expression. Images obtained with ApE © software.

32

Due to loss of part of the CMV promoter sequence in plasmids carrying the E1A targeting sequence during

cloning procedures in a previous work, it was necessary to correct the constructed plasmids pVAX-N3-GFP-E1A

and pVAX-N3-GFP-E1A-LAMP. So with the aim of studying the target sequence E1A and the result of its

combination with LAMP, these new DNA vaccine prototypes were constructed by biomolecular procedures and

further studied in in vivo and in vitro studies along with the plasmids mentioned above: pVAX-GFP, pVAX-N3-

GFP and pVAX-N3-GFP-LAMP.

III.1.1. Molecular cloning

III.1.1.1. Culture of DH5α / Production of plasmid vector

Escherichia coli (E.coli) DH5 α cell banks harboring plasmid vectors pVAX-N3-GFP and pVAX-N3-GFP-

LAMP were cultivated overnight at 37⁰C and 250 rpm in 5 mL of 20 g/L Luria Bertani (LB) selective media

(NaCl- 5 g/L; Tryptone 10 g/L; Yeast Extract- 5 g/L) (Sigma-Aldrich) supplemented with 5 µL of kanamycin

(30 µg/mL) in an orbital shaker model AGITORB 200 (ARALAB). The culture was grown until an OD600nm of

approximately 2.5 was reached, the beginning of the stationary phase (Appendix II). The cultured volume was

centrifuged in an Eppendorf bench-top Centrifuge 5810 R with a rotor A-4-62 at 3,220 g and 4⁰C, for

10 minutes. Once the pellet was obtained, plasmid DNA proceeded to be purified with High Pure Plasmid

Isolation Kit (Roche). The purified DNA plasmids obtained were eluted in 100 µL elution buffer (Tris-HCl Buffer,

pH 8.5) and stored at 4⁰C or -20⁰C (long term storage), for posterior cloning procedures.

III.1.1.2. Synthetic oligonucleotide design of E1A sequence and annealing

The reverse and forward oligonucleotide sequences of the antigen-targeting sequence adenovirus e1a

(E1A) were obtained from Stab Vida Inc., Portugal. Restriction sites, selected through the software ApE

software®, were added to both extremities of the oligonucleotides, in order to perform insertion of the

sequence through biomolecular techniques. The final sequence was determined as the one presenting minimal

risk of hairpin formation, ideal melting temperature and GC content between 50-60%. The construction was

verified for quality parameters, such as Tm and GC content, through the bioinformatic tools OligoEvaluatorTM

(Sigma-Aldrich®) and OligoAnalyzer version 3.1. (Integrated DNA Technologies ®).

The nucleotide insert used for cloning procedures was obtained by performing annealing of the

complementary oligonucleotides, according to the “Protocol for Annealing Oligonucleotides” by Sigma-Aldrich

Co., USA and “Anneal complementary pairs of oligonucleotides” protocol by the Thermo Fisher Scientific Inc.,

USA. Both complementary oligonucleotides were resuspended at the same molar concentration (100 pmol/µL)

in an Annealing Buffer (10 mM Tris, 50 mM NaCl, 1mM EDTA, pH 7.5-8.0). A volume of 5 µL of each

complementary oligonucleotide was mixed together in a PCR tube, both with a final equimolar concentration

33

of 10 pmol/ µL, with 40 µL of annealing buffer, resulting in a final volume of 50 µL. The annealing procedure

was performed in T gradient thermocycler (Biometra®), programmed to heat to 95⁰C and maintain this

temperature for 5 minutes. The temperature was reduced to 25⁰C at a rate of 2⁰C per minute. The annealed

sample was stored at 4⁰C or -20⁰C in case of long term storage.

The success of the annealing method used was assessed by comparison of single-stranded forward

and reverse oligonucleotides and the double-stranded obtained from the annealing procedure in an agarose

gel electrophoresis (2% and 4%). Further confirmation was obtained by performing melting curve analysis of

the single-stranded forward and reverse oligonucleotides and the double-stranded oligonucleotide. This

procedure was carried out in Roche LightCycler detection systemTM

using the FastStart DNA Master SYBR Green

I Kit (Roche Diagnostics), with 2 µL of SYBR Green reagent, 1 µL of MgCl2 and 15 µL of PCR grade water per

sample. All samples were analyzed at 5 mM and 1 mM. The melting curve analysis started with a temperature

of 95oC, followed by a period of 30 seconds at 55

oC and heating up to 95

oC. The sample was cooled to 40

oC for

30 seconds and melting curves for the three samples were acquired and analyzed.

III.1.1.3. DNA Restriction

A double digestion with restriction endonucleases BstEII (restriction site G^GTGACC) (Promega) and

NheI (restriction site G^CTAGC) (Promega) was performed in order to create cohesive ends in both the plasmid

DNA vectors and the E1A insert sequence. Each digestion procedure, for both insert and vectors, consisted of

3 µg of DNA, 0.5-0.8 µL of each enzyme (10 u/µL), 10 % of 10X Multicore Buffer (ideal activity for conjugated

digestion with both enzymes) and filtered sterilized milliQ water to a determined final volume (20-35 µL). All

mixtures were incubated for 3.5 h at 37⁰C.

III.1.1.4. Purification of insert and vector

After the digestion process and considering the fragment’s small size (58 bp), the total volume of the

final E1A sequence was purified with the GenEluteTM

PCR Clean-Up Kit (Sigma-Aldrich, USA) and 50 µL of

purified DNA were recovered and stored at 4⁰C or at -20⁰C until subsequent cloning into the plasmid vector.

On the other hand, the Qiagen Gel Extraction Kit (Qiagen) was used in order to purify the now

linearized plasmid DNA vectors (pVAX-N3-GFP and pVAX-N3-GFP-LAMP) from agarose gel. Firstly, since

exposure to ethidium bromide is hazardous for DNA and decreases cloning efficiency, a method by analogy was

used in order to extract the vector from the gel. A 1% agarose gel electrophoresis was run for 1h30 at 120 volts,

with 5 µL of the digestion mixture in the first lane next to the marker (NZY DNA ladder III, NYZtech), and the

rest of the sample three lanes apart. Only the side of the gel with the 5 µL sample was stained, for 20 minutes,

with ethidium bromide. After observation of the gel under UV light, the 5 µL sample was removed from the gel.

The unstained part of the gel with the rest of the sample was put side by side with the stained one and a band

corresponding to the plasmid vector was excised from the gel by comparison without exposure to ethidium

bromide and UV light.

34

Concentrations of both purified vector and insert were determined by NanoVue Plus

Spectrophotometer (GE Healthcare®) in order to perform further calculations for the ligation of insert and

vector. The quality of both DNA samples was also assessed by the values of A260/A280 and A260/A230 ratios.

III.1.1.5. Ligation

The final step in the construction of the desired plasmids consisted in ligating the E1A sequence into

the complementary digested vector backbones. For the cloning experiments, the masses of the vectors and the

E1A sequence were determined according to a 3 insert: 1 vector and a 6 insert: 1 vector molar ratios (equation

1). The required volumes of vector and insert were mixed together with 2 µL of T4 DNA ligase enzyme

(Promega), which catalyses the formation of phosphodiester bonds that link nucleotides together, and the

corresponding T4 ligase buffer (300 mM Tris-HCl (pH 7.8), 100 mM MgCl2, 100 mM DTT 10 mM ATP), to a final

volume of 20 µL. A negative control, consisting of ligase and vector alone, was always added, in order to ensure

the correct digestion of the vector and check for vector re-circularization. The mixtures were then incubated

for 3h at room temperature. After incubation, 10 µL were immediately transformed into DH5 α E. coli cells,

while the remaining 10 µL of the mixture were incubated overnight at 4⁰C and then used for bacterial

transformation.

III.1.1.6. Transformation into E. coli DH5α cells

In order to create chemically competent cells, a 5 mL of 20 g/L LB culture with E. coli DH5 α cells was

grown overnight, at 37⁰C and 250 rpm. The culture was then diluted into 30 mL of fresh LB media with a

starting OD600nm of 0.1, according to the equation 2. Cells were grown, in the same conditions, to an OD600nm of

approximately 0.5, harvested into 15 mL falcon tubes and centrifuged in an Eppendorf bench-top Centrifuge

5810 R with a rotor A-4-62 for 10 minutes (1811 g, 4⁰C). The resulting pellet was resuspended in 500 µL of

filtered TSS buffer (1/10 of the starting culture volume) (5% Dimethyl sulfoxide (DMSO), 50 mM MgCl2, 10 %

PEG 8000 (w/v) (pH 6.5), LB (20 g/L)). The suspension was kept on ice for 10 minutes and then split into 100 µL

aliquots in previously chilled eppendorf tubes. Cryovials were left in ice for 10 minutes and immediately stored

at -80⁰C.

, in which “i” represents the inoculum and “f” represent the final volume and OD.

Equation 1

Equation 2

35

Transformation of plasmids into the bacterial cells was achieved by thermal shock. After thawing

100 µL DMSO-competent E. coli DH5 α cells on ice, 10 µL of the previously obtained pDNA were added and

gently mixed. This suspension was left on ice for 30 minutes, incubated for 1 minute at 42⁰C and once again

placed on ice for 2 minutes. 1 mL of sterile LB medium was added to the cells and after incubation at 37⁰C for

1h, the suspension was centrifuged at 3,820 g for 2 minutes in an Eppendorf bench-top centrifuge 5810 R with

a rotor F45-30-11. The pellet resuspended in residual 100 µL of supernatant was plated in kanamycin

(30μg/mL) selective LB agar plates (35 g/LB; tryptone 10 g/L; yeast extract 5 g/L; NaCl 5 g/L and Agar 15 g/L

(Sigma-Aldrich ®)). Transformation plates were incubated overnight at 37⁰C.

III.1.2. Confirmation of clones by enzymatic digestion

Successful transformant colonies were selected for confirmation of correct plasmid DNA construction

and cultivated overnight (OD600nm ≈3.5) in 5 mL of LB media (Sigma-Aldrich®) supplemented with 5 µL of

kanamycin (30 µg/mL). The cultured volume was centrifuged in an Eppendorf bench-top Centrifuge 5810 R with

a rotor A-4-62 at 3,220 g and 4oC, for 10 minutes. Once the pellet was obtained, plasmid DNA was subsequently

purified with High Pure Plasmid Isolation Kit (Roche), according to the manufacturer’s instructions.

In order to confirm the correct insertion of the E1A sequence into the plasmid backbones, two

restriction enzymes, MluI and NheI, were selected, using the ApE software®. Due to the small size of the

fragment (58 bp) and the difficulty in observing in an agarose gel such short sequence, one of the enzymes did

not immediately flank the sequence, but instead its restriction site was localized upstream the nucleotide

sequence in the plasmid. Therefore, in order to confirm the construction of pVAX-E1A-N3-GFP and pVAX-E1A-

N3-GFP-LAMP, 1 µg of pDNA was digested with 0.5 µL of each endonuclease and 2 µL of Buffer C (Promega), to

a final volume of 20 µL. The resulting double-digested fragments were analyzed in 1 % agarose gel.

III.1.3. Confirmation of clones by automated sequencing

Plasmid DNA constructions, with the expected fragments obtained in restriction confirmation, were

sequenced. 1 µL of pDNA, 2 µL of buffer, 0.5 µL of the primer (table 3) and 4 µL of PCR mix were mixed in a PCR

microtube. Sterilized water was added in order to achieve a total volume of 20 µL. The gene fragment was

confirmed in a CFX96 TouchTM

Real-Time PCR Detection System (Bio-Rad®), by heating the PCR mixture at 96⁰C

for 1 minute, followed by 25 cycles of 10 seconds at 96⁰C, 15 seconds at 55⁰C and 1 minute at 60⁰C. After the

amplification step, samples were cooled to 20⁰C. In order to precipitate DNA, 2 µL of 125 mM EDTA, 2 µL of

3 M Sodium Acetate and 50 µL of 95 % ethanol were added to the PCR product. The mixture was vortexed and

centrifuged for 30 minutes, at maximum speed and 4⁰C. The supernatant was carefully removed, 200 µL of

70 % ethanol were added to the pellet and once again the mixture was vortexed, followed by a 10 minute

centrifugation in the same conditions previously stated. Supernatant was removed and the pellet carefully

36

dried. The precipitated DNA was resuspended in 20 µL of formamide and the mixture was loaded onto a 96-

well reaction plate, carefully covered with a rubber septa. The plate was finally loaded into the Genetic

Analyzer 3130 (Applied Biosystems®). Sequencing data was acquired and analyzed by the SeqScape® version

2.5.

Table 3- Primers used in automated sequencing of plasmid DNA constructs

Primers Sequence

T7 Forward 5’ – TAATACGACTCACTATAGGG – 3’

N3 Reverse 5’ – CCCAATCGTATTGCATTGTCC - 3’

III.1.4. Preparation of E. coli DH5 α cell banks for pVAX-E1A-N3-GFP and pVAX-E1A-

N3-GFP–LAMP

After confirmation, the colonies harboring the correct plasmid construction were grown overnight at

37⁰C and 250 rpm in 5 mL of LB media (20 g/L) supplemented with 5 µL of Kanamycin (30 µg/ mL). A

determined amount of cell suspension (equation 2) was then transferred to a 100 mL Erlenmeyer with 30 mL of

LB media supplemented with 30 µL of kanamycin and incubated, in the same conditions, until an OD600nm of

approximately 1.5 (3/4 of the exponential phase) was reached. Cell banks of the constructed plasmids (figure

13) were prepared by mixing 80 µL of the cell suspension with 20 µL of sterile 99% glycerol (Sigma-Aldrich ®)

and posteriorly stored at -80⁰C.

Figure 13- Schematic illustration of the two designed DNA vaccine vectors, pVAX-E1A-N3-GFP and pVAX-E1A-N3-GFP-LAMP,

constructed by insertion of the adenovirus e1a endoplasmic reticulum sequence in the previously constructed plasmids

pVAX-N3-GFP and pVAX-N3-GFP-LAMP.Images obtained with ApE © software.

37

III.2. Plasmid Production and purification

III.2.1. Alkaline lysis and hydrophobic interaction and size exclusion

chromatographies (HIC-SEC)

III.2.1.1. Production of alkaline lysate

E. coli DH5α cells harboring plasmids of interest, previously stored at -80oC, were cultivated in

100 mL Erlenmeyer flasks containing 30 mL of 20 g/L Luria Bertani (LB) selective medium (NaCl- 5 g/L; Tryptone

10 g/L; Yeast Extract- 5 g/L) (Sigma-Aldrich) supplemented with of 30 µL kanamycin (30 µg/mL), overnight at

37⁰C and 250 rpm in an orbital shaker model AGITORB 2000 (ARALAB). A determined volume of cell suspension

calculated using equation 2 for a starting OD600nm ≈ 0.2 was then added to 250 mL of LB media, in 2000 mL

flasks, supplemented with 250 µL of kanamycin. Cells were grown at 37⁰C and 250 rpm for about 7 hours until

the late phase of stationary phase, corresponding to an OD600nm ≈3.5-4.5, measured in U-2000

spectrophotometer (Hitachi®) was reached. Culture media was then centrifuged at 6,000 g for 15 minutes at

4⁰C with a SLA 3000 rotor in a Sorvall RC 6 centrifuge (Milford, MA, USA).

Required volumes of P1 (50 mM glucose, 25 mM Tris-HCl, 10 mM EDTA, pH 8.0), P2 (0.2 M NaOH, 1%

(m/v) SDS) and P3 (5M potassium acetate, 6.8 M glacial acetic acid) solutions were calculated with the

previously measured OD of the cell culture and equation 3. The supernatant was discarded and the pellet

resulting from cell harvesting was resuspended in buffer P1 using vortex. Alkaline lysis was performed by

adding the P2 solution, followed by an incubation of 10 minutes at room temperature. The P3 solution was

added followed by gentle homogenization, in order to stop the lysis and precipitate genomic DNA, and the

mixture was left on ice for 10 minutes. The alkaline lysate was centrifuged at 20,000 g for 30 minutes, at 4⁰C, in

a Sorvall RC6 centrifuge with a SS-34 rotor, in order to remove genomic DNA, proteins and cell debris. The

supernatant was placed in new centrifuged tubes and centrifuged with the same settings as before to ensure

complete removal of cell debris.

III.2.1.2. Plasmid DNA primary purification

In order to precipitate all nucleic acids (pDNA, genomic DNA and RNA) a previously calculated volume

(70% volume of the lysate volume) of isopropanol (100 % v/v) (equation 4) was added to the alkaline lysate,

followed by careful and gentle homogenization and a resting period of 4 hours. The mixture was centrifuged

for 30 minutes at 4⁰C and 20,000 g and the supernatant was discharged. The pellet was washed with 4 mL of

Equation 3

38

ethanol 70 % (v/v) and once again centrifuged with same parameters stated previously. The supernatant was

discharged and the tubes with the pellet were inverted in order to remove the remaining ethanol. The pellet

was dissolved overnight in 1000 µL of Tris-HCl (10 mM, pH 8.0). The resulting solution was placed in eppendorf

tubes with 0.337 g of ammonium sulphate, homogenized and left on ice for 15 minutes to allow removal of

impurities by precipitation. The samples were centrifuged in an Eppendorf bench-top Centrifuge 5417 R with a

rotor F45-30-11 at 17,950 g and 4⁰C for 30 minutes and the supernatant (≈ 1000 µL) transferred to a new

eppendorf tube. Samples not immediately processed were stored at -20⁰C until further use.

III.2.2.3. Preparation of chromatography columns

Previously prepared Bio-Rad Econ-Pac columns, packed with 10 cm height hydrophobic interaction

Phenyl Sepharose 6 Fast Flow resin (GE Healthcare Bio-Sciences) was stored at 4⁰C with 20 mL of 20 % ethanol.

Before chromatographic procedures, the columns were always washed with 30 mL of H2O milliQ and

equilibrated with 30 mL of 1.5 M ammonium sulphate (in 10 mM tris-HCl (pH 8.0)).

III.2.2.4. Hydrophobic interaction chromatography (HIC)

A sample of 500 µL of clarified pDNA was injected in the column and isocratic elution was performed

with 20 mL of 1.5 M ammonium sulfate (in 10 mM Tris-HCl pH 8.0), with recovery of 500 µL aliquots of

flowthrough pDNA fractions. The remaining more hydrophobic molecules (RNA, genomic DNA, denatured

pDNA) were eluted by injection of 30 mL of 10 mM Tris-HCl, which reduces the ionic strength of the ligations

between impurities and matrix .The column was washed with 30 mL of H2O milliQ and stored with 20 mL of

20% ethanol. The concentration and quality of samples recovered from the procedure was determined in

NanoVue Plus spectrophotometer (GE Healthcare®) and the fractions with higher concentrations were selected

for further purification in the SEC step.

III.2.2.5. Size exclusion chromatography (SEC)

The column was washed with 30 mL of H2O milliQ and equilibrated with 30 mL of 10 mM Tris-HCl pH

8.0. Samples of pDNA obtained by HIC with similar concentrations were pooled together and injected in the

SEC column (≈ 2mL per injection). Elution of pDNA was performed with 10 mL of 10 mM Tris-HCl. Aliquots of

500 µL were collected and concentration of pDNA was determined by NanoVue Plus spectrophotometer (GE

Healthcare®) and analyzed by gel electrophoresis to confirm presence of supercoiled pDNA. 10 mL of 10 mM

Tris-HCl was injected in the column in order to elute bound species. The quality of the pDNA samples was also

Equation 4

39

assessed by the values of A260/A280 and A260/A230 ratios. The criteria used were an optical density ratio

260:280nm > 1.8 and 260:230nm > 2.0. Samples were stored at -20⁰C until further use.

After final use, the resin was cleaned (Amersham Protocol) with 20 mL of 1 M NaOH (Fisher Chemical),

40 mL of distilled water and 40 mL of ethanol 70 % (v/v) and was stored in 20 % ethanol (v/v) at 4⁰C.

III.2.3. HiSpeed Plasmid Midi and Maxi Kit (Qiagen)

30 µL of cell banks of E. coli DH5α cells harboring the plasmids pVAX-GFP, pVAX-N3-GFP, pVAX-E1A-

N3-GFP, pVAX-N3-GFP-LAMP and pVAX-E1A-N3-GFP-LAMP were cultivated and grown overnight in 150 mL of

LB media (20 g/L) supplemented with 150 µL of Kanamycin (30 µg/mL), at 37⁰C and 250 rpm in an orbital

shaker model AGITORB 2000 (ARALAB) until a cell density of 3-4 x 109 cells was reached (OD600nm≈3,5-4.5). Cell

culture was split into 50 mL falcon tubes and centrifuged at 6000 g and 4⁰C, for 15 minutes in Eppendorf

bench-top Centrifuge 5810 R with a rotor A-4-62. Plasmid DNA was afterwards purified with the Hispeed

Plasmid Purification Midi and Maxi Kit (Qiagen) and purified pDNA was eluted in 1 mL of TE buffer (10 mM Tris-

Cl (pH 8.0) and 1 mM EDTA) and stored at -20⁰C until use. The quality of the pDNA samples was assessed by

agarose gel electrophoresis and by the values of A260/A280 and A260/A230 ratios obtained with NanoVue Plus

spectrophotometer (GE Healthcare®). The criteria used were optical density ratios of 260:280nm > 1.8 and

260:230nm > 2.0.

III.3. In vitro assays in CHO cells

Plasmids pVAX-GFP, pVAX-N3-GFP, pVAX-E1A-N3-GFP, pVAX-N3-GFP-LAMP and pVAX-E1A-N3-GFP -

LAMP were tested for plasmid copy number, transfection efficiency and protein expression in a series of in vitro

tests in CHO (Chinese hamster ovary) cells, a stable and reproducible mammalian cell line. Each essay consisted

of three wells of transfect cells for each one of the plasmids and always included three replicates of a negative

control, consisting of cells exposed to the lipofectamine 2000TM

reagent but not to pDNA. In vitro assays were

carried out in INIAV and transfected cells were further analyzed by flow cytometry, RT-PCR and RRT-PCR

analysis.

III.3.1. Culture and transfection of CHO cells

One vial of previously frozen CHO Cells (1 mL of cells and 10 % DMSO) destined for in vitro testing was

seeded in 75 cm2 T flasks using 2.5 mL of fetal bovine serum (FBS- 10 % v/v) (Gibco, UK) and 22.5 mL of F-12

(Ham) nutrient mixture (Gibco, UK), supplemented with 1% of MEM non-essential amino acids 100X (Gibco,

UK), 1% of antibiotic-antimycotic (Gibco, UK), 1% of sodium pyruvate 100 mM (Gibco, UK) and 0.1% of

40

Gentamicin (50 mg/µL) (Gibco,UK). The culture was incubated at 37⁰C in 5 % CO2 humidified environment, in

order to obtain the ideal confluence (80-90%) and cell density for passage to 24-well plates. Upon reaching

such parameters, the supplemented F-12 medium in the T-flask was discarded and cells were washed with

8 mL of PBS, to ensure total removal of the growth media, which can inhibit the action of trypsin. Cells were

trypsinized with 4 mL of trypsin and incubated at 37⁰C for 5 minutes. Afterwards, 6 mL of PBS were added to

the trypsin and the total volume was centrifuged at 240 g (4⁰C) for 8 minutes. Supernatant was discarded and

the pellet was resuspended in 5 mL of PBS. A 10-fold dilution was performed and cells were subsequently

counted in a Neubauer chamber, in order to calculate the volume necessary to obtain a cell density of 2 x 105

cells per well. After centrifugation of the required volume, cells were seeded into 24-well plates, in a final

volume of 500 µL of F12 medium without antibiotics and 10 % FBS, per well. Plates were incubated for 24 hours

(37⁰C, 5% CO2) until 80-90 % confluence was reached. Cells were transfected with a mixture obtained by the

conjugation of two solutions: 1 µg of pDNA with F-12 incomplete medium (to a final volume of 50 µL) and 2 µL

of Lipofectamine® 2000 reagent (Invitrogen™) added to 48 µL of F-12 incomplete medium, incubated for 5

minutes at room temperature. Both solutions were mixed and the 100 µL transfection mixture was allowed to

equilibrate for 20 minutes at room temperature, giving rise to the lipoplexes with Lipid/pDNA. After removal of

250 µL of F-12 medium, 100 µL of transfection mixture was added to each one of the wells and the plates were

incubated at 37⁰C and 5 % CO2 humidified environment for 4 hours. After this incubation time, the medium

with the transfection mixture and non-internalized pDNA was replaced by complete medium with antibiotics

and 10 % FBS to a final volume of 1000 µL per well. Cells were harvested after a 48h incubation period.

III.3.2. Quantitative Real-time PCR for determination of plasmid copy number in

transfected CHO cells

Preparation of samples for RT-PCR was carried out by harvesting transfected cells and negative

controls after 48 h. Medium was removed from the wells, cells were washed with 800 µL of PBS and

subsequently 200 µL of trypsin were added to each well. Plates were incubated at 37⁰C humidified

environment for 5 minutes. After three additional washing steps with 1.0 mL of PBS, cells were centrifuged at

240 g and 4⁰C for 8 minutes. The pellet was resuspended in 2 mL of PBS and a sample of this suspension was

counted in a Neubauer chamber. A second centrifugation was performed, with the same settings stated above,

and the pellets were resuspended in the necessary volume of sterilized and filtered water, in order to obtain a

concentration of 6250 cells/µL in each suspension. The samples were stored at -20⁰C until the quantitative RT-

PCR analysis was performed.

For determination of plasmid content in transfected CHO cells, Quantitative Real-Time PCR

amplification and analysis was carried out in a Roche LightCycler™ detection system using the FastStart DNA

Master SYBR Green I Kit (Roche Diagnostics) by amplification of a 108 bp fragment of the eGFP gene. Each

41

capillary was prepared with 16 µL of a reaction mixture containing 2 µL of SYBR Green, 1 µL of the forward and

the reverse eGFP primers (10 µM) (table 4), 1.6 µL of MgCl2 (3.0 mM) and 10.4 µL of PCR Grade water.

Firstly, serial dilutions, in sterilized and filtered MilliQ water, of previously purified pDNA by the HIC-

SEC procedure were used to construct the calibration curves. 2 µL of these dilutions, with concentrations

ranging from 2.5 to 25,000 pg/µL, were added to a 2 µL suspension of non-transfected CHO cells (12,500 cells

per capillary) and to 16 µL of the PCR reagents mixture described above. The standard curves were produced

for each one of the five plasmids, in duplicate.

Two negative controls, each one performed in duplicate, were added to the analysis. The first control

consisted of 4 µL of PCR grade water, in order to detect undesired contamination, and the second control

consisted of 2 µL of PCR grade water and 2 µL of non-transfected CHO cells (exposed to lipofectamine, but not

pDNA). Both controls contained also 16 µL of PCR reaction mixture described above. Determination of pDNA

concentration in the transfected cells was always done in triplicate, for each analysis and performed by adding

2 µL transfected CHO cells and 2 µL of PCR grade water, with 16 µL of the PCR reaction mixture.

The reaction consisted of a 10 minute incubation at 95⁰C to lyse the cells and activate the FastStart

DNA polymerase, followed by the amplification step, consisting of 30 cycles of 10 seconds at 95⁰C

(denaturation), 5 seconds at 55⁰C (annealing) and 7 seconds at 72⁰C (elongation), adapted from Carapuça et al.

(2007) (Carapuça et al. 2007). After the completion of the cycles, samples were maintained at 70⁰C for 30

seconds, followed by a melting curve analysis from 70⁰C to 95⁰C, at a gradient of 0.1⁰C/s, in order to confirm

the amplification of specific products. The reaction was finalized with a cooling step at 40⁰C for 30 seconds.

The threshold cycle (CT) was calculated by the “Fit points method” in the LightCycler software version

3.5 (Roche Diagnostics). Standard curves obtained in the reaction provide the relation between plasmid copy

number and the CT, an absolute quantification method.

III.3.3. Flow cytometry analysis

Cells were harvested after 48 hours of transfection. Medium was removed from the wells; cells were

washed with 800 µL of PBS and trypsinized with 200 µL of trypsin. Plates were incubated at 37⁰C humidified

environment for 5 minutes. 1.0 mL of PBS was added to each well and the cells were centrifuged at 240 g (4⁰C)

Table 4 - Real-time PCR forward and reverse primer sequences for assessment of eGFP gene presence.

eGFP Primers Sequence

Forward 5’-TCGAGCTGGACGGCGACGTAAA-3’

Reverse 5’-TGCCGGTGGTGCAGATGAAC- 3’

42

for 8 minutes. Pellets were resuspended in 800 µL of 2% paraformaldehyde (PFA) (Sigma®) and samples were

stored in 4⁰C in the dark until flow cytometry analysis.

Flow cytometry for detection of GFP expression levels of transfected and non-transfected cells was

performed up to a maximum of 4 days after transfection. Samples were analyzed in a FACscan Scalibur flow

cytometer (Becton-Dickinson, Franklin Lakes, NJ). This analysis allowed for the selective visualization of cells

from unwanted particles, such as dead cells and cell debris, in a process referred to as gating, according to their

individual forward scatter (FSC) versus side scatter (SSC) characteristics, which relate to cell size and cell

complexity, respectively. Detection of light emitted by fluorescence channel FL1, which detects emission from

the GFP protein, allowed for the determination of transfection efficiency. Background FL1 from non-

transfected cells was subtracted from the total cell population and GFP expression levels was determined by

CellQuest Pro Software (Becton-Dickinson, USA). Only samples with a minimum of 10000 events (cells)

registered were considered statistically significant data.

III.3.4. Real-time reverse transcription PCR (RRT-PCR)

CHO cells transfected with pDNA and negative controls were harvested after 48 h incubation,

following the same procedure performed in collection for flow cytometry analysis (section III.3.3). 1x106 cells

per plasmid and negative control were resuspended in 200 µL of sterile PBS and the High Pure RNA Isolation Kit

(Roche®, Germany), suited for total RNA extraction from cultured cells, was carried out, including DNase I

treatment. RNA was eluted in 50 µL of elution buffer (PCR grade water) and samples were stored at -80⁰C, until

further processing. First strand DNA was synthesized using First Strand cDNA Synthesis Kit for RT-PCR (AMV)

(Roche ®, Germany) with oligo-p(dT)15 primer and 1 µg of RNA per sample. Real-time PCR was performed in the

same procedure previously described in section III.3.2., using 2 µL of cDNA of each sample. Calibration curves

for each plasmid were constructed with serial dilutions (2.5 to 25,000 pg/µL) added to a 2 µL of sterilized and

filtered water. The RT-PCR determination of mRNA content in transfected cells was performed for each

plasmid, with triplicate samples. The negative controls included 4 µL of PCR-grade water and 2 µL of cDNA

obtained from non-transfected cells.

III.3.5. Data and statistical analysis

All values were expressed as mean ± standard deviation (SD). Experimental data obtained in in vitro

assays (mean fluorescence, transfection efficiency, plasmid copy number and mRNA copy number) was

statistically analyzed by performing an one way analysis of variance (ANOVA). For all tests, P < 0.05 was

considered statistically significant. Data analysis was performed with Microsoft Excel.

43

III.4. In vivo assays in chickens

III.4.1. DNA vaccination of chickens

For the immunization trial, 15 four-week-old chickens were divided into 6 isolated groups: plasmids

pVAX-N3-GFP, pVAX-E1A-N3-GFP, pVAX-N3-GFP-LAMP and pVAX-E1A-N3-GFP-LAMP were inoculated into

three chickens each, 2 chickens were immunized with pVAX-GFP and one chicken was not immunized.

20 µL of Lipofectamine 2000® reagent (Invitrogen™) and 100 µL of sterile PBS were mixed and

incubated at room temperature for 30 minutes, to allow formation of lipossomes. 100 µg of purified pDNA was

added to PBS to a final volume of 280 µL. Both solutions were mixed together and the 400 µL vaccine inoculum

was equilibrated at room temperature for 15 minutes.

In every immunization, chickens received two intramuscular injections of 200 µL on each breast

muscle. After the first inoculation, two booster doses were administered at two week-intervals. Two weeks

after the last immunization, at day 52, chickens were inoculated with 100 µL of purified N3 protein.

Before every immunization procedure and at days 7, 42, 52, 66, 78 and 90 from the first immunization,

the chickens were bled. The blood samples collected from the wing veins of chickens were kept at 37⁰C for 30

minutes and then centrifuged at 2400 g for 10 minutes at 4⁰C. Serum was stored at -20⁰C until performance of

ELISA assays.

III.4.2. N3 protein production and purification

N3 protein production and purification was performed at INIAV by Ana Margarida Mourão. The N3

gene was cloned in plasmid pET-28a. E.coli JM109 cells harboring plasmid pET-28-N3 were cultivated in 4 mL of

LB media supplemented with kanamycin (30 µg/mL) and grown overnight at 37oC. The culture volume was then

transferred to 200 mL of fresh LB media supplemented with kanamycin and grown in the same conditions for

3 hours. Isopropylthio-β-galactoside (IPTG 1M) was added to the culture for protein expression induction, at

37oC for 4 hours. The protein N3 was subsequently purified by a method based in a purification methodology

described elsewhere (Lu, Z. et al 2007).

III.4.3. Enzyme Linked Immunosorbent Assay (ELISA)

The preparation of the 96-well ELISA plates consisted of a coating of 100 µL of previously diluted 1:100

N3 protein, in carbonate-bicarbonate buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6), in each well. The ELISA

plate was covered and incubated overnight at 4⁰C. After incubation, the plate was washed four times with a

44

PBS/Tween 0.05% solution (950 mL H2O, 50 μL PBS 20x and 500 μL of polyoxyethylene sorbitan monolaurate

(Sigma ®)).

20 µL of the serum samples, collected from chickens of the same plasmid test group and in the same

dates, were mixed together in order to form a pool from each group. Each chicken pool serum sample and the

positive controls (anti-H5N3) were diluted 1:50 in blocking buffer (2% of milk, 5% of Newborn serum, 5% E.coli

lysate in sterile PBS) and incubated at 37oC for 30 minutes. 100 µL of the diluted samples and control were

added to each well and the plate was incubated at 22⁰C for 1h. After incubation, the plate was washed four

times as previously described. 100 µL of a previously prepared anti-chicken serum IgG (Fc):HPr (Abd serotec ®)

dilution in blocking buffer (1:10000) was added to the wells. The plate was incubated once again at 37⁰C for 30

minutes and washed again four times. Generation and detection of a signal was performed by the addition of

100 μL of peroxidase substrate TMB (tetramethylbenzidine (Sigma ®)) to each well. The reaction was stopped

after 30 minutes by adding a stopping solution of 100 μL 2M sulfuric acid. Data was analyzed in a SUNRISE

Tecan ® reader machine, at 450 nm.

III.4.4. Data and statistical analysis

Experimental data obtained in in vivo assays (absorvance values in the ELISA) were statistically

analyzed by performing the unpaired two-tailed Student’s t-test. For all tests, P < 0.05 was considered

statistically significant. Data analysis was performed using GraphPad Prism © version 5.0.

45

I.V. Results and discussion

IV.1. DNA vaccine construction

DNA prototype vaccines that were tested and evaluated during the experimental work of this thesis

were previously constructed by biomolecular techniques, which included the insertion of the N3 gene

sequence and targeting sequences E1A and LAMP in base vectors. The resulting plasmids consisted of pVAX-

GFP, always used as a control, pVAX-N3-GFP, pVAX-E1A-N3-GFP, pVAX-N3-GFP-LAMP and pVAX-E1A-N3-GFP-

LAMP. These DNA vaccines were previously tested for protein expression in mammalian cells and antibody

titter production in an in vivo essay in immunized chickens (Gil 2011) (Freitas 2012). During the course of such

work, it was noted that the sequence of the CMV promoter in plasmids harboring the E1A sequence was

incomplete. The pCMV sequence consists of 558 bp, with an enhancer sequence located between the 27th

and

431st

nucleotides. In the E1A-plasmids, there is an evident and considerable reduction - of 225 bp - in the size of

the promoter. Such event occurred most likely due to a misperformed cloning experiment, since the E1A

sequence was inserted immediately after a restriction site for SnaBI (TAC^GTA) in the middle of the CMV

promoter, which resulted in the deletion of part of the enhancer sequence. The correct expression of the

protein of interest, in this case the N3 protein fused with GFP, is under the control of the CMV promoter, which

promotes a high level of constitutive protein expression in several mammalian cell lines, transcribing higher

levels of mRNA than alternative promoters (Williams 2013) (Kutzler & Weiner 2008) and higher antigen

expression in vivo (Laddy & Weiner 2007). It was therefore essential to correct the sequence of the two

plasmids carrying the E1A target sequence.

The annealing of forward and reverse synthetic oligonucleotides of the E1A sequence and posterior

cloning into base vectors was the approach selected to reach such goal. The sequence was flanked with two

restriction sites, for enzymes BstEII and NheI, in order to obtain cohesive ends after cleavage to clone the insert

in the correct position in both plasmids, downstream from T7 promoter and upstream the antigen N3

sequence. To allow the correct ligation of the restriction enzymes and correct cleavage, six adenines were also

added to both oligonucleotides. The choice of the number and particular nucleotide was based in the quality of

all the possible options with bioinformatics tools. The final sequence (table 5) was the one that presented

minimal risk of secondary structures formation (hairpins), melting temperature of 79,25oC and GC content

between 50-60% to ensure stability (in this case 56,6%).

Table 5- Sequences of forward and reverse oligonucleotides used for the construction of the E1A insert.

Primer Sequence

E1A_Forward 5'- AAAAAAGGTGACCATGCGCTATATGATTCTGGGCCTGCTGGCGCTGGCGGCGGTGTGCAG CGCGGCTAGCAAAAAA-3’

E1A_Reverse 5'- TTTTTTGCTAGCCGCGCTGCACACCGCCGCCAGCGCCAGCAGGCCCAGAATCATATAGCGC ATGGTCACCTTTTTT-3'

46

The annealing procedure was carried out with different concentrations of single-stranded

oligonucleotides (ss oligos) and optimized to a final concentration of 10 µM double-stranded oligonucleotide

(ds oligo). The final ds oligo mixture, with a size of 76 bp, was confirmed in 2-4% agarose gels (figure 14).

The results were inconclusive. While it is clear that ss oligos and the annealed ds oligos show a

different pattern of migration through the agarose gel, it would be expected to see the bands corresponding to

the ds oligos above in the gel, corresponding to a slower migration caused by its higher molecular weight. This

pattern is mostly visible in the 2% agarose gel. It is important to notice that ethidium bromide, used to stain the

agarose gels during this work, has more affinity for ds DNA by intercalating with its double-helix, while leading

to poor resolution when staining RNA and ss DNA. The smear presented in the several gels probably

corresponds to the incorrect ligation of ethidium bromide to ss DNA, formation of hairpins or aggregations of ss

DNA that led to a slower migration and the presence of salt in the mixture, since the annealing buffer

contained NaCl. Due to the fact that the annealing was not 100% efficient, the final solution may also be

contaminated with unannealed oligos that form hairpins, and thus appear has a band of lower molecular

weight in the gel. In order to further confirm the success of the annealing procedure, Real-time PCR with SYBR

Green I melt peak analysis was performed for both the forward and reverse ss oligos and the annealed ds oligo.

SYBR Green binds non-specifically to double-stranded DNA by intercalation (Varga & James 2006), so this dye

fluoresces when intercalated into double-stranded DNA but not into single-stranded DNA (Winder et al. 2011).

Indeed, the double-stranded oligos showed a clear melting peak, while SYBR Green didn’t bind to single-

stranded oligos and did not produce melting peaks (figure 15). With this result, it was possible to move forward

with the cloning procedures to construct the desired plasmids.

M 1 2 3 M 1 2 3 4 5 6

Figure 14- Agarose gel electrophoresis, 4% (A) and 2% (B), for the confirmation of annealing procedure. A:

forward ss oligo (lane 1), reverse ss oligo (lane 2), annealed oligos (lane 3). B: forward ss oligo 10 µM (lane

1), reverse ss oligo 10 µM (lane 2), Annealed sequence 2 µM (lane 3), 10 µM (lane 4) and 20 µM (lane5),

annealed sequence 10 µM after purification (lane 6). M- DNA molecular weight marker- GeneRuler 50bp

DNA Ladder (Bioline).

A B

47

IV.1.1. Construction of pVAX-E1A-N3-GFP and pVAX-E1A-N3-GFP-LAMP

In order to construct the corrected vectors pVAX-E1A-N3-GFP and pVAX-E1A-N3-GFP-LAMP, the base

vectors pVAX-N3-GFP and pVAX-N3-GFP-LAMP and the insert E1A were double digested with BstEII and NheI,

in order to create compatible cohesive ends in both structures. Due to the difference of optimal temperature

between restriction enzymes, NheI at 37oC and BstEII at 60

oC, the digestion conditions had to be optimized

throughout a series of cloning experiments. Finally, the digestion procedures were accomplished at 37oC for 3.5

hours.

Following restriction digestions, the base vectors were purified, as described in detail in chapter III

section 1.1.4., by 1% agarose gel. The excision of the vector-corresponding band was performed through a

comparison method between a 5 µL sample of the digested vector stained with ethidium bromide, and the

remainder of the sample unstained with this DNA intercalator and unexposed to UV light, ensuring that cloning

and ligation efficiencies were not compromised and there was no damage caused to the DNA sample. The

agarose extracts containing the linearized vectors were subsequently purified with the Qiagen Gel Extraction

Kit (Qiagen).

Figure 15 - Melt peak analysis of single-stranded forward (A) and reverse (B) oligonucleotides

and double-stranded annealed oligonucleotide (C). The Tm obtained for the ds oligo was

88,77oC. Images obtained with LightCycler software version 3.5 (Roche Diagnostics).

A

B

C

48

Due to the small size of the E1A sequence and the fact that purification of DNA fragments through

excision from agarose gel lead to low recovery yields in the vector purification (32%), the double-digested E1A

fragment was purified through a PCR Clean-Up Kit that allows for the DNA to be bound on a silica membrane,

cleaned and finally eluted, with an average recovery yield of about 76% during the experimental work.

Once the vectors and the insert fragment were purified, it was possible to perform ligation between

them with T4 DNA ligase. Different molar ratios of molecules of insert and vector were used in the ligation

experiments. Both 3:1 and 6:1 molar ratios of Insert:Vector produced successful ligation results, with clones

harboring the desired constructions. The ligation experiment was carried out for 3h at room temperature and

3h at room temperature followed by an overnight period at 4oC. The DNA ligase, which catalyses the joining of

the 3′-OH end to the 5′-phosphate end of another molecule, has an optimal temperature of 25oC. On the other

hand, lower temperatures, which translate into lower mobility of molecules, are ideal for bringing the DNA

molecules together. The two methods used are therefore a balance between contradicting ideal temperatures.

Too low temperatures would affect DNA ligase activity, while too high temperatures would affect the cloning

efficiency by melting the annealed DNA ends and by increasing molecular movement in the reaction (Lund et

al. 1996), so a room temperature incubation followed by an overnight period at 4oC would favor the reaction.

Both ligation mixtures and negative controls were transformed by heat shock into E. coli DH5 α competent cells

and plaqued in kanamycin-supplemented LB agar plates.

Several resulting colonies were selected and purified by miniprep High Pure Plasmid Isolation Kit

(Roche) and screened by restriction analysis. In order to verify the presence of the correct plasmid

construction, double digestions with MluI and NheI were performed in the resulting DNA and also base vectors

used as starting material in the cloning experiments, in order to compare the fragment sizes.

Plasmids with the correct fragments (table 6) observed in agarose gel electrophoresis (figure 16) were

then sequenced with primers flanking the insertion place for E1A, for further confirmation. Automatic

sequencing confirmed the correct, total and in frame cloning of the E1A sequence in the base vectors, giving

rise to correct pVAX-E1A-N3-GFP and pVAX-E1A-N3-GFP-LAMP, now harboring the complete CMV promoter.

Plasmid DNA vectors Expected fragment sizes (bp)- Digestion MluI + NheI

pVAX-N3-GFP (control) 4430 / 667

pVAX-E1A-N3-GFP 4430 / 711

pVAX-N3-GFP-LAMP (control) 4541 / 667

pVAX-E1A-N3-GFP-LAMP 4541 / 711

Table 6 - Expected fragment sizes of correct plasmid constructions, obtained by ligation of E1A sequence and base vectors, after digestion with restriction enzymes MluI and NheI.

49

IV.2. Production of pDNA vaccines

After construction of the DNA vaccine vectors, it was necessary to produce and purify the plasmids for

both in vitro and in vivo assays.

For the in vitro assays, the chosen pDNA purification approach was the hydrophobic interaction

chromatography (HIC) and the size-exclusion chromatography (SEC) combined procedures, a method

correlated with more stable lipoplexes and higher plasmid uptake and GFP expression (La Vega et al. 2013). For

this purpose, E. coli DH5 α cells harboring the different plasmids were grown until an O.D.600 nm of

approximately 3.5 was reached. Cells were harvested by centrifugation, disrupted by alkaline lysis, followed by

concentration with isopropanol, to reduce the volume of the solution containing the pDNA (Diogo et al. 2001)

and ammonium sulphate precipitation, an essential step for the removal of contaminants such as proteins,

endotoxins and RNA and also to increase the ionic strength of the solution, suitably conditioning the sample for

the HIC column (Trindade et al. 2005) (Diogo et al. 2001). Finally, the pDNA sample was purified through the

combination of two chromatography procedures, hydrophobic interaction and size exclusion. Samples of

500 µL obtained through gravity-flow HIC were quantified in Nanodrop through spectrophotometry at 260 nm,

in which salt and protein contamination are also analyzed by the 260/230 and 260/280 absorvance ratios,

respectively. The quality of the purified pDNA solution and evaluation of the separation of plasmid from

impurities was assessed through agarose gel electrophoresis. From these samples, the ones with the higher

pDNA content were pooled together and submitted through the SEC procedure. The SEC procedure, carried out

A

B

B

Figure 16 - Confirmation in 1% agarose gel electrophoresis of constructed plasmids

pVAX- E1A-N3-GFP-(A) and pVAX-E1A-N3-GFP -LAMP (B). Lanes 1A and 1B correspond to

control plasmids pVAX-N3-GFP and pVAX-N3-GFP-LAMP, respectively. Lanes 2A and 3B

correspond to clones further sequenced and confirmed to be the correct constructions.

M- NYZDNA ladder III (NYZTech).

M 1 2 3 M 1 2 3 4

800 bp

5.0 kb

50

in the same matrix, allows buffer exchange, a process essential for the desalting of HIC fractions, which contain

high concentrations of ammonium sulfate, and for final purification of sc pDNA from other plasmid

topoisomers (Ferreira et al. 2000).

Figure 17 shows the several selective processes during pDNA purification of pVAX-N3-GFP. The same

assessment of purification success and plasmid quality was performed for all the plasmids (results not shown).

Before the performance of HIC, it is evident the presence of a high amount of RNA contaminating the pDNA

sample. During the purification process, supercoiled pDNA degradation is likely to occur due to alkaline lysis,

non-optimal pH and temperature and several centrifugations, so other configurations of pDNA, such as open

circular and linear, are often present in the plasmid samples. Such damage decreases the sc pDNA quantity, a

parameter of plasmid purity and also essential for optimal transfection. Nevertheless, it is possible to observe a

clear and well defined band, representing the sc pDNA isoform, in much more quantity than the other

isoforms. Other bands, present in pre-purification samples, represent concatemers and genomic DNA (gDNA), a

higher molecular weight form. Complete removal of RNA and absence of gDNA were achieved. Purified pDNA

was stored at 4oC during the in vitro assays.

Due to the requirements of high amounts of pDNA necessary for the in vivo assays, 100 µg in 280 µL

per chicken per immunization (900 µg of total mass per plasmid), the pDNA was purified with the Plasmid

Purification Midi and High Speed Maxi Kit (Qiagen) and eluted in TE buffer (10 mM Tris-Cl, pH 8.0; 1mM EDTA),

ideal for DNA storage, since it keeps DNA at a defined pH and suppresses DNA degradation by chelating Mg2+

M 1 2 3 4 5 6 7

Figure 17- Analysis of purity and quality of pVAX-N3-GFP by 1% agarose gel electrophoresis.

Plasmid samples were obtained after neutralization of the lysate (lane 1 and 2), precipitation with

isopropanol and clarification with ammonium sulphate (lanes 3 and 4) and purification through

HIC-SEC (lanes 5, 6 and 7). Lane M: Molecular-weight marker (NYZ DNA Ladder III,NYZTech).

Purified pDNA samples in lanes 5, 6 and 7 correspond to 20.25 µg, 110.5 µg and 65 µg per total

sample (500 µL), respectively.

51

and other divalent metal ions, essential for the activity of DNases. Quality of the purified pDNA was assessed

by agarose gel electrophoresis (Figure 18A). To confirm the correct constructions and labeling of the plasmids,

the pDNA was double digested with KpnI and XbaI restriction enzymes and the obtained fragment sizes were

compared with the sizes expected, confirming the correct plasmid constructions (Table 7, Figure 18B). The DNA

vaccine prototypes were stored at -20oC for long-term storage and at 4

oC once the in vivo assays initiated, in

order to avoid damage by repeated cycles of freeze-thawing.

Table 7 - Expected fragment sizes, obtained with ApE software, after double enzymatic digestion with restriction enzymes

KpnI and XbaI, for confirmation of correct pDNA vaccine vectors.

Plasmid DNA constructions Expected Fragment sizes (bp) – XbaI+KpnI digestion

pVAX-GFP 2925 / 772

pVAX-N3-GFP 3070 / 1257 / 770

pVAX-E1A-N3-GFP 3114 / 1257 / 770

pVAX-N3-GFP-LAMP 3070 / 1257 / 881

pVAX-E1A-N3-GFP -LAMP 3114 / 1257 / 881

M 1 2 3 4 5 M 1 2 3 4 5

800 bp

3.0 kb

Figure 18 – Agarose gel electrophoresis (1%): (A): Assessment of quality of plasmids purified by High Speed Maxi

Kit (Qiagen), where is possible to detect a high amount of sc pDNA, with traces of linear and open circular isoforms.

(B): Confirmation of plasmid constructions by enzymatic digestion with restriction endonucleases XbaI and KpnI.

pVAX-GFP (1A, 1B); pVAX-N3-GFP (2A, 2B); pVAX-E1A-N3-GFP (3A, 3B); pVAX-N3-GFP-LAMP (4A, 4B); pVAX-E1A-

N3-GFP -LAMP (5A, 5B). M- Molecular-weight marker (NYZ DNA Ladder III,NYZTech).

A B

52

IV.3. In vitro assays

CHO cells were transfected with the plasmids pVAX-GFP, pVAX-N3-GFP (N3), pVAX-E1A-N3-GFP (E1A-

N3), pVAX-N3-GFP-LAMP (N3-LAMP) and pVAX-E1A-N3-GFP-LAMP (E1A-N3-LAMP), with lipofection, used as

the gene delivery technique, in order to improve this non-viral strategy of cell transfection. Positively charged

liposome/DNA complexes (lipoplexes), formulated with the reagent Lipofectamine 2000TM

, have been widely

used as a safe transfection methodology. In vitro transfection assays have demonstrated to be improved by the

use of cationic liposomes, due to its protective role of pDNA (Sun et al. 2013) (Ribeiro et al. 2012) against

nuclease attack, also enhancing cellular uptake via endocytosis (La Vega et al. 2013).

The ultimate goal of transfection is gene expression. Efficient gene expression, one of the setbacks of

non-viral vectors, is dependent on several factors, such as plasmid uptake, degradation rate of plasmid copies

inside the cell, plasmid access to the nucleus through successful intracellular trafficking and, finally,

transcription and translation efficiencies. In the context of DNA vaccination, one of the principal setbacks is the

efficient delivery of pDNA to cell nucleus (Carapuça et al. 2007). Furthermore, localization of the expressed

antigen within the cell has an effect on both the cellular immune response and the production of antibodies,

through the availability for the MHCI or MHCII pathways (Boyle et al. 1997) (Dobaño et al. 2007a). Therefore,

the behavior of transfect pDNA constructs harboring two protein targeting sequences, E1A, LAMP and their

combination, were tested in several assays, in order to achieve a quantitative understanding of the effect of

such sequences in intracellular trafficking and protein expression.

Expression of the antigen, neuraminidase 3 (Appendix V), fused with the reporter protein was

measured by mean fluorescence obtained through flow cytometry. Plasmid copy number and mRNA level

determination, assessed by the most sensitive and precise techniques available, RT-PCR and RRT-PCR

respectively, are essential to obtain an insight on plasmid stability and also to evaluate transfection and

transcription efficiencies. The transfection efficiency is also assessed by the percentage of GFP positive cells

(number of transfected cells) obtained through cytometry analysis.

IV.3.1. Flow cytometry analysis - Transfection efficiency

Cell transfection rates were quantified by flow cytometry, as estimated by percentages of GFP positive

(GFP+) cells and the level of reporter protein expression. The analysis of GFP positive cells (figure 19), compared

with control non-transfected cells exposed to the same amount of lipofectamine, confirmed the expected

results regarding the most efficient plasmid uptake. pVAX-GFP, always used as a positive control plasmid,

encoding the reporter gene GFP, had a significantly higher (P < 0.05) transfection efficiency than all the

plasmids harboring the N3 sequence. Such transfection efficiency is explained by the smaller size of the pVAX-

GFP plasmid, when compared with the N3 constructs. Plasmid size is an important characteristic regarding

transfection efficiency, as in the number of cells presenting fluorescence, and plasmid copy number (Ribeiro et

al. 2012). Significant smaller sizes are correlated with higher transfection efficiencies (Ribeiro et al. 2012)

53

(Kamiya et al. 2002). Plasmids harboring the N3 gene showed consistently and significantly decreased

transfection efficiencies, as expected. These plasmid DNA constructs, N3, E1A-N3, N3-LAMP and E1A-N3-LAMP,

expressed the fusion protein N3-GFP, which resulted in less fluorescent cells than the ones expressing only the

GFP protein. Among these plasmids, there are no significant differences (P > 0.05) regarding percentage of

GFP+ cells between the N3 plasmid and the N3-E1A plasmid, hinting that there was no influence of the E1A

sequence on plasmid entry in the cell. The N3-LAMP and E1A-N3-LAMP plasmids showed significantly lower

transfection efficiencies (P < 0.05) when compared to the N3 and E1A-N3 plasmids, and no significant

differences between themselves (P > 0.05). These results are confirmed by the mean fluorescence, discussed

below.

IV.3.2. Flow cytometry analysis- Mean fluorescence of transfected cells

Flow cytometry provides an accurate method to quantify protein expression, an alternative and more

precise method than fluorescence microscopy. A comparison between the expression of N3 fused to GFP and

the different targeting sequences helps to provide an understanding of the effect of targeting signals in protein

expression and transport to the targeted cell compartments (figure 20).

As expected the control plasmid, pVAX-GFP, coding for a much smaller protein GFP, had significantly

higher levels (P < 0.05) of fluorescence than the N3-plasmids. Cells expressing different fusion proteins had

lower fluorescence, up to 100 times, than cells expressing the reporter gene GFP alone. Differences can be

0

5

10

15

20

25

30

pVAX N3 E1A-N3 N3-LAMP E1A-N3-LAMP

Tran

sfe

ctio

n E

ffic

ien

cy (%

)

Figure 19 - Evaluation of transfection efficiency assessed by flow cytometry 48h post-transfection. Results

from one experiment with triplicates for each plasmid are displayed. Differences between transfection

efficiency of different plasmids were compared by performing the one way analysis of variance test ANOVA

(P < 0.05). Error bars represent standard deviation between triplicates.

54

observed in the plasmids coding for N3 protein associated with the different targeting sequences. When

compared to the other N3 constructs, the mean fluorescence of cells transfected with the N3-LAMP and N3-

E1A-LAMP was significantly lower (P < 0.05). Fluorescence associated with the plasmid harboring the

combination of the two target sequences, E1A and LAMP, was higher than the N3-LAMP plasmid (P < 0.05).

Regarding the E1A-N3 plasmid, the pattern of fluorescence revealed significant differences between the

constructs harboring the LAMP sequence, with higher levels of fluorescence and also significantly different

when compared to the N3 construct (P < 0.05), though only slightly higher. These results are consistent with

the transfection efficiency, or number of GFP+ cells, shown in the previous section. The fluorescence levels

associated with each plasmid can be correlated to the expressed protein levels and confirm the expected and

correct transport to specific intracellular compartments. The LAMP sequence or its combination with the E1A

sequence promotes the sorting of the translated protein to the lysosome, where protein degradation occurs,

which is translated in a decrease of fluorescence levels, corroborated with the presented results. The higher

fluorescence presented with the plasmid E1A-N3-LAMP when compared to the plasmid N3-LAMP suggests that

the addition of the E1A sequence affects the protein sorting to the lysosome, resulting in less degradation.

Furthermore, a protein associated with the E1A sequence is transported to the endoplasmic reticulum, a

destination that does not result in protein degradation. Therefore, no decrease in fluorescence is likely to be

observed, as presented in the flow cytometry data obtained. As mentioned before, the differences of mean

fluorescence between cells transfected with N3 and E1A-N3 are indeed significant, which might indicate that

targeting of the protein to the ER instead of the cytosol, had a protective effect against degradation.

0

20

40

60

80

100

120

140

N3 E1A-N3 N3-LAMP E1A-N3-LAMP

Me

an F

luo

resc

en

ce (A

U)

Figure 20- Mean fluorescence assessed by flow cytometry analysis, 48h post-transfection. Results from one

experiment with triplicates for each plasmid are displayed. Differences between mean levels of fluorescence

of cells transfected with different plasmids were compared by performing the the one way analysis of

variance test ANOVA (P < 0.05). pVAX-GFP, used as a positive of control on the trasfection experiments had a

mean fluorescence value of 1081 ± 350 (MF ± SD). AU- arbitrary units. Error bars represent standard

deviation between triplicates.

55

IV.3.3. Analysis of plasmid copy number and mRNA content

In order to quantify plasmid uptake and stability and GFP mRNA transcription in transfected CHO cells,

quantitative real-time PCR and quantitative reverse transcription RT-PCR methods were performed. An RT-PCR

methodology, based on the work of Carapuça et al. 2007 (Carapuça et al. 2007), was established in order to

quantify the number of plasmid copies inside the cells. This method has a high sensitivity, detecting quantities

as low as 100 copies/cell in CHO cells. In this protocol, whole cells are used, so the values obtained correspond

to plasmid copies inside the nucleus and the cytosol (Ribeiro et al. 2012). It is important to mention that these

experiments were always performed 48h post-transfection. Half-life of naked plasmid in the cytosol has been

determined in a time-window between 50 min and 5h (Ribeiro et al. 2012). On the other hand, this value

improves when cationic lipids are used, increasing the half-life of plasmid DNA to approximately 20h (Carapuça

et al. 2007). The following quantification assessments were performed 48h after transfection, so we can

assume that the results correspond to plasmid molecules in the nucleus, which can explain the low number of

plasmid copies detected by quantitative RT-PCR analysis.

The determination of plasmid copy number was achieved by an absolute quantification approach, in

which calibration curves of serial dilutions of pDNA standards (5 pg; 50 pg; 500 pg; 5,000 pg; 50,000 pg) were

established in a suspension of non-transfected CHO cells, showing a linear response (Appendix III). The addition

of non-transfected cells to the calibration curves is essential in order to discount the effect of cell components

in the amplification. Each sample analyzed in the RT-PCR assays, consisting of the same number of cells,

resulted in a Ct value (cycle threshold), which corresponds to the number of amplification cycles necessary for

the fluorescent signal to surpass the background level (threshold). Therefore, the Cts and the quantity of the

target gene are inversely proportional (Ribeiro et al. 2012). Absolute quantification determines the exact copy

concentration of the target gene (GFP) by relating the Ct value to the obtained standard curve (Lee et al. 2006).

Plasmid DNA copies per cell were determined taking into account the size and molecular weight of each

plasmid (660 daltons in 1 bp) and the number of cells per reaction in each sample (12,500 cells).

Data analysis shows that all plasmids (figure 21), including pVAX-GFP and with the exception of N3

plasmid, showed similar numbers of plasmid copies (P > 0.05), suggesting that size was not a factor

contributing to plasmid degradation by cytosolic nucleases. The values obtained were significantly lower than

plasmid copy numbers obtained in other studies for pVAX-GFP (Carvalho et al. 2010) (Azzoni et al. 2007) (La

Vega et al. 2013) and also other plasmids (Carapuça et al. 2007), in CHO cells. Such studies documented tens of

thousands of plasmid copies internalized by the cells. Once inside the cells, pDNA tends to be aggregated in

cellular compartments, never reaching the nucleus, and thus explaining the often low amount of cells

expressing the GFP protein.

56

Carvalho et al. (2010) studied the effect of addition of genes (ISG) to the pVAX-GFP plasmid backbone

and also of targeting sequences in plasmid uptake and stability. Their results suggest that the addition of the

ISG gene and the targeting sequences did not introduce sensitivity regions in the plasmid construct to nuclease

attack and therefore did not affect the plasmid copy number. Despite this, it has been reported that the

addition of DNA sequences to a plasmid backbone can not only affect its stability, but also promote a higher

nuclease degradation and contribute to a lower number of plasmid copies that reach the nucleus and are

further transcribed (Carvalho et al. 2010). Therefore, the results here obtained were not expected, since the

similar behavior was expected for all plasmids carrying the N3 sequence. When the lipoplexes, cationic lipids

carrying the plasmid, are internalized by the cell, they must reach the perinuclear region in order to release the

pDNA into the nucleus. Once inside the cells, their structure is endocytized in vesicles that eventually lead to

their release before maturation into lysosomes (Madeira et al. 2010). In the cytoplasm, pDNA is susceptible to

attack by cytosolic nucleases, which will reduce the number of intact pDNA copies that eventually reach the

nucleus. Due to similar experimental conditions, such as cell line, lipofection and pDNA size (between 5097 bp

and 5252 bp), the half-life of N3 plasmids was expected to be similar. In all the experiments performed, the N3

plasmid showed, consistently, a significantly lower number of plasmid copies (P < 0.05), while plasmids carrying

this gene and targeting sequences had no significant differences when compared to pVAX-GFP and to each

other. Therefore, it was essential to check plasmid integrity, by agarose gel electrophoresis, to determine if

pDNA degradation was the cause of the N3 plasmid results.

0

500

1,000

1,500

2,000

2,500

3,000

3,500

4,000

pVAX N3 E1A-N3 N3-LAMP E1A-N3-LAMP

Pla

smid

co

py

nu

mb

er

/ ce

ll

Figure 21- Plasmid copy number per cell determined 48h post-transfection, by quantitative Real-Time PCR.

Data displayed represents results from two independent assays, with three replicates for each plasmid

construct. In order to compare pDNA copies in cells transfected with the different plasmids, one way ANOVA

was performed, with a level of confidence of P < 0.05. Error bars represent standard deviation between

replicates.

57

Figure 22 represents the configurations of each pDNA vector used in the analysis. The amount of pDNA

analyzed was the same used in the transfection experiments (1 µg). The quality assessment of sc pDNA

integrity explains the decreased plasmid copies in the case of the N3 plasmid. It is visible that there is a

significant degradation of the sc isoform. This conformation is essential to yield high rate of plasmid entry in

the cell, and also for the promotion of the necessary strand separation during replication and transcription

(Sousa et al. 2008). Therefore, it is safe to assume that the low quantities of supercoiled pDNA, which occurred

overtime during long term storage, are the cause of the low number of plasmid copies per cell regarding

transfection with the N3 plasmid.

Copy numbers obtained by RT-PCR do not always result into proportional transcription rates and

protein expression, as it will be further addressed. Since plasmid uptake and transfection efficiency does not

always translate in gene expression, it was essential to perform analysis of mRNA content in order to

understand gene expression associated with each plasmid. The GFP mRNA content of CHO cells transfected

with each plasmid construct was determined by quantitative reverse transcription RT-PCR. 1 µg of Total RNA,

extracted from the cells, for each plasmid was used in the synthesis of cDNA. Quality of the extracted RNA, an

essential requirement to obtain meaningful gene expression data, was determined by its A260/A280 ratio,

which was around 2.1 for all samples, indicating a good quality of starting RNA (Fleige & Pfaffl 2006). This

parameter is very important, since after extraction RNA is unstable and very sensitive to degradation by

RNAses (Fleige et al. 2006). 2 µL of each cDNA sample was used in the quantitative RT-PCR. As for plasmid copy

number determination, calibration curves of serial dilutions of pDNA (5 pg to 50 000 pg) were constructed

(Appendix IV). The mRNA copy number per ng of total RNA was determined by applying the resulting Cts to the

Open circular pDNA

Supercoiled pDNA

Circular single-stranded pDNA

Figure 22- Plasmid DNA quality assessment by 1% agarose gel electrophoresis: pVAX-GFP

(lane 1), pVAX-N3-GFP (lane 2), pVAX-E1A-N3-GFP (lane 3) and pVAX-E1A-N3-GFP-LAMP

(lane 4). M- DNA Molecular weight Marker Hypperladder I (Bioline).

M 1 2 3 4

58

calibration curves, taking into account the total RNA mass present in each 2 µL sample applied in the real-time

PCR reaction.

Table 8- Determination of mRNA content per ng of total RNA, 48h post-transfection of CHO cells. Results presented are the

mean of three replicates per plasmid in one quantitative reverse transcription RT-PCR assay. SD- Standard Deviation

between triplicates.

The results of the assessment of gene expression (table 8) are coherent with the values obtained in

the flow cytometry analysis. A higher expression (P < 0.05), translated in significantly more GFP transcripts, was

obtained for pVAX-GFP, in accordance to previous works (Carvalho et al. 2010), in spite of similar plasmid copy

content obtained with the E1A-N3, N3-LAMP and N3-E1A-LAMP (Figure 21). This plasmid codes for a much

smaller protein, which might result in higher transcription rates and transcript stability (Carvalho et al. 2010).

On the other hand, the N3 construct had significantly less transcripts (P < 0.05) than the N3 constructs

harboring targeting sequences, highlighting the less quantity of plasmid used in the transfection experiment,

already evident with plasmid copy number results and confirmed by the agarose gel electrophoresis analysis.

Nonetheless, it is important to establish a relation between plasmid copy number (PCN) and mRNA copy

number, in order to understand the level of transcription achieved with each plasmid. In fact, despite

presenting low PCN and mRNA transcripts, the N3 plasmid demonstrated to have a high mRNA/PCN ratio (8.0)

which is related to high levels of transcription. The control plasmid, pVAX-GFP, gave rise to a high content of

mRNA transcripts, which reveals a high level of transcription, since its transcript content is significantly higher

(P < 0.05) than the other plasmids. pVAX-GFP was indeed expressed at high levels, with a mRNA/PCN ratio of

74.3. The N3 constructs harboring the targeting sequences E1A and LAMP had lower transcription levels, 3.8

and 6.0 respectively. These results might have suggested that indeed the addition of targeting sequences to the

N3-fusion protein affects the transcription process in the nucleus. Contrary to this tendency, the N3-E1A-LAMP

construct had a higher level of gene transcription in relation to the number of plasmids present in the cell,

evident by the ratio between mRNA copy number and plasmid copy number (11.8). In other works, plasmids

harboring signal sequences or no signals had similar transcription efficiencies, hinting that indeed the addition

of such sequences and the size of the pDNA molecules has no effect on the level of gene transcription. It is

GFP mRNA Transcription

Plasmid mRNA GFP copies/ ng total RNA SD mRNA GFP copies/ PCN

pVAX-GFP 178,299 3,694 74.3

pVAX-N3-GFP 2,292 161 8.0

pVAX-E1A-N3-GFP 8,806 140 3.8

pVAX-N3-GFP-LAMP 12,275 401 6.0

pVAX-E1A-N3-GFP-LAMP 23,983 2,329 11.8

59

important to mention that the results obtained for GFP mRNA content are based on one experiment, which

obviously affects confidence in the results.

IV.3.4. Effect of gene and target sequence addition to plasmid stability,

transcription and GFP expression

Gene expression comparison can be achieved by multiplying the values of mean fluorescence intensity

by the percentage of GFP+ cells (Ribeiro et al. 2012). GFP expression confirms the correct sorting of the

translated proteins to different cellular compartments (table 9). The E1A sequence, directs the newly

synthesized N3-GFP fusion protein to the ER. This pathway does not result in protein degradation. This type of

directing of the protein has the potential of increasing the antigen loading into the MHC I pathway. On the

contrary, the targeting signal LAMP, that directs the protein to the lysosome, contributes to the degradation of

the protein in this vesicle, which results in the presentation of endogenous antigen through the MHC II

pathway, a pathway that mostly processes exogenous antigens. The addition of these targeting signals can

contribute to the modulation of the immune response, resulting in the generation of potentiated humoral and

cellular responses. The level of biological activity, as measured by fluorescence levels of GFP, confirms that

indeed correct sorting of proteins was achieved, which translates in decreased fluorescence of cells transfected

with plasmids harboring the LAMP sequence and higher levels of fluorescence in the plasmid harboring the E1A

sequence. Regarding plasmid copy number/cell, a parameter that helps understanding plasmid stability and

sensitivity to cytosolic nucleases, pVAX-GFP and the other plasmids had similar results, which suggests that

increase in size and the addition of sequences to the plasmid backbone did not result in susceptibility to

nuclease attack or a decrease in plasmid stability in the cell. The N3 plasmid was an exception to this trend due

to degradation of the supercoiled isoform prior to transfection, resulting in lower plasmid content. As

expected, transcription levels were much higher for the control plasmid pVAX-GFP. A smaller and more stable

transcript resulted in a greater mRNA content. Plasmids encoding the N3-GFP fusion protein had significant

differences in the larger and more unstable transcript.

Table 9 – Comparison of parameters obtained by in vitro experiments for the five studied plasmids: Transfection efficiency and mean

fluorescence, obtained by flow cytometry analysis; Plasmid copy number and mRNA content, assessed by RT-PCR.

In vitro results

Plasmid Transfection Efficiency (%)

Mean Fluorescence

(AU)

GFP Expression (%T x AU)

Plasmid Copy Number/ cell

mRNA/ng total RNA

pVAX-GFP 24.2 1,082 26,130 2,400 178,299

pVAX-N3-GFP 6.1 89 543 288 2,292

pVAX-E1A-N3-GFP 7.3 119 865 2,332 8,806

pVAX-N3-GFP-LAMP 2.0 10 21 2,442 12,275

pVAX-E1A-GFP-LAMP 3.2 41 130 2,617 23,983

60

One of the objectives of this work was to correct the CMV promoter sequence on plasmids harboring

the E1A targeting signal and evaluate such changes in the context of plasmid stability, gene transcription and

protein translation and intracellular sorting. Comparison of results obtained during this work with a previous

work (Freitas 2012) establishes that indeed there was an improvement in the level of transcription and in

protein expression for the E1A-N3 and E1A-N3-LAMP vectors. The levels of GFP expression (%T x AU) presented

a progress of 552 to 865.33 and of 102 to 130.4, for E1A-N3 and E1A-N3-LAMP respectively. Most importantly,

in this work there was no degradation of protein associated with the E1A-N3 plasmid when compared to the

N3 construct, an observation that was not achieved previously. Transcription levels also presented enhanced

results, since mRNA/PCN ratios were higher during this work, indicating that for the same quantity of plasmid

molecules more transcripts were generated, a fact that can be directly attributed to the correction of the CMV

promoter which resulted in superior transcription efficiencies.

IV.4. In vivo DNA vaccination

In order to understand the effect of the designed DNA vector vaccines in modulating the immune

system, groups of chickens were immunized by intramuscular injection with the pDNA/lipofectamine

formulations. Liposome-based transfection has shown promise in enhancing the DNA delivery efficacy and

immune response caused by DNA vaccines in vivo leading to an increase in the plasmid copy number at the

injection site in immunized animals (Sun et al. 2013) and increased antibody response (Henriques et al. 2009)

(Kutzler & Weiner 2008). A heterologous prime-boost strategy was implemented, consisting of a prime

immunization followed by two boost vaccinations with the pDNA vaccines, with intervals of two weeks (days 0,

14 and 28). These strategies have shown potential in enhancing the immune response. After the three

vaccinations, a recombinant protein boost was applied at day 52. Contact with the relevant antigen following

DNA immunization with the same epitope induces the production and proliferation of pre-existing memory T-

cells specific for the antigen, focusing the immune response (Carvalho et al. 2009) (Henriques et al. 2007). This

DNA-prime-protein-boost strategy has the specific focus on giving rise to important protective antibody

responses and establishing a T-cell response to a dominant epitope (Woodland 2004) (Fioretti et al. 2010).

A total of nine blood samples were collected per chicken, at days 0, 7, 14, 28, 42, 52, 66, 78 and 90. In

order to characterize the humoral response triggered by the DNA immunizations, pools of serum from each

chicken group were analyzed by several ELISA assays, in plates coated with the N3 antigen, for detection and

quantification of antibodies. The negative groups were comprised of one chicken not immunized and two

chickens inoculated with the empty vector pVAX-GFP. Normalization of results in each assay was achieved by

dividing the absorvance values obtained by the average of the values obtained at point 0 in each chicken group.

Normalized values correspond to antibody production. Absorvance values were lower than expected, due to

variations and optimization problems intrinsic to the ELISA assay. Nonetheless, results indicate that

61

pDNA/liposome formulation was uptaken by the cells, the N3 antigen was correctly expressed and targeted to

intracellular compartments and several conclusions can be drawn regarding improvement of the DNA vaccines.

It is important to observe and draw conclusions of the immune response resulting from the different

vaccines in a timeline perspective (figure 23). Antibody production titre has a clear rise following the first DNA-

prime immunization (measured in day 7), but this event is not repeated after the two subsequent

immunizations. The pattern of antibody levels is irregular between plasmids, with a downward tendency until

day 66. From day 0 till day 52, three immunizations were performed. In this timeline, only the N3 pDNA vaccine

displayed a significantly higher antibody titter (P < 0.05), when compared with the negative control chicken

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

0 10 20 30 40 50 60 70 80 90 100

Ab

sorv

ance

OD

45

0 n

m

Time (days)

CN

pVAX

N3

E1A-N3

N3-LAMP

E1A-N3-LAMP

Figure 23 - Antibody production, measured by absorbance at 450 nm, against N3 antigen in chickens immunized

intramuscularly with N3, E1A-N3, N3-LAMP and E1A-N3-LAMP constructs (groups of 3 chickens). A non-immunized chicken

(CN) and two chickens immunized with pVAX-GFP vector (pVAX) represent the negative controls. Immunizations with 100

µg of the DNA vaccines were carried out at days 0, 14 and 28 ( ). Protein boost with 100 µL of purified N3 protein was

administered at day 52 ( ). Data is representative of five independent ELISA assays. Vertical bars represent standard

deviation between measurements of each data point. Antibody titers of each DNA vaccine were compared with the

negative control and between each other. Significant differences were analyzed by the unpaired two-tailed Student’s t-test.

P < 0.05 was considered statistically significant.

62

antibody levels. The other vaccines, including the other negative control vector pVAX-GFP, showed a

statistically similar (P > 0.05) antibody response. The protein boost at day 52 did indeed change the antibody

levels produced by the immune system, confirming that the DNA prime-protein boost is an important and

effective vaccination strategy of focusing the humoral response, which also shows promise in eliciting cellular

responses (H. Gao et al. 2013). After administration of the antigen at day 52, when compared to the negative

control, the overall antibody levels related to plasmids N3, E1A-LAMP and LAMP were significantly higher (P <

0.05), as predicted. Unexpectedly, pVAX-GFP also had a significantly higher (P < 0.05) when compared to the

not-immunized chicken, probably due to a sudden and unanticipated rise in antibody response in day 78. The

E1A-N3 construct was unresponsive throughout the in vivo study, showing no significant differences (P > 0.05)

in the humoral response when compared to the negative control.

In order to understand the effect of the addition of targeting sequences to the DNA vectors, it is

important to compare the overall antibody production in chickens immunized with the N3 plasmid with those

immunized with the other constructs harboring the antigen sequence and different targeting sequences. The

antibodies levels during the entire assay in chickens inoculated with the E1A-N3 plasmid are significantly lower

(P < 0.05) than the elicited by the N3 plasmid alone. On the other hand, plasmids E1A-LAMP and LAMP didn´t

elicit antibody production with significant differences (P > 0.05). Day 66, two weeks after the challenge of

chickens with the N3 protein, is an important point in the study, since the variations between different vaccines

are more clear and accentuated and an understanding of immunological memory can be correlated with the

results. Of the intracellular targeting sequences tested, it is possible to observe that the N3-LAMP construction

displayed the greatest humoral response, at day 66 after inoculation with the N3 antigen and until the end of

the assay. Furthermore, the antibody levels at day 66 are significantly higher than the ones displayed by the

group of chickens immunized with the N3 pDNA vaccine alone (P < 0.05). The level of humoral response elicited

by N3 and the E1A-N3-LAMP was similar after protein boost (P > 0.05). The antibody response in chickens

immunized with N3-LAMP tripled after administration with the N3 protein, maintaining a high value until the

end of this experiment, which might suggest a greater stimulation of memory cells. These results, especially

when compared with the results achieved with the negative controls (non-immunized and pVAX-GFP), confirm

that the N3, N3-LAMP and E1A-N3-LAMP vectors did indeed potentiate the humoral immune response, eliciting

the development and production of anti-N3 antibodies and stimulation of immunological memory upon

secondary encounter with the antigen.

IV.4.1. Effect of Intracellular targeting sequences on the immune response

Induction of immunity is influenced by site and procedure used for injection, since DNA delivery

methods affect cell types that are transfected. This distinction is clear in intramuscular delivery versus gene gun

(intradermal) delivery for example. While gene gun tends to directly transfect APCs in the skin (Langerhans

cells) with the DNA construct that behave has the source of antigen presentation, intramuscular delivery is

associated with cross-presentation as the major priming route. Delivery of the DNA vaccine through

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intramuscular injection, the delivery method used throughout the in vivo experiments, predominantly leads to

the transfection of myocytes (Fioretti et al. 2010) (Donnelly et al. 2000) and, to a lesser extent, transfection of

resident APCs (Kutzler & Weiner 2008). Myocytes lack the MHC II pathway, which is typically present on APCs,

which are rare on muscle tissue. Professional APCs, such as dendritic cells, are responsible for the priming of

the immune system, presenting in the lymph nodes vaccine-derived peptides in the MHC I molecules (either by

direct transfection or cross-presentation) or in MHC II molecules (by capturing exogenous antigen secreted

from transfect cells). Once in the draining lymph nodes, DCs activate and expand CD8+ and CD4

+ T lymphocytes,

stimulating them to develop into CTLs and Th cells, respectively. The bias towards a MHC I pathway by DNA

vaccination, through the processing of endogenous antigen, is one of the characteristics of this type of

immunization. Although DNA vaccines can produce antibody responses and CD4+ T cell helper responses, they

are indeed more prone to stimulate CD8+ T cells.

Most infectious diseases require both “arms” of the immune system, requiring protection from a

robust CD4+ and CD8

+T cell and antibody response. Modulating the immune system through the addition of

targeting signals in the plasmid DNA backbone is an effective way to manipulate antigen presentation and,

therefore, engineer the type of response desired, cellular or humoral or both.

In this in vivo study, the LAMP and E1A targeting sequences were tested for their effect on the

antibody response to the N3 antigen of AIV. The LAMP sequence targets the endogenously produced antigen

for degradation by the endosomal/lysosomal pathway, presentation at the surface in MHC II molecules and

leads to the enhancement of CD4+ T cells and antibody responses. The antibody response, monitored by ELISA,

elicited by the N3-LAMP construct was stronger when compared to the other plasmids. This confirms the

successful improvement of CD4+

T cell response, through the sorting of the antigen that was produced after

DNA vaccination to the MHC II pathway of APCs, which in turn activates antigen-specific CD4+ T cells more

effectively than other constructs. Moreover, the secondary antibody response following exposure to the

antigen indicates that the plasmid had an effect on immunological memory. Once CD4+ T helper cells become

activated, they can differentiate into Th2 or Th1 profiles through cytokine stimulation. These subsets of cells

have an effect on antigen-specific CD8+ T cell response and also activate B cells, through cell-to-cell interaction

and secretion of specific cytokines (for example IL-2 and IL-4) (Luckheeram et al. 2012) (Dobaño et al. 2007).

Furthermore, once T cells are activated in the draining lymph nodes they can migrate and be further stimulated

and expanded at the site of immunization by transfected muscle cells (Kutzler & Weiner 2008). As other studies

have demonstrated, the addition of LAMP sequence has not only an effect on B cell induction antibody

response; it is also determinant in the improvement of the antigen-specific CD8+ T cell response (Kim et al.

2003) (Marques et al. 2003) (Chikhlikar et al. 2004), which requires the help provided by CD4+ T cells (Th1)

(Dobaño et al. 2007). It is possible to conclude that the inclusion of LAMP did indeed improve the humoral

response of the DNA vaccine carrying the N3 antigen.

The E1A sequence targets the antigen to the endoplasmic reticulum, where the epitopes are

complexed with the newly synthesized MHC I molecules, leading to an even more accentuated bias towards

the MHC I pathway and, consequently, to a hypothesized predominant cellular response. The E1A-N3-LAMP

plasmid, which leads the antigen through the ER to a final destination in the lysosome, presented a similar

64

humoral response to the N3 construct, suggesting that the addition of the E1A sequence had a deleterious

effect on the sorting of antigen to the lysosome/endosome by LAMP. The antibody response in chickens

immunized with the E1A-N3 plasmid confirms this hypothesis. ER-targeting resulted in the lowest antibody

response elicited by the N3-harboring plasmids after DNA immunization and protein boost, following a

response pattern similar to the negative control. This suggests that targeting to the ER was achieved and

antigen sorting was led to a predominantly MHC I- restricted pathway. This targeting strategy is a prerequisite

for the induction of CTL responses to intracellular pathogens (Xu et al. 2005) (Spackman 2008), not studied in

this work.

The secondary antibody response elicited by the DNA vaccine N3 construct, without targeting

sequences, was also significant. The protein N3 is characterized by a transmembrane portion, between amino

acids 7 and 29 (Appendix V). APCs are responsible for presentation in MHC II molecules, which in turn

endogenously produced proteins, as is the case, usually cannot gain access to. The pathway that leads to

presentation of endogenously synthesized antigens in MHC II molecules is not very well defined (Dissanayake

et al. 2005). Even so, such response was probably due to intracellular digestion of the newly synthesized N3

protein in the cytosol that accessed the endosome by autophagy or, in the transport to the membrane, this

transmembranar protein entered the endocytic pathway (Paludan et al. 2005) (Dissanayake et al. 2005)

(Villadangos & Schnorrer 2007), and therefore gained access to the MHC II pathway. Likewise, APCs can present

peptides at the MHC II molecules, after capturing protein antigens shed from transfected cells and processing it

through the endocytic pathway (Kutzler & Weiner 2008) (Ingolotti et al. 2010). Antibody response could be

improved by administration of the vaccine through a different route and location. As discussed before,

immunization with gene gun elicits higher antibody levels due to the direct transfection of APCs in the skin

(Ullas 2012) (Laddy & Weiner 2007).

Acquired immunity is vital against viral infection and it is comprised of two essential components,

cytotoxic-T-lymphocyte and antibody responses (Sawai et al. 2008). Regarding influenza virus infection, it is still

unclear which type of response is of more critical importance (Sawai et al. 2008). While antibodies response is

essential for neutralization and prevention of virus infection, CTLs recognize virus-infected cells and activate

anti-viral pathways leading to cytolysis and clearance of virus-infected cells (Sawai et al. 2008) (Xu et al. 2011).

A Th1 –biased response is an important and desired feature of vaccines aiming at controlling intracellular

pathogens (Yang et al. 2008) but, on the other hand, exclusive priming of CD4+ T cell responses can result in the

suppression of CD8+ T cell responses (Woodland 2004). Nonetheless, CD8

+ T cell responses require CD4

+ T-

helper cells (Th1) for secondary expansion and development of memory (Woodland 2004) (Marques et al.

2003) (Luckheeram et al. 2012). Furthermore, cellular responses target internal proteins, which are less

variable and therefore generate cross-subtype CTL-mediated protection (Xu et al. 2011). An effective, and

protective against a broad range of avian influenza virus subtypes, DNA vaccine would probably include a

combination of both types of immune response. Investigation of cellular response elicited by the DNA

prototype vaccines, namely by flow cytometry analysis of T-cell sub-populations (Lim et al. 2012) (Reemers et

al. 2012) (Xu et al. 2011), cytokine gene expression analysis by real-time quantitative RT-PCR (Chikhlikar et al.

2004), ELISA (Naderi et al. 2013)(Chikhlikar et al. 2004) and intracellular staining assays (Dobaño et al. 2007)

65

(Kim et al. 2003), would greatly benefit the conclusions regarding their overall efficacy in modulating the

immune system. The E1A-N3 construction, despite null antibody production, probably elicited a CD8 +

T cell

response. Furthermore, the plasmids associated with the LAMP sequence, by priming CD4+ T lymphocytes,

might also have had a role in the expansion and maintenance of a cellular response, leading to the stimulation

of both arms of the immune system, one of the greatest strengths of DNA vaccines.

66

67

IV. Final remarks and future work

The present work aimed at studying the effect of targeting sequences on DNA vaccine potency and

efficacy. Because the AIV is prone to genetic reassortment and new influenza A virus periodically emerge, DNA

vaccines constitute an attractive alternative. Low immunogenicity and consequent need for large doses of DNA

vaccines can be overcome by targeting the endogenously produced antigen to specific intracellular

compartments and consequently to the major presentation pathways. This strategy has shown potential in

modulating the immune response outcome, with focus on the production of antibodies, although results have

been varied (Rice et al. 2008).

The DNA vaccine prototypes, pVAX-N3-GFP, pVAX-E1A-N3-GFP, pVAX-N3-GFP-LAMP, pVAX-E1A-N3-GFP-

LAMP and control vector pVAX-GFP, were tested in a series of in vitro assays in CHO cells and through in vivo

testing in intramuscularly immunized chickens.

Some conclusions can be inferred from in vitro results. Firstly, plasmids harboring the E1A sequence

were corrected due to incomplete CMV promoters. This correction generated higher transcription efficiency

and protein expression. In vitro assays helped interpreting the effects of addition of additional sequences to

the plasmid vector and the behavior of pDNA vaccines in eukaryotic cells. Furthermore, correct sorting to

cellular compartments was correctly achieved: LAMP promotes sorting to the lysosome, leading to protein

degradation. Levels of GFP expression, correlated with the mean fluorescence values, of both N3-LAMP and

E1A-N3-LAMP confirm this outcome. The conjugation of the E1A and LAMP sequences lead to less protein

degradation, when compared to LAMP alone, which further confirms the antagonist effects of these sequences

in terms of protein fate in the cell. On the other hand, the E1A sequence, targeting the protein to the

endoplasmic reticulum, led to a stable and non-degraded protein resulting from expression of the E1A-N3

plasmids. Overall, transfection with cationic liposomes was successful. The addition of the N3 antigen sequence

and targeting sequences did not result in lower plasmid copy numbers per cell, when compared to the positive

control. In spite of this, mRNA content and protein expression, as expected, was much higher for pVAX-GFP

when compared to the cells transfected with the N3-GFP protein encoding plasmids. This highlights the less

stable transcripts and protein expressed and also the difficulty of lipofectamine complexation with much larger

plasmids, leading to reduced transfection efficiency. Plasmid supercoiled conformation proved to be

determinant in the results, since damage to the N3 plasmid affected transfection, and therefore, the ability to

draw more definitive conclusions regarding addition of targeting sequences to the plasmid backbone.

A correlation can be drawn between the observed effect of plasmids in vitro and in vivo. The N3-LAMP

construction led to a significant higher production of antibodies against the N3 antigen, following immunization

with the purified protein. The enhanced degradation observed in vitro increased the amount of peptides

available for presentation through the MHC II pathway by the APCs. Such results confirm the targeting of the

antigen to the endocytic route, which in this work was translated in decreasing levels of protein present in

transfected cells in vitro and higher presentation to CD4+ T cells in vivo, leading to a stimulation of antibody

production and hypothetically eliciting immunological memory. This is an important improvement of DNA

vaccine efficiency, since epitopes of endogenous proteins, after expression in the cytoplasm of transfected

68

cells, tend to be presented by MHC I molecules. N3 and E1A-N3-LAMP elicited a similar humoral response in

immunized chickens, leading to the conclusion that, despite higher degradation in in vitro experiments, the

combination of both sequences was not translated in a higher stimulation of antibody production when

compared to the antigen expressed in the cytosol alone. The E1A-N3 pDNA vector had a null effect on humoral

response, probably because transport to the ER was achieved correctly, inducing a bias towards the MHC I

pathway and leading to the absence of peptides available for the endocytic pathway, a hypothesis

corroborated with in vitro observations. Nonetheless, it is clear that the immunization strategy, DNA prime

with protein boost combined with the targeting sequence LAMP elicited a considerable immune response, and

improved the humoral response of the N3 plasmid with no targeting signal.

Future work

As mentioned before, it would be of great importance to evaluate the effect of targeting sequences in

the cellular immune response to DNA vaccines. Not only the E1A-N3 construct might have produced an

enhanced CD8+ T cell response, but also the constructs E1A-N3-LAMP and N3-LAMP could have produced a

synergetic immune response, both humoral and cellular. Furthermore, only challenge of the chickens with the

virus could determine the real effect of the DNA vaccines in the protection and enhancement of immunological

memory to AIV. Regarding in vitro assays, a repetition of some experiments, namely mRNA content, would be

valuable in the generation of more confident conclusions.

Improvement of DNA vaccine performance could also be achieved by combining other antigen sequences,

such as internal proteins or hemagglutinin, in the plasmid backbone, changing route of administration to a

more effective technique, such as gene-gun, or co-administration with genetic adjuvants.

69

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VI. Appendix

Appendix I- plasmid pVAX1/lacZ features

Weight: 6050 bp

- CMV promoter: bases 137-724

- T7 promoter/priming site: bases 664-683

- LacZ ORF: bases 773-3829

- BGH reverse priming site: bases 3874-3891

- BGH polyadenylation signal: bases 3880-4104

- Kanamycin resistance gene: bases 4277-5071

- pUC origin: bases 5371-6044

Figure 24- Schematic diagram of pVAX1/LacZ. Source:

http://www.lifetechnologies.com/order/catalog/product/V26020

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Appendix II- Growth curves of vectors used in molecular cloning pVAX-N3-GFP and pVAX-N3-

GFP-LAMP

0

0.5

1

1.5

2

2.5

3

0 2 4 6 8 10

OD

60

0 n

m

Time (h)

Growth curve pVAX-N3-GFP-LAMP

0

0.5

1

1.5

2

2.5

3

0 2 4 6 8 10

OD

60

0 n

m

Time (h)

Grow curve pVAX-N3-GFP

Figure 25- Growth curves for E.coli DH5α cells harboring pVAX-N3-GFP-LAMP and pVAX-N3-GFP.

Cell culture in Erlenmeyer flasks, under orbital agitation of 250 rpm and 37oC.

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Appendix III- Calibration curves used for pDNA copy number quantification in transfected CHO

cells, by RT-PCR.

y = -3.216x + 19.422 R² = 0.9905

0

2

4

6

8

10

12

14

16

18

20

0 1 2 3 4 5

Thre

sho

ld c

ycle

(C

t)

Log [pg/µL]

pVAX-GFP

y = -3.0775x + 18.185 R² = 0.997

0

2

4

6

8

10

12

14

16

18

0 1 2 3 4 5 Th

resh

old

cyc

le

Log [pg/µL]

pVAX-N3-GFP

y = -3.418x + 21.116 R² = 0.9945

0

5

10

15

20

25

0 1 2 3 4 5

Thre

sho

ld c

ycle

Log [pg/µL]

pVAX-N3-GFP-LAMP

y = -3.1437x + 20.315 R² = 0.9915

0

5

10

15

20

25

0 1 2 3 4 5

Thre

sho

ld c

ycle

Log [pg/µL]

pVAX-E1A-N3-GFP

y = -3.355x + 20.409 R² = 0.9988

0

5

10

15

20

25

0 1 2 3 4 5

Thre

sho

ld c

ycle

Log [pg/µL]

pVAX-E1A-N3-GFP-LAMP

Figure 26- Standard curves for the absolute quantification of plasmid copy number in transfected CHO cells, obtained using pDNA masses ranging from 5 pg to 50000 pg per reaction, spiked with 12500 non-transfected cells. Regression analysis is presented by r

2 values.

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y = -3.175x + 17.858 R² = 0.9948

0

2

4

6

8

10

12

14

16

18

0 1 2 3 4 5

Thre

sho

ld c

ycle

Log [pg/µL]

pVAX-E1A-N3-GFP-LAMP

Appendix IV- Calibration curves used for mRNA copy number quantification in transfected CHO

cells, by RRT-PCR

y = -3.4065x + 19.141 R² = 0.999

0

2

4

6

8

10

12

14

16

18

20

0 1 2 3 4 5

Thre

sho

ld c

ycle

Log [pg/µL]

pVAX-GFP

y = -3.145x + 18.422 R² = 0.9909

0

2

4

6

8

10

12

14

16

18

20

0 1 2 3 4 5

Thre

sho

ld c

ycle

Log [pg/µL]

pVAX-N3-GFP

y = -3.348x + 19.038 R² = 0.9992

0

2

4

6

8

10

12

14

16

18

20

0 1 2 3 4 5

Thre

sho

ld c

cyle

Log [pg/µL]

pVAX-E1A-N3-GFP

y = -3.447x + 18.45 R² = 0.9994

0

5

10

15

20

0 1 2 3 4 5

Thre

sho

ld c

ycle

Log [pg/µL]

pVAX-N3-GFP-LAMP

Figure 27- Standard curves for the absolute quantification of mRNA copy number in transfected CHO cells, obtained using pDNA masses ranging from 5 pg to 50000 pg per reaction. Regression analysis is presented by r

2

values.

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Appendix V- N3 antigen gene and protein sequences and characteristics

N3 gene sequence (obtained with ApE ©software):

atgaatcctaatcagaagatcataacaattggtgtagtgaatactactctatcaacaatagcccttcttattggaattggaaatctggttttcaacactgttatacat

gagaaaataggggaccaccaaactgtggtatatccaacagtaacagccccggtggtaccaaactgcagtgacaccataataacatacaataacactgtggtaa

acaacataacaacaacaataataactaaagcggaaacgcacttcaagccctcattgccactgtgccccttccgaggtttcttcccctttcacaaggacaatgcaa

tacgattgggtgaaaccaaagacgtaatagtcacaagggagccttatgtcagttgtgacaatgatgattgctggtcctttgctcttgcccaaggggctctgctggg

gaccaaacacagcaatggaaccatcaaagacaggacaccatatagatcgctaatccggttcccaatagggactgccccagtactgggcaattacaaggagata

tgtgttgcttggtcaagtagcagttgcttcgatggaaaggaatggatgcatgtttgcatgactgggaacgacaatgatgcaagtggccaaataatgtatgcaggg

aaaatgacagactccattaaatcatggagaaaggatatactaagaactcaagagtctgaatgtcaatgcattgatgggacctgtgttgtcgctgttacagatggt

cctgcagctaatagtgcagaccaccgaatttattggatacgggaagggaagataataaagtatgagaacattcccaagacaaagatacaacatttggaggagt

gctcttgttatgtggacatcgatgtgtactgcatatgtagggacaattggaaaggttccaacaggccttggatgaggatcaacaatgagaccatattagaaacag

ggtatgtatgtagtaaattccattcagatacccccaggccagccgatccttcaacagtatcgtgtgattctccgagtaacgtcaatggagggcctggagtcaaag

gttttggcttcaaaacgggtaatgatgtatggttgggaaggactgtatcaactagtggaagatcaggctttgaaatcatcaaagtcacagaggggtggattaact

cccccaatcatgctaaatcagttacacaaacattagtgtcaaacaatgattggtcaggttattcagggagtttcattgttgagaacaatggctgttttcagccctgc

ttctatattgagcttatacgggggaagcccaataagaatgatgacgtttcttggacaagcaatagtatagtcactttctgtgggctagacaatgaacctggatcgg

gaaattggcctgatggttccaacattgggttcatgcccaagtaa

N3 protein sequence (obtained with translation tool web.expasy.org/translate/):

MNPNQKIITIGVVNTTLSTIALLIGIGNLVFNTVIHEKIGDHQTVVYPTVTAPVVPNCSDTIITYNNTVVNNITTTIITKAETHFKPSL

PLCPFRGFFPFHKDNAIRLGETKDVIVTREPYVSCDNDDCWSFALAQGALLGTKHSNGTIKDRTPYRSLIRFPIGTAPVLGNYKEIC

VAWSSSSCFDGKEWMHVCMTGNDNDASGQIMYAGKMTDSIKSWRKDILRTQESECQCIDGTCVVAVTDGPAANSADHRIY

WIREGKIIKYENIPKTKIQHLEECSCYVDIDVYCICRDNWKGSNRPWMRINNETILETGYVCSKFHSDTPRPADPSTVSCDSPSNV

NGGPGVKGFGFKTGNDVWLGRTVSTSGRSGFEIIKVTEGWINSPNHAKSVTQTLVSNNDWSGYSGSFIVENNGCFQPCFYIELI

RGKPNKNDDVSWTSNSIVTFCGLDNEPGSGNWPDGSNIGFMPK

Figure 28- Analysis of functional domains of the N3 protein structure. Intracellular, extracellular and transmembranar domains are discriminated. Source: http://www.cbs.dtu.dk/services/TMHMM/

82

Appendix VI- Molecular Weight Markers and Agarose gel electrophoresis analysis

Agarose Gel electrophoresis

In the course of this work, agarose gel electrophoresis was the most used analytical method,

performed in an electrophoresis system composed of a GE Healthcare/Amersham Pharmacia Electrophoresis

EPS 3501 XL Power Supply and two submarine electrophoresis units: an Hoefer HE 99 for the 20 cm gels and an

Hoefer HE 33 for the 10 cm gels. The samples were mixed with 6X DNA loading buffer (16g of sucrose

(Panreac), 100 ng of bromophenol blue (Sigma)) and analyzed in 1 to 4 % agarose horizontal gels (LE agarose

SeaKem) in 1X TAE buffer (40 mM Tris base, 20 mM acetic acid and 1 mM EDTA, pH 8.0). 20 cm gels were run at

120 V for 1.5h and 10 cm gels were run at 90 V for 1h. 5 µL of a DNA ladder (NZYDNA ladder III from NYZtech or

HypperLaderTM I from Bioline (Appendix VI)) was also added to the run as a DNA molecular weight marker. After

the completion of the electrophoretic run, gels were stained with ethidium bromide (0.5mg/mL) for 20 minutes

and results were visualized in the transiluminator EagleEye II Video Imaging System (Stratagene).

Figure 30- DNA molecular weight marker NZYDNA Ladder III (NZYTech®). Source and further product details: https://www.nzytech.com/site/vmchk/DNA-Markers/NZYDNA-Ladder-III

Figure 29- DNA molecular weight marker HypperLadder TM

50 bp (Bioline®). Source and further product details: http://www.bioline.com/h_prod_detail_ld.asp?itemid=152

83