Development of recombinant modified vaccinia Ankara ... · MVA-BN-WEEV could provide 100% protection to the mice against WEEV Fleming strain and 80% to WEEV 71V-1658 strain. Meanwhile,

Development of recombinant modified vaccinia Ankara-Bavarian Nordic (MVA-BN)-based vaccines against encephalitic alphaviruses

Wei-Gang Hu Les P. Nagata DRDC – Suffield Research Centre Robin Steigerwald Marcus Kalla David Noll Bavarian Nordic GmbH, Fraunhoferstrasse 13 D-82152 Martinsried, Germany Prepared for: Canadian Forces Health Services, Operational Medicine

Defence Research and Development Canada Scientific Report DRDC-RDDC-2017-R039 March 2017

IMPORTANT INFORMATIVE STATEMENTS This research has been done in collaboration between Defence Research and Development Canada and Bavarian Nordic (an international biotechnology company developing and manufacturing novel vaccines for infectious diseases). In conducting the research described in this report, the investigators adhered to the 'Guide to the Care and Use of Experimental Animals, Vol. I, 2nd Ed.' published by the Canadian Council on Animal Care.

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© Her Majesty the Queen in Right of Canada, as represented by the Minister of National Defence, 2017

© Sa Majesté la Reine (en droit du Canada), telle que représentée par le ministre de la Défense nationale, 2017

DRDC-RDDC-2017-R039 i

Abstract

The three encephalitic alphaviruses, Venezuelan, western and eastern equine encephalitis viruses (VEEV, WEEV, and EEEV) are endemic throughout North, Central and South America. Horses and humans are normally exposed after being bitten by infectious mosquitoes and the infection can develop into encephalitis with high rates of morbidity and mortality. These viruses are potential biological threat agents, being highly infectious through the aerosol route of exposure, easily produced in large amounts, and relatively stable in the environment. Currently, there is no licensed vaccine to the three encephalitic alphaviruses, and efforts to move vaccine candidates forward into clinical trials have not been successful. In this report, the development of recombinant modified vaccinia Ankara-Bavarian Nordic (MVA-BN)-based vaccines for the three encephalitic alphaviruses is described. The recombinant vaccines MVA-BN-VEEV, MVA-BN-WEEV, and MVA-BN-EEEV were constructed and produced. The protective efficacy of these vaccines was evaluated in vivo. Female Balb/c mice were immunized subcutaneously with 108 tissue culture infective dose 50 (TCID50) of single MVA-BN encephalitic alphavirus vaccine or a triple mixture of three vaccines at a four-week interval. The mice were then instilled intranasally with 1×103 to 1×104 plaque forming units of VEEV, WEEV, or EEEV 14 days after the second immunization. The MVA-BN-VEEV or MVA-BN-EEEV-immunized mice fully survived the corresponding virus challenge without any signs of infection or weight loss while all the control mice died. The MVA-BN-WEEV could provide 100% protection to the mice against WEEV Fleming strain and 80% to WEEV 71V-1658 strain. Meanwhile, the triple mixture of three vaccines could also provide 100% protection to the mice against WEEV and VEEV challenges, but 60% protection against EEEV challenge. These data suggest that MVA-BN-EEEV, MVA-BN-WEEV, and MVA-BN-VEEV are potential vaccine candidates against the encephalitic viruses and the three vaccines can be given in a mixture without significantly reducing efficacy.

Significance to defence and security

The three encephalitic alphaviruses, VEEV, WEEV, and EEEV can infect humans by infectious mosquitoes with high rates of morbidity and mortality; furthermore, these viruses are highly infectious to humans via respiratory exposure, easily produced in large quantities, and relatively stable in the environment. As such, the three alphaviruses are potential agents of biological warfare (BW) interest. In fact, VEEV was actually weaponized by both the United States and by the former Soviet Union as an incapacitating agent. Currently, no commercialized medical countermeasures exist to combat infections with these viruses. The development of vaccines against the three alphaviruses will provide important protection against these viruses for both biodefence and public health.

ii DRDC-RDDC-2017-R039

Résumé

Les trois alphavirus causant l’encéphalite, le virus de l’encéphalite équine du Venezuela, de l’encéphalite équine de l’Ouest et de l’encéphalite équine de l’Est (VEEV, VEEO et VEEE) sont endémiques dans toute l’Amérique du Nord, l’Amérique centrale et l’Amérique du Sud. Les chevaux et les humains y sont normalement exposés par la piqûre d’un moustique contaminé et l’infection peut évoluer vers une encéphalite présentant un taux élevé de morbidité et de mortalité. Ces virus sont des agents potentiels de menace biologique parce qu’ils sont très infectieux en cas d’exposition à des aérosols, qu’ils sont facilement produits en grande quantité et qu’ils sont relativement stables dans l’environnement. À l’heure actuelle, il n’existe aucun vaccin homologué contre les trois alphavirus causant l’encéphalite et les tentatives visant à amener les vaccins éventuels à l’étape des essais cliniques ont échoué. Le présent rapport décrit la mise au point de vaccins à base d’un virus recombinant de la vaccine de souche modifiée Ankara – Bavarian Nordic (MVA-BN) – contre les trois alphavirus causant l’encéphalite. Les vaccins recombinants MVA-BN-VEEV, MVA-BN-VEEO et MVA-BN-VEEE ont été fabriqués et produits. L’efficacité de la protection conférée par ces vaccins a été analysée in vivo. Des souris femelles Balb/c ont été immunisées par voie sous-cutanée au moyen de 108 DICT50 (dose infectant 50 % d’une culture de tissus) d’un seul vaccin MVA-BN contre un alphavirus causant l’encéphalite ou d’un mélange des trois vaccins à quatre semaines d’intervalle. Les souris ont ensuite reçu par voie nasale entre 1 × 103 et 1 × 104 unités formatrices de plages du VEEV, du VEEO ou du VEEE 14 jours après la deuxième immunisation. Les souris immunisées au moyen du MVA-BN-VEEV ou du MVA-BN-VEEE ont toutes survécu à l’attaque du virus correspondant et n’ont présenté aucun signe d’infection ni perte de poids, tandis que toutes les souris témoins sont mortes. Le MVA-BN-VEEO pourrait protéger les souris à 100 % contre la souche Fleming du VEEO et à 80 % contre la souche 71V-1658 du VEEO. Entre-temps, le mélange des trois vaccins pourrait aussi protéger les souris à 100 % contre le VEEO et le VEEV, mais à 60 % contre le VEEE. Ces données portent à croire que les MVA-BN-VEEE, MVA-BN-VEEO et MVA-BN-VEEV sont des vaccins possibles contre les virus causant l’encéphalite, et les trois peuvent être administrés dans un mélange sans entraîner de baisse significative de l’efficacité.

Importance pour la défense et la sécurité

Les trois alphavirus causant l’encéphalite, les VEEV, VEEO et VEEE, peuvent infecter les humains par l’intermédiaire de moustiques contaminés et présenter un taux élevé de morbidité et de mortalité. En outre, ces virus sont très infectieux pour les humains qui y sont exposés par les voies respiratoires. Ils sont facilement produits en grande quantité et sont relativement stables dans l’environnement. Comme tels, les trois alphavirus sont des agents potentiels de guerre biologique présentant un intérêt. Dans les faits, le VEEV at été utilisé comme arme tant par les États-Unis que par l’ancienne Union soviétique à titre d’agent incapacitant. À l’heure actuelle, il n’existe pas dans le commerce de contre-mesure médicale pour combattre les infections causées par ces virus. La mise au point de vaccins contre les trois alphavirus fournira une importante protection contre ces virus tant pour la défense biologique que pour la santé publique.

DRDC-RDDC-2017-R039 iii

Table of contents

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i Significance to defence and security . . . . . . . . . . . . . . . . . . . . . . i Résumé . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ii Importance pour la défense et la sécurité . . . . . . . . . . . . . . . . . . . . ii Table of contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . iii List of figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iv

List of tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . vi 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

2 Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . 4

2.1 Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

2.2 Cells and viruses . . . . . . . . . . . . . . . . . . . . . . . . . 4

2.3 Construction of MVA-BN-based alphavirus vaccines . . . . . . . . . . . 4

2.4 Antigen expression characterization of MVA-BN-based alphavirus vaccines . . . 5

2.5 Efficacy evaluation of MVA-BN-based alphavirus vaccines . . . . . . . . . 6

2.6 Serum anti-alphavirus neutralization titration assay . . . . . . . . . . . . 6

3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

3.1 Construction and characterization of MVA-BN-based alphavirus vaccines . . . 8

3.2 Protective efficacy study . . . . . . . . . . . . . . . . . . . . . . 9

3.2.1 MVA-BN-VEEV vaccine . . . . . . . . . . . . . . . . . . . 9

3.2.2 MVA-BN-WEEV vaccine . . . . . . . . . . . . . . . . . . 10

3.2.3 MVA-BN-EEEV vaccine . . . . . . . . . . . . . . . . . . . 13

3.3 Serum ANT . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

iv DRDC-RDDC-2017-R039

List of figures

Figure 1: Construction of recombinant MVA-BN-based alphavirus vaccines. . . . . . 5

Figure 2: Experiment layout for efficacy evaluation of MVA-BN-based alphavirus vaccines. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Figure 3: Serum anti-alphavirus neutralization titration assay. . . . . . . . . . . . 7

Figure 4: Antigen expression analysis by FACS. . . . . . . . . . . . . . . . . 8

Figure 5: MVA-BN-VEEV immunized mice against 5×103 pfu of VEEV TrD challenge. 9

Figure 6: Two immunization routes of MVA-BN-VEEV immunized mice against 104

pfu of VEEV TrD challenge. . . . . . . . . . . . . . . . . . . . . 10

Figure 7: MVA-BN-WEEV immunized mice against 5 × 103 pfu of WEEV 71V challenge. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Figure 8: MVA-BN-WEEV immunized mice against 103 pfu of WEEV 71V challenge. . 12

Figure 9: MVA-BN-WEEV immunized mice against 5 × 103 pfu of WEEV Flem challenge. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Figure 10: MVA-BN-EEEV immunized mice against 5 × 103 pfu of EEEV PE6 challenge. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

DRDC-RDDC-2017-R039 v

List of tables

Table 1: Anti-alphavirus neutralization titers in the serum of immunized mice. . . . . 15

vi DRDC-RDDC-2017-R039

Acknowledgements

The authors wish to acknowledge the technical assistance of Ms. Nicole Barabe as a member of the Project Team. The technical assistance of the Containment Level 3 and animal care support groups, in particular provided by Mr. Grant Hennes at DRDC SRC to the project is also greatly appreciated.

DRDC-RDDC-2017-R039 1

1 Introduction

The alphaviruses comprise a group of about 28 enveloped viruses with a positive sense, non-segmented single-stranded RNA genome of around 11–12 kb [1, 2]. They form enveloped virions with icosahedral symmetry and are 60–70 nm in diameter. All alphaviruses share basic structural, sequence, and functional similarities, including a genome with two polyprotein gene clusters [2]. The three encephalitic alphaviruses, Venezuelan, western and eastern equine encephalitis viruses (VEEV, WEEV, and EEEV) are highly pathogenic for both equines and humans and have caused periodic epizootics throughout North, Central, and South America [1, 2]. Human infection with VEEV typically results in a systemic incapacitating disease characterized by severe flu-like symptoms, such as fever, headache, lymphopenia, myalgia, and malaise. Neurological involvement is less frequent in humans, but fatal encephalitis (<1% case fatality rate) can result from the virus across the cerebral vascular endothelium or the olfactory epithelium [3]. WEEV and EEEV are neurotrophic and can cause severe neurotrophic infection, with case fatality rates of up to 10% and 50%, respectively.

Although the three viruses are naturally transmitted by mosquitoes, accidental laboratory infections with these viruses [4] and experimental studies in animals [5] have demonstrated that all three alphaviruses are highly infectious by the aerosol route. Furthermore, alphavirus infections via the aerosol route seems to develop much faster than the conventional natural route since the aerosol route likely allows more virus to contact with olfactory neurons, thus facilitating the earlier viral invasion to the brain [6]. In addition, the alphavirus virulence seems to be enhanced. For example, the mortality rate in laboratory accidents involving aerosol exposure to WEEV was up to 40% [7], as compared to 8–15% by natural infection route [8]. Besides, these alphaviruses could also be easily produced in large quantities and unlike many other pathogenic viruses, these viruses are relatively stable (either liquid or dry) in the environment [9]. Additionally, the size of these viruses is relatively small and susceptible to genetic manipulations, which may enhance its infectiousness and virulence for biological weapons purposes [9]. These characteristics have made the three viruses suitable for weaponization. As such, the three alphaviruses are potential agents of BW interest [10]. And in fact, VEEV had previously been weaponized by both the United States and the former Soviet Union in multi-ton quantities as an incapacitating agent. There are no commercialized vaccines against these viruses available for humans yet, although a commercialized, trivalent inactivated vaccine is available for horses against the three viruses [11]. Therefore, it is a high priority to develop vaccines against the three viruses.

Currently, personnel at risk of exposure to VEEV are recommended by the Centers for Disease Control and Prevention (CDC), to be vaccinated with a live attenuated vaccine TC-83 (an Investigational New Drug, IND) followed by booster vaccination with formalin-inactivated TC-83 vaccine (also known as C-84) [11, 12]. Formalin-inactivated EEEV and WEEV vaccines were also developed and can be used as INDs for at-risk personnel. The major drawback of TC-83 is its high reactogenicity rate (about 20% in vaccinees) [12], which is unacceptable for being licensed as a human vaccine. The formalin-inactivated vaccines for VEEV, WEEV, and EEEV have better safety profiles; however, the immunogenicity is low for these vaccines, requiring three doses for immunization and annual boosters to maintain protective immune response. Several approaches have been used to develop safer and more effective vaccines for VEEV, WEEV, and EEEV [11, 12]. A reverse genetics approach to introduce specific mutations

2 DRDC-RDDC-2017-R039

to the viral sequence was used by Davis et al. to develop a live-attenuated VEEV vaccine candidate. This vaccine candidate was designated as V3526 from a full-length cDNA clone of the Trinidad Donkey (TrD) strain by deleting a furin cleavage site from the envelope protein precursor 2 (PE2) and inserting a single amino acid mutation in the envelope protein 1 (E1) [13]. V3526 was protective against aerosol or subcutaneous challenge of various subtypes of VEEV in rodents and nonhuman primates [14–16]. The success of pre-clinical studies made V3526 a leading candidate of VEEV vaccines to move forward to the safety and immunogenicity study in a Phase 1 clinical trial. Unfortunately, V3526 caused headache, fever, malaise and sore throat in a significant number of vaccinees although the vaccine is able to induce strong immune responses. These adverse effects prompted the discontinuation of the clinical trial for V3526 as a live attenuated vaccine for VEEV. Another approach for overcoming the problems of the traditional live attenuated VEEV vaccines is through the construction of chimeric Sindbis virus (SINV) expressing structural proteins of VEEV, WEEV or EEEV. To construct live attenuated chimeric SIN/VEE viruses, the genes encoded the replicative enzymes and the cis-acting RNA elements of SINV were ligated with the genes encoding the structural proteins of VEEV TC-83 strain. Mouse studies showed the chimeric virus highly attenuated and immunogenic [17, 18]. A similar approach was used to make chimeric SIN/EEE and SIN/WEE viruses conferring complete protection against intraperitoneal challenge of a homologous strain of EEEV [19], and WEEV [20], respectively in a mouse model, and against EEEV in nonhuman primates [21].

Alternative approaches utilized the structural proteins to elicit immune protection either as subunit vaccines, or expressed from viral vectors. Dupuy et al. demonstrated that codon optimization and intramuscular electroporation delivery improved immunogenicity and efficacy of a VEEV DNA vaccine, where the structural polyprotein was expressed from a mammalian expression vector [22]. Mice injected with the vaccine by intramuscular electroporation generated a similar high level of VEEV-neutralizing antibody which was observed in mice given the live-attenuated VEEV vaccine TC-83 [23]. Viruses, such as vaccinia virus and adenovirus, can be modified to deliver genes encoding protective antigens of VEEV, WEEV or EEEV. Several studies demonstrated that an adenovirus-vectored WEEV vaccine encoding E3-E2-6K-E1 structural proteins of the 71V-1658 (71V) strain of WEEV conferred rapid and complete protection [24, 25]. However, pre-existing immunity to the human adenovirus vector was thought to reduce the immune response in human adenovirus-vectored vaccines. Another vaccine was made based on a vaccinia virus vector expressing the structural proteins of VEEV TrD [26]. The vaccine provided protection of mice against peripheral challenge of various subtypes of VEEV. However, only partial protection was achieved against aerosol challenge.

Modified vaccinia virus Ankara-Bavarian Nordic (MVA-BN) is an attenuated vaccinia virus, which is derived from its replication competent ancestor chorioallantois virus Ankara by over 570 passages on chicken embryo cells [27]. It is approved as a smallpox vaccine in Canada and the EU (under the trade names IMVAMUNE® and IMVANEX® respectively). It is also a replication-deficient viral vector, which is a proprietary and patented vaccine platform technology of BN. In the recent years, MVA-BN received a lot of attention as a recombinant vaccine vector. MVA-BN has intrinsic adjuvant capacities to induce both humoral and cellular immune responses [27, 28]. In animal models, MVA-based vaccines have been found to quickly elicit protective immune response against various infectious agents. Clinical data demonstrated the MVA-based vaccines have an excellent safety profile in humans and protection of the general environment. In addition, the impact of pre-existing vector immunity to MVA is limited, unlike viral vectors such as adenovirus-based vaccines [29]. MVA is also capable to encode one or more foreign antigens


and thus functions as a multivalent vaccine. Finally, MVA-based vaccines are very stable over time in ambient temperature, enabling shipment to remote areas with limited cold-chain maintenance. As such, MVA-BN has been used as a vector for many different vaccines ranging from infectious diseases to various cancers.

In this report, recombinant MVA-BN vaccines for the three encephalitic alphaviruses, VEEV, WEEV, and EEEV were constructed and produced. The protective efficacies of vaccines in single and triple mixture (three vaccines) formats were evaluated in vivo.


2 Materials and methods

2.1 Reagents

All cell culture reagents were purchased from GIBCO (Fisher Sci., Ottawa, ON). Virus DNA for PCR was purified using the NucleoSpin Blood QuickPure Kit (Macherey und Nagel, Düren, Germany) and RNA for reverse transcription (RT)-PCR was purified using the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany). For PCR and RT-PCR amplification of the inserted transgenes, the One Taq Polymerase (NEB, Frankfurt, Germany) and the RT-Polymerase (VWR) were used respectively.

2.2 Cells and viruses

Vero (CCL-81) and HeLa (CCL-2) cells were obtained from the ATCC (Manassas, MD). The cells were maintained in Dulbecco’s modified Eagle media containing 5% (Vero) or 10% (HeLa) heat-inactivated fetal bovine serum (FBS). WEEV strain Fleming (Flem) was purchased from the ATCC. Strain 71V was kindly provided by Dr. Nick Karabatsos (CDC, Fort Collins, CO) and EEEV PE6 and VEEV TrD were kindly provided by Dr. George Ludwig (U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD). Seed stocks of VEEV TrD were made by inoculation of suckling mice and harvesting the brain as a 10% suspension. Stocks of WEEV or EEEV were made by inoculation of Vero cell monolayers with WEEV or EEEV at a multiplicity of infection of 0.1. Supernatants from infected cells were aliquoted and stored at -70°C. The titers of the alphaviruses were determined by plaque titration on Vero cells. MVA-BN and derived recombinant vaccines were grown in primary chicken embryo fibroblast (CEF) cells. CEF cells were prepared from 11 day-old embryonated chicken eggs and cultured in virus production-serum free medium (GIBCO) for virus stock production or in Roswell Park Memorial Institute medium supplemented with 7% FBS for titration assays.

2.3 Construction of MVA-BN-based alphavirus vaccines

The recombinant vaccines encoding the structural proteins E3-E2-6K-E1 of VEEV (strain TrD for vaccine MVA-BN-VEEV), WEEV (strain 71V for vaccine MVA-BN-WEEV), and EEEV (strain FL93-939NA for vaccine MVA-BN-EEEV) were respectively constructed on the MVA-BN vector shown in Fig. 1, deposited at the European Collection of Cell Cultures, Salisbury, UK under number V00083008. Primary CEF cells used for recombinant MVA-BN-based vaccine generation and production of research grade stocks were prepared from embryonated eggs and maintained in serum free conditions.

The cDNAs for the structural protein genes were codon optimized and adapted to avoid large stretches of identity or repeated sequences between the genes and synthesized by GeneArt, Regensburg, Germany. For optimal expression, the individual cDNAs were combined with suitable vaccinia early or early/late promoters. For the expression of VEEV envelop protein, the synthetic pHyb promoter was applied [30], whereas for the expression of WEEV and EEEV envelope proteins, the native MVA-BN tandem repeat promoter Pr13.5-long was used [31]. These genes were cloned into recombination plasmids for the subsequent targeted integration into


Table 1: Anti-alphavirus neutralization titers in the serum of immunized mice.

Vaccines Immunization routes Strains

Neutralizing titers (reciprocal)*

Pre-serum 14 days 41 days

MVA-BN-VEEV S.C. TrD <60 <60 2,160 MVA-BN-VEEV I.M. TrD <60 <60 6,480 Mixture of 3 Vaccines S.C. TrD <60 <60 <60 MVA-BN S.C. TrD <60 <60 <60 MVA-BN-WEEV S.C. 71V <60 180 360 Mixture of 3 Vaccines S.C. 71V <60 60 180 MVA-BN S.C. 71V <60 <60 <60 MVA-BN-WEEV S.C. Flem <60 180 720 Mixture of 3 Vaccines S.C. Flem <60 60 360 MVA-BN S.C. Flem <60 <60 <60 MVA-BN-EEEV S.C. PE6 <60 60 720 Mixture of 3 Vaccines S.C. PE6 <60 <60 180 MVA-BN S.C. PE6 <60 <60 <60


4 Discussion

The recombinant MVA-BN-based encephalitic alphavirus vaccines were designed to include the E3/E2/6k/E1 coding sequences for VEEV, WEEV, or EEEV respectively. The codon usage was adapted for the high expression in humans. The choice of promoters for expression of antigens in a recombinant vector plays a critical role for the success of a vaccine. In all three recombinant MVA-BN-based alphavirus vaccines, strong promotors were selected for the expression of antigens to start very early after infection of cells with the vaccines. This may favour the generation of antigen specific T-cells, which have not been investigated in this report and the high levels of antibodies elicited as well. Meanwhile, the expression of antigens is expected to reach the peak level after the booster [33, 35]. All three vaccines were confirmed to express the E3-E2-6K-E1 transgenes of EEEV, WEEV, and VEEV respectively by FACS analysis with antibodies that are strain specific. Each vaccine or the mixture of three vaccines were then evaluated in vivo against the corresponding virus. In general, MVA-BN-based alphavirus vaccines did an excellent job of protecting mice. Surviving mice showed no signs of infectivity or weight loss. MVA-BN-VEEV and MVA-BN-EEEV provided 100% protection to the mice against the corresponding virus challenge. MVA-BN-WEEV was designed by using a more recent isolate, 71V; however, older WEEV strains isolated in the 1930s–1040s, such as Flem, are more virulent, being capable to cause mouse death within 4–5 days as compared to 9–12 days for 71V in mouse models [36]. MVA-BN-WEEV demonstrated to provide 80% protection to the mice against homologous 71V challenge and 100% protection against heterologous Flem challenge.

Since all the three single vaccines expressed the E3-E2-6K-E1 transgene of EEEV, WEEV and VEEV respectively in equivalent amounts via FACS analysis, a triple mixture of three vaccines was made by adding 108 TCID50 of each and administered in a total volume of 150 μL per mouse. This group was compared to the single MVA-BN alphavirus vaccine to examine if the triple mixture would affect the effective efficacy. The triple mixture demonstrated to provide 100% protection against VEEV or WEEV challenges without any signs of infection, indicating no interference between MVA-BN-WEEV and MVA-BN-VEEV when administered together. However, on the other side, the triple mixture only provided 60% protection to the mice against EEEV challenge. The lower level of potency of MVA-BN-EEEV in the triple mixture is not understood yet and needs further investigation.

For MVA-BN-VEEV, the immunization routes of S.C. and I.M. were compared. No significate difference was observed between the two routes of administration. Both gave 100% protection against even a higher challenge dose of VEEV Trd (104 pfu).

In order to investigate the mechanism of protective efficacy of MVA-BN-based alphavirus vaccines, the serum samples from the vaccinated mice were evaluated in the ANT. In general, MVA-BN-based single alphavirus vaccines did elicit anti-alphavirus neutralizing antibodies. An immunization booster increased the titers of neutralizing antibodies. However, the triple mixture of three vaccines only elicited neutralizing antibodies against WEEV Flem and EEEV PE6, not against VEEV TrD. Further, the triple mixture underperformed single vaccines in terms of titers of neutralizing antibodies. Although neutralizing antibodies played a pivotal role in protective efficacy against alphavirus [24, 37, 38], T-cell immunity also played roles in the protection [39]. MVA-BN vector is good at eliciting both humoral and T-cell immunities [40]. Unfortunately, in


this report, it was not investigated whether T-cell immunity was elicited by the recombinant MVA-BN-based alphavirus vaccines. The triple mixture of three vaccines did affect neutralizing antibody titers as compared to the single vaccine, but it was unknown whether the triple mixture would affect T-cell immunity. Taken together, anti-alphavirus neutralizing antibodies play an important role in the protection against encephalitic alphavirus-mediated infections in the mice vaccinated with MVA-BN-based alphavirus vaccines. However, the anti-alphavirus T-cell immunity might also play roles in the protection, particularly for the mice immunized with the triple mixture of vaccines, which for VEEV and WEEV Flem, provided equivalent protection to the single vaccines. More studies are required before further conclusions can be made.


5 Conclusion

The three recombinant MVA-BN-based encephalitic alphavirus vaccines, MVA-BN-VEEV, MVA-BN-WEEV, and MVA-BN-EEEV demonstrated highly protective efficacy against encephalitic alphaviruses in lethal alphavirus mouse challenge models when given by either the single vaccine or the triple mixture of vaccines, indicating the MVA-BN-based vaccine may be a promising approach against alphavirus infections for biodefence and public health.


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DOCUMENT CONTROL DATA (Security markings for the title, abstract and indexing annotation must be entered when the document is Classified or Designated)

1. ORIGINATOR (The name and address of the organization preparing the document. Organizations for whom the document was prepared, e.g., Centre sponsoring a contractor's report, or tasking agency, are entered in Section 8.) DRDC – Suffield Research Centre Defence Research and Development Canada P.O. Box 4000, Station Main Medicine Hat, Alberta T1A 8K6 Canada

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UNCLASSIFIED

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(NON-CONTROLLED GOODS) DMC A REVIEW: GCEC DECEMBER 2013

3. TITLE (The complete document title as indicated on the title page. Its classification should be indicated by the appropriate abbreviation (S, C or U) in

parentheses after the title.) Development of recombinant modified vaccinia Ankara-Bavarian Nordic (MVA-BN)-based vaccines against encephalitic alphaviruses

4. AUTHORS (last name, followed by initials – ranks, titles, etc., not to be used) Hu, W-G.; Nagata, L.P.; Steigerwald, R.; Kalla, M.; Noll, D.

5. DATE OF PUBLICATION (Month and year of publication of document.) March 2017

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33

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e.g., interim, progress, summary, annual or final. Give the inclusive dates when a specific reporting period is covered.) Scientific Report

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13. ABSTRACT (A brief and factual summary of the document. It may also appear elsewhere in the body of the document itself. It is highly desirable that the abstract of classified documents be unclassified. Each paragraph of the abstract shall begin with an indication of the security classification of the information in the paragraph (unless the document itself is unclassified) represented as (S), (C), (R), or (U). It is not necessary to include here abstracts in both official languages unless the text is bilingual.)

The three encephalitic alphaviruses, Venezuelan, western and eastern equine encephalitis viruses (VEEV, WEEV, and EEEV) are endemic throughout North, Central and South America. Horses and humans are normally exposed after being bitten by infectious mosquitoes and the infection can develop into encephalitis with high rates of morbidity and mortality. These viruses are potential biological threat agents, being highly infectious through the aerosol route of exposure, easily produced in large amounts, and relatively stable in the environment. Currently, there is no licensed vaccine to the three encephalitic alphaviruses, and efforts to move vaccine candidates forward into clinical trials have not been successful. In this report, the development of recombinant modified vaccinia Ankara-Bavarian Nordic (MVA-BN)-based vaccines for the three encephalitic alphaviruses is described. The recombinant vaccines MVA-BN-VEEV, MVA-BN-WEEV, and MVA-BN-EEEV were constructed and produced. The protective efficacy of these vaccines was evaluated in vivo. Female Balb/c mice were immunized subcutaneously with 108 tissue culture infective dose 50 (TCID50) of single MVA-BN encephalitic alphavirus vaccine or a triple mixture of three vaccines at a four-week interval. The mice were then instilled intranasally with 1×103 to 1×104 plaque forming units of VEEV, WEEV, or EEEV 14 days after the second immunization. The MVA-BN-VEEV or MVA-BN-EEEV-immunized mice fully survived the corresponding virus challenge without any signs of infection or weight loss while all the control mice died. The MVA-BN-WEEV could provide 100% protection to the mice against WEEV Fleming strain and 80% to WEEV 71V-1658 strain. Meanwhile, the triple mixture of three vaccines could also provide 100% protection to the mice against WEEV and VEEV challenges, but 60% protection against EEEV challenge. These data suggest that MVA-BN-EEEV, MVA-BN-WEEV, and MVA-BN-VEEV are potential vaccine candidates against the encephalitic viruses and the three vaccines can be given in a mixture without significantly reducing efficacy.

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Les trois alphavirus causant l’encéphalite, le virus de l’encéphalite équine du Venezuela, de l’encéphalite équine de l’Ouest et de l’encéphalite équine de l’Est (VEEV, VEEO et VEEE) sont endémiques dans toute l’Amérique du Nord, l’Amérique centrale et l’Amérique du Sud. Les chevaux et les humains y sont normalement exposés par la piqûre d’un moustique contaminé et l’infection peut évoluer vers une encéphalite présentant un taux élevé de morbidité et de mortalité. Ces virus sont des agents potentiels de menace biologique parce qu’ils sont très infectieux en cas d’exposition à des aérosols, qu’ils sont facilement produits en grande quantité et qu’ils sont relativement stables dans l’environnement. À l’heure actuelle, il n’existe aucun vaccin homologué contre les trois alphavirus causant l’encéphalite et les tentatives visant à amener les vaccins éventuels à l’étape des essais cliniques ont échoué. Le présent rapport décrit la mise au point de vaccins à base d’un virus recombinant de la vaccine de souche modifiée Ankara – Bavarian Nordic (MVA-BN) – contre les trois alphavirus causant l’encéphalite. Les vaccins recombinants MVA-BN-VEEV, MVA-BN-VEEO et MVA-BN-VEEE ont été fabriqués et produits. L’efficacité de la protection conférée par ces vaccins a été analysée in vivo. Des souris femelles Balb/c ont été immunisées par voie sous-cutanée au moyen de 108 DICT50 (dose infectant 50 % d’une culture de tissus) d’un seul vaccin MVA-BN contre un alphavirus causant l’encéphalite ou d’un mélange des trois vaccins à quatre semaines d’intervalle. Les souris ont

ensuite reçu par voie nasale entre 1 × 103 et 1 × 104 unités formatrices de plages du VEEV, du VEEO ou du VEEE 14 jours après la deuxième immunisation. Les souris immunisées au moyen du MVA-BN-VEEV ou du MVA-BN-VEEE ont toutes survécu à l’attaque du virus correspondant et n’ont présenté aucun signe d’infection ni perte de poids, tandis que toutes les souris témoins sont mortes. Le MVA-BN-VEEO pourrait protéger les souris à 100 % contre la souche Fleming du VEEO et à 80 % contre la souche 71V-1658 du VEEO. Entre-temps, le mélange des trois vaccins pourrait aussi protéger les souris à 100 % contre le VEEO et le VEEV, mais à 60 % contre le VEEE. Ces données portent à croire que les MVA-BN-VEEE, MVA-BN-VEEO et MVA-BN-VEEV sont des vaccins possibles contre les virus causant l’encéphalite, et les trois peuvent être administrés dans un mélange sans entraîner de baisse significative de l’efficacité.

14. KEYWORDS, DESCRIPTORS or IDENTIFIERS (Technically meaningful terms or short phrases that characterize a document and could be helpful in cataloguing the document. They should be selected so that no security classification is required. Identifiers, such as equipment model designation, trade name, military project code name, geographic location may also be included. If possible keywords should be selected from a published thesaurus, e.g., Thesaurus of Engineering and Scientific Terms (TEST) and that thesaurus identified. If it is not possible to select indexing terms which are Unclassified, the classification of each should be indicated as with the title.) Vaccines; encephalitic alphaviruses; efficacy

Documents

Development of recombinant modified vaccinia Ankara ... · MVA-BN-WEEV could provide 100% protection to the mice against WEEV Fleming strain and 80% to WEEV 71V-1658 strain. Meanwhile,