SPECIAL POINTS OF INTEREST: VIROLOGY MATTERS NRF RCA …

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NRF RCA 1

Welcome 2

Graduates 3

Visitors 7

M&G top 200 8

Publications 9

Conferences 18

Events 20

Congratula-

tions

21

VIROLOGY MATTERS O C T O B E R 2 0 1 4

SPECIAL

POINTS OF

INTEREST:

NRF RCA

HIV R4P

INSID E THIS

EDITION

NRF Research Career Advancement Fellowships

Tracy Meiring’s NRF RCA study is under the

mentorship of Prof A-L Williamson. They are investi-

gating the complex communities of microorganisms

(viruses, bacteria, fungi, protozoans) in the genital

tract and the effect of HIV and/or HPV infection on

these communities.

Denis Chopera’s study under the men-

torship of Carolyn Williamson will utilise a

longitudinal cohort of HIV infected individ-

uals to assess the impact of immune-

driven HIV viral evolution on viral replica-

tion in subtype C and how this is associ-

ated with disease progression.

Lindi Mason, under the guidance of Jo-Ann

Passmore, will investigate potential causes of

genital inflammation, which we have previously

shown is associated with increased risk of HIV

acquisition, in young South African women dur-

ing her fellowship.

We welcome 3 fellows to Medical Virology:

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V I R O L O G Y M A T T E R S

Welcome

Godfrey Dzhivhuho

I am a PhD candidate in Medical Virology , investigating the impact of Schistoso-miasis on the immunogenicity of candidate HIV vaccines in mice models. My ap-proach evaluates how cellular, humoral and mixed responses are impacted by Schistosomiasis. My passion for science has led me to take a multidisciplinary approach to the scientific community. I investigated the role of cytokine gene pol-ymorphisms in the susceptibility to HIV infection during my honors and worked on microsatellite markers for human identification during my Masters. I also worked in the Cancer Genomics Department with a team in the University of Vir-

ginia, USA. As for my Doctoral studies, I plan to explore more avenues and broaden my spectrum of scientific knowledge in medical research to be a better scientist in the nearest future. I am a born again Christian.

Dr Rakiya Saidu is a specialist Gynaecologist from University of Ilor-in, Nigeria. Started PhD 2014. Working on the prognostic importance of immune cells in vulva cancer in the context of HIV infection. I will work with archiv-al tissue blocks for immunohistochemistry and HPV typing and fresh sam-ples to be collected from vulvar cancer and vulva intraepithelial neoplasia (VIN) patients for IHC and flow cytometry. Co-supervised by Jo-Ann Passmore. Hopes to pursue a career in re-search in the area of tumour immunology and immunotherapy.

Fatima Abrahams I have recently joined Anna-lise Williamson’s group as the Lab manager. I am very happy to be part of this welcoming and supportive environment. I look for-ward to building an effective and beneficial relationship with the group.

Ms Robin W. Njenga Robin is visiting Anna-Lise’s lab on internship from the University of Heidel-berg in Germany. She is a MSc student in Molecular Biosciences and she will be on attachment in our lab for 9 weeks (from 22 September to 21 No-vember 2014). She has been participating in a project titled “Development of a novel cytokine-based method for assessment of candidate HIV vaccines in nonhuman primate models”, in which we are developing a panel of ELISpot-based cytokine assays.

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in the GEMS group to a PhD.

Shameem Jaumdally’s thesis examines the impact of heterosexual partner HIV status on immune acti-vation and inflammation in blood and at the genital mucosa on susceptibly of HIV infection in the HIV negative partner; and HIV-1 disease progression in HIV positive individuals with partners who are also HIV positive (HIV serocon-cordant) or partners who are HIV negative (HIV serodiscordant) in South African couples. His work suggests that immune acti-vation and inflammation detected in blood are likely to be important in HIV sus-ceptibility and pathogene-sis in seroconcordant cou-ples, and that higher viral loads in blood of HIV sero-concordant individuals, driven by the immune acti-vation and inflammation, lead to more HIV shedding at the genital tract. Lower levels of immune activation and inflammation in HIV negative serodiscordant individuals in this study could account for their re-sistance to HIV infection. These findings imply that partner status is an im-portant determinant in both risk for HIV in uninfected individuals and rate of HIV disease progression in in-fected individuals.

He is presently doing his Post Doctoral Fellowship in the GEMS group and con-currently enrolled for a Master of Public Health at UCT.

and South Africa. He found that the central nature of consensus sequences re-sults in their broad recogni-tion both in a highly diverse HIV epidemic (Cameroon) and a homogeneous epi-demic (South Africa), but observed a profound lack of immunodominance. Marcel also showed that new rea-gent sets encompassing epitope variants to broaden the detection of T cell re-sponses do not improve im-mune recognition in a popu-lation with highly divergent viruses. These data inform the testing of current candi-date HIV vaccines in differ-ent regions and have im-portant implications for vac-cine development.

Shameem Z Jaumdally

Thesis Title: Impact of im-mune activation and inflam-mation on the susceptibility to HIV infection and disease progression in HIV serodis-cordant and seroconcordant couples.

Supervisor: Prof Jo-Ann Passmore

Shameem was awarded his PhD in June 2014. Shameem joined the Divi-sion for his BSc(Hons) in Infectious Diseases and Im-munology doing his project in the HIV Diversity and Pathogenesis group. In 2010, he upgraded his MSc degree that he had started

Marcel Tongo Passo

Thesis Title: Immunology and Virology of HIV Infection in Cameroon

Supervisor: Dr Wendy Burgers

Co-Supervisor: Dr Darren Martin

Marcel Tongo’s PhD thesis characterised HIV genetic diversity in Cameroon and HIV-specific immunity. An effective HIV vaccine is the main hope for controlling the pandemic and is a global health priority. Whether a universal vaccine that would be effective against all the clades of HIV is possible, is unknown. Marcel character-ised the diversity of HIV gag and nef genes and nine new full length viruses of Came-roonian HIV-1 isolates. He demonstrated the predomi-nance of HIV-1 CRF02_AG viruses alongside viruses belonging to multiple known and unknown group M line-ages. This provides further evidence that Cameroon is a major hotspot of HIV diversi-ty, and highlights the need for continued molecular epi-demiological surveillance. Marcel investigated T cell responses to centralised rea-gents such as consensus M, and compared immunity in individuals from Cameroon

PhD Graduate

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a potent attractor of neu-trophils, involved in early control of Mtb. Further-more, we also detected enhanced responsiveness of monocytes to LPS stim-ulation during HIV infec-tion. This may reflect spe-cific TLR4 cross-talk from ongoing activation of the innate immune system by HIV-encoded TLR ligands or by the circulating prod-ucts of microbial transloca-tion from the gut. Thus, the increased susceptibil-ity of HIV-infected individu-als to TB (and indeed oth-er gram positive bacteria) may be due to impairment of cytokine responses by monocytes. The results of this study provide further insight into how HIV af-fects innate immunity to Mtb, which is important for a better understanding of how a protective immune response develops against Mtb.

Daniel Sheward

Thesis Ti-tle: Neu-tralizing Antibody Responses in HIV-1 Dual Infec-tion: Les-sons for

Vaccine Design

Supervisor: Prof Car-olyn Williamson

A better understanding of how neutralizing antibody responses develop during natural HIV-1 infection, and the factors that aug-ment them, would be in-valuable in informing vac-cine design and test-ing. The use of polyvalent

MSc Graduates Narjis Thawer

Thesis Title: In-vestigating defects in monocyte re-sponses to Myco-bacterium tuber-culosis (Mtb) in HIV-TB co-infection

Supervisor: Dr Wendy Burgers

HIV-infected persons are more susceptible to TB, and the rea-sons for this are not fully un-derstood. HIV infection leads to CD4+ T cell depletion, com-promising adaptive immunity to Mtb, however less is known regarding the effect on innate immunity. Monocytes play a key role in innate immune de-fense and are the precursors of macrophages. These cells sense pathogens through toll-like receptors (TLR), which leads to the release of pro-inflammatory mediators, trig-gering innate and adaptive im-mune responses to infection, critical events for the control of Mtb. The aim of this study was to investigate whether HIV in-fection induced functional de-fects in monocytes, impairing their ability to respond to Mtb. The focus was on TLR func-tioning in monocytes, by exam-ining whether infection with HIV altered cytokine produc-tion in response to TLR stimu-lation.

Overall, our data demonstrate that HIV co-infection impaired the ability of monocytes to se-crete the key chemokine IL-8, in response to mycobacterial lipoarabinomannan (LAM), a TLR2-stimulating mycobacteri-al cell wall component. IL-8 is

immunogens is a common strategy used in an attempt to enhance the breadth of vaccine-elicited immune re-sponses and HIV dual infec-tion (infection by >1 distinct HIV strain) provides a unique in vivo model to characterize how the im-mune system responds to multiple HIV strains.

In the CAPRISA 002 cohort from KwaZulu Natal, South Africa eleven cases of dual infection were detected, of whom eight were co-infected at the first sero-positive visit and three were superinfected (infected with a second strain following seroconversion to an initial infection). To establish the clinical consequences of dual infection, we compared the viral loads, and CD4+ T cell counts of dual infected participants to 18 singly in-fected participants from the same cohort. Neither dual infection, nor envelope di-versity in early infection was significantly associated with accelerated disease pro-gression. Further, a multi-variate analysis suggested that infection with Neisseria Gonorrhoeae was associat-ed with multiple infection, suggesting N. Gonorrhoeae increased the susceptibility to multiple infection, alt-hough this did not reach statistical significance (p=0.0566).

Single genome amplification (SGA) derived sequences of the early superinfecting viral quasispecies were generated. In all three su-perinfected participants identified, a single founder variant likely established superinfection. Similar to


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male genital tract com-pared to blood. She found that the majority of T-cells in the genital tract were found to be effector memory T-cells (TEM), while the majority of T-cells in blood were naïve and effector memory CD4+ T-cells and terminally-differentiated CD8+ T-cells. HAART did not influ-ence the predominance of TEM cells in the genital tract, although the frequen-cy of the central memory T-cell (TCM) population within the genital tract did increase with initiation of HAART. Higher frequen-cies of T-cells from the genital tract were activated (measured by expression of CD38 and HLA-DR) than those in blood from HIV-infected women on HAART. Initiation of HAART resulted in a signif-icant reduction in the level of T-cell activation in blood but not in the genital tract, where activation levels were slightly increased compared to before initia-tion of HAART. T-cells col-lected from the genital tract of women on HAART were significantly more exhaust-ed than those in blood (measured by CD57 ex-pression); and 1 month on HAART resulted in an in-creased population of ex-hausted T-cells in the geni-tal tract, particularly for TCM and TEM subsets. CD4:CD8 ratios in blood of HIV-infected women on long-term HAART (1:1.4) showed evidence of partial CD4 immune reconstitution

transmitted/founder viruses from primary HIV infection, these superinfecting viruses had significantly shorter, less glycosylated variable regions compared to viruses sam-pled at three years post in-fection from participants in the same cohort.

Neutralization breadth data at three years post infection was available for 11 of the dual infected participants, as well as for 16 singly infected controls . Comparing the breadth revealed that dual infection was not significantly associated with greater neu-tralizing antibody breadth, even after controlling for viral loads and CD4+ T cell counts. However, one su-perinfected participant, CAP256, developed a broad, and extremely potent neu-tralizing antibody re-sponse . Using computa-tional methods, the epitope targeted by CAP256 was predicted to be highly de-pendent on residues 166 and 169, overlapping epitopes recognized by mon-oclonal antibodies PG9/PG16. This was later vali-dated, and the epitope com-prehensively mapped . Lon-gitudinal sequencing of env revealed that escape from this potent and broad anti-body response occurred via multiple pathways, and was accelerated by recombina-tion. Further, we show that the targeted epitope was not conserved in the primary in-fecting virus, suggesting that superinfection did not focus the neutralizing antibody re-sponse onto an epitope con-served in both infecting vi-ruses.

Taken together, these re-sults reiterate that sexual-ly transmitted infections can enhance susceptibil-ity to HIV infection. Fur-ther, they show that expo-sure to two divergent HIV variants is not sufficient to broaden the neutralizing antibody response, and raise doubts in the poten-tial efficacy of polyvalent HIV immunogens. They also suggest the clinical consequences of dual in-fection, if any, are not se-vere. However, larger studies will be required to definitively address the clinical and immunologi-cal consequences of HIV co- and superinfection, and to address the corre-lates of risk of HIV super-infection.

Smritee Dabee

Thesis Title: Role of HAART in reconstituting T-cell function and HIV inhibitory activity in the female genital tract during chronic HIV infection.

Supervisor: Prof Jo-ann Passmore

Her study investigated the impact of HAART on the levels of inflammatory cytokines and immune reconstitution (measured by immune activation, proliferation and exhaus-tion of T-cells) in the fe-

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ing at the susceptibility factors of adolescent girls to HIV acquisition.

Emmanuel Margolin

Thesis Title: An Investi-gation into Improved HIV-1 Subtype C Envelope Based Vaccine Design

Supervisor: Prof Anna-Lise Williamson

Co-Supervisor: Dr Ros Chapman

This study served to de-velop a heterologous DNA prime-protein boost vac-cine combination by ex-ploiting existing genetic immunization and plant expression platforms de-veloped by our research group. An antigenically promising HIV-1 envelope, with known sensitivity to prototype broadly neutral-izing monoclonal antibod-ies, was selected for de-velopment into 3 vaccine immunogens. The first an-tigen, gp150, comprised of a truncated derivative of the full length envelope protein designed to en-hance expression and the exposure of antibody sen-sitive epitopes. The re-maining 2 antigens, gp120-HA2 and gp140-HA2tr, comprised of chimeric HIV envelope antigens fused to portions of the influenza haemagglutinin transmem-

compared to other studies, alt-hough genital tract CD4:CD8 ratios (1:0.8) were significantly lower. While complete viral suppression in both plasma and in genital secretions was evident as early as 1 month following initiation of HAART, immune reconstitution (measured by CD4:CD8 ratios) was not significantly higher. Lastly, cytokine concentrations in the genital tract were gener-ally higher than those in blood. Viral suppression seen early after HAART initiation was as-sociated with significantly low-er IP-10 concentrations in plas-ma as well as the level of acti-vation of T-cells in blood. In contrast, inflammatory cytokine concentrations in genital secre-tions were largely unchanged after initiation of HAART alt-hough the anti-inflammatory cytokine IL-10 was detected at significantly lower concentra-tions.

In conclusion, initiation of HAART during HIV infection had a more pronounced impact on T-cell activation and inflam-mation in blood than in the genital compartment, which remained inflamed and highly activated. Being on HAART for longer periods of time was still accompanied by higher levels of genital T-cell activation and inflammation in genital tract than blood, despite the fact that most women had fully sup-pressed blood viral loads. On-going inflammation in the geni-tal tract and local immune acti-vation during HAART would potentially place women at in-creased risk for HIV genital shedding despite full suppres-sion of viraemia systemically.

She is now enrolled for her PhD in the GEMS group look-

brane subunit. DNA vac-cines encoding each of the immunogens were con-structed using a plasmid which exploits a porcine circovirus type 1 enhancer element to drive high levels of antigen expression. High levels of expression of all three immunogens were ob-served by both western blotting and immunostaining of HEK 293 cells transfect-ed with the DNA vaccines.

Recombinant strains of Agrobacterium tumefa-ciens were generated for each immunogen and antigen expression opti-mized in plants. The most successful approach in-volved infiltrating high den-sities of the bacterial culture into plant leaves and ex-tracting protein directly from the apoplast. The co-expression of a post-transcriptional gene silenc-ing suppressor also had a profound influence on ex-pression, enabling the dura-tion and magnitude of ex-pression to be improved. The use of denaturing con-ditions to extract protein proved successful for gp140-HA2tr antigen only but this approach was deemed im-practical as it would pre-clude the recovery of intact protein. Variation in antigen expression was seen when experiments were repeated highlighting the influence of plant development on re-combinant protein expres-sion. At best low level ex-pression of the recombinant antigens were achieved in plants that was insufficient for further characterization or immunization studies.


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Dr Mario Roederer from the Vaccine

Research Center, NIH visited Wendy

Burgers lab and gave a seminar entitled

‘Antigenic Heterogeneity of Clonal

Envelopes: A Novel Immune Escape

Mechanism’ in October

Visitors Dr Erica Andersen-Nissen

from the Cape

Town HVTN

Immunology

Laboratory

(CHIL) pre-

sented on De-

fining Innate Immune respons-

es to HIV Vaccines in April

Prof David Katzenstein

(Stanford Uni-

versity) visited

in August and

presented a

seminar enti-

tled “The end

of AIDS? A

long and winding road”.

Gianguido C. Cianci, Post Doctoral Fellow at the Hope HIV Laboratory at Northwest-ern University, Chicago, Illi-nois.

Gianguido visited the GEMS lab for 2 weeks in September to advise and train the group members with image analy-sis.


Anna Ursula Happel, Master

student in Molecular Medicine at

the Friedrich-Alexander-

University, Erlangen-Nuremberg,

Germany. She is with the GEMS

group for 3 months (September

to November) doing an

internship with a the future goal

of doing her Master dissertation

in the field of HIV immunology

when she goes back to

Germany.

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a next generation of vaccines.”

Born and raised in Durban, Sheward studied for a BSc in biomedical science at the Uni-versity of KwaZulu-Natal be-fore mov-

ing to Cape Town for postgraduate studies at UCT. His academic career shows a certain sin-gle-mindedness: honours in infectious diseases and immu-nology, master’s in medical virology, training at the Na-tional Institute for Communicable Dis-eases in Johannes-burg and now a doc-torate wrestling with HIV.

“I was passionate about science and simply wanted to be involved in an im-portant field. Infec-tious diseases sounded reward-ing. South Africa had been hit hard with HIV and Aids so I started out in an HIV lab at UCT and haven’t looked back.”

The 27-year-old has some role models. “It is often a few people who improve the lives of millions, who change the world for the better. It’s those people who inspire me. People like Maurice Hilleman, John Enders, Louis Pas-teur, Edward Jenner and many others.

“I also have a huge amount of respect for Bill Gates – I hope to be able to do a fraction of the good he is doing.”

Despite his involve-ment in research projects that have been published in Nature Medicine and Nature, Sheward says he’s still working on rack-ing up outstanding achievements.

Asked where he aims to be in 10 years’ time, he says: “Wherever I am, I hope to have a bet-ter answer to: ‘What would you say are your greatest achievements?’”

University of Cape Town (UCT) doctor-al student Daniel Sheward of the HIV

Diversity Group has been an integral part of a pioneering study aimed at beat-ing South Africa’s bête noire, HIV.

“Vaccines represent the best tools we have to control in-fectious diseases and have saved hundreds of millions of lives.

“However, HIV has proven to be an un-precedented chal-lenge and has sent vaccinologists back to the drawing board,” he says. “In broad terms, I am trying to understand the immune re-sponse, particularly the development of antibodies, to HIV in infected individuals, and to use this to inform the design of


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Mariba Lebeko

,

V I R O L O G Y

M A T T E R S


Kidzeru EB1, Hesseling AC, Passmore JA, Myer L, Gamieldien H,

Tchakoute CT, Gray CM, Sodora DL, Jaspan HB.

In-utero exposure to maternal HIV infection alters T-cell immune re-

sponses to vaccination in HIV-uninfected infants.

AIDS. 2014 Jun 19;28(10):1421-30.

OBJECTIVE:

In sub-Saharan Africa, HIV-exposed uninfected (HEU) infants have

higher morbidity and mortality than HIV-unexposed infants. To evaluate

whether immune dysfunction contributes to this vulnerability of HEU

infants, we conducted a longitudinal, observational cohort study to as-

sess T-cell immune responses to infant vaccines (Mycobacterium bovis

BCG and acellular pertussis) and staphylococcal enterotoxin B (SEB).

In total, 46 HEU and 46 HIV-unexposed infants were recruited from

Khayelitsha, Cape Town.

RESULTS:

HEU infants demonstrated elevated BCG-specific CD4 and CD8 T-cell

proliferative responses at 14 weeks (P = 0.041 and 0.002, respective-

ly). These responses were significantly increased even after adjusting

for birth weight, feeding mode and gestational age. Similar to BCG, in-

creased CD4 and CD8 T-cell proliferation was evident in response to

SEB stimulation (P = 0.004 and 0.002, respectively), although pertus-

sis-specific T cells proliferated comparably between the two groups.

Within HEU infants, maternal CD4 cell count and length of antenatal

antiretroviral exposure had no effect on T-cell proliferation to BCG or

SEB. HIV exposure significantly diminished measurable cytokine poly-

functionality in response to BCG, Bordetella pertussis and SEB stimu-

lation.

CONCLUSION:

These data show for the first time, when adjusting for confounders, that exposure to HIV in utero is associated with significant alterations to CD4 and CD8T-cell immune responses in infants to vaccines and non-specific antigens.

Recent Virology Publications

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Rametse CL1, Olivier AJ, Masson L, Barnabas S, McKinnon LR,

Ngcapu S, Liebenberg LJ, Jaumdally SZ, Gray CM, Jaspan HB,

Passmore JA.

Role of semen in altering the balance between inflammation and toler-

ance in the female genital tract: does it contribute to HIV risk?

Viral Immunol. 2014 Jun;27(5):200-6

Abstract

While the main reproduction aim of semen is the transport of spermato-

zoa to the female genital tract, seminal plasma is a complex fluid that

also carries a broad array of immunologically active molecules. Seminal

plasma has been shown to contain a diverse array of anti-inflammatory

and pro-inflammatory soluble mediators that regulate immune responses

within the female reproductive tract than can facilitate fertilization. Since

the natural inflammatory response to semen deposition in the female

genital tract may result in recruitment of activated HIV target cells into

the female genital mucosa, we discuss the constituents of semen that

may increase the risk for HIV infection in women

Burgers WA, Ginbot ZG, Shen YJ, Chege GK, Soares AP, Müller

TL, Bunjun R, Kiravu A, Munyanduki H, Douglass N, Williamson

AL.

The novel Capripoxvirus vector Lumpy Skin Disease Virus (LSDV) effi-

ciently boosts MVA HIV responses in rhesus macaques.

J Gen Virol. 2014 May 27. Abstract

Poxvirus vectors represent promising HIV vaccine candidates and were

a component of the only successful HIV vaccine efficacy trial to date.

We tested the immunogenicity of a novel recombinant Capripoxvirus

vector, Lumpy Skin Disease Virus (LSDV) in combination with Modified

Vaccinia Ankara (MVA), both expressing genes from HIV-1. Here, we

demonstrate that the combination regimen was immunogenic in rhesus

macaques, inducing high magnitude, broad and balanced CD4+ and

CD8+ T cell responses, and transient activation of the immune re-

sponse. These studies support further development of LSDV as a vac-

cine vector

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Offerman K, Carulei O, van der Walt AP, Douglass N, Williamson AL.

The complete genome sequences of poxviruses isolated from a penguin

and a pigeon in South Africa and comparison to other sequenced avipox-

viruses.

BMC Genomics. 2014 Jun 12;15(1):463.

BACKGROUND:

Two novel avipoxviruses from South Africa have been sequenced, one

from a Feral Pigeon (Columba livia) (FeP2) and the other from an African

penguin (Spheniscus demersus) (PEPV). We present a purpose-

designed bioinformatics pipeline for analysis of next generation sequence

data of avian poxviruses and compare the different avipoxviruses se-

quenced to date with specific emphasis on their evolution and gene con-

tent.

RESULTS:

The FeP2 (282kbp) and PEPV (306kbp) genomes encode 271 and 284

open reading frames respectively and are more closely related to one an-

other (94.4%) than to either fowlpox virus (FWPV) (85.3% and 84.0% re-

spectively) or Canarypox virus (CNPV) (62.0% and 63.4% respectively).

Overall, FeP2, PEPV and FWPV have syntenic gene arrangements; how-

ever, major differences exist throughout their genomes. The most striking

difference between FeP2 and the FWPV-like avipoxviruses is a large de-

letion of ~16kbp from the central region of the genome of FeP2 deleting a

cc-chemokine-like gene, two Variola virus B22R orthologues, an N1R/p28

-like gene and a V-type Ig domain family gene. FeP2 and PEPV both en-

code orthologues of vaccinia virus C7L and Interleukin 10. PEPV contains

a 77 amino acid long orthologue of Ubiquitin sharing 97% amino acid

identity to human ubiquitin.

CONCLUSIONS:

The genome sequences of FeP2 and PEPV have greatly added to the

limited repository of genomic information available for the Avipoxvirus ge-

nus. In the comparison of FeP2 and PEPV to existing sequences, FWPV

and CNPV, we have established insights into African avipoxvirus evolu-

tion. Our data supports the independent evolution of these South African

avipoxviruses from a common ancestral virus to FWPV and CNPV


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Mariba Lebeko


Chapman R, Bourn WR, Shephard E, Stutz H, Douglass N,

Mgwebi T, Meyers A, Chin'ombe N, Williamson AL.

The Use of Directed Evolution to Create a Stable and Immunogenic

Recombinant BCG Expressing a Modified HIV-1 Gag Antigen.

PLoS One. 2014 Jul 25;9(7):e103314.

Numerous features make Mycobacterium bovis BCG an attractive

vaccine vector for HIV. It has a good safety profile, it elicits long-

lasting cellular immune responses and in addition manufacturing

costs are affordable. Despite these advantages it is often difficult to

express viral antigens in BCG, which results in genetic instability and

low immunogenicity. The aim of this study was to generate stable re-

combinant BCG (rBCG) that express high levels of HIV antigens, by

modification of the HIV genes. A directed evolution process was ap-

plied to recombinant mycobacteria that expressed HIV-1 Gag fused to

the green fluorescent protein (GFP). Higher growth rates and in-

creased GFP expression were selected for. Through this process a

modified Gag antigen was selected. Recombinant BCG that ex-

pressed the modified Gag (BCG[pWB106] and BCG[pWB206]) were

more stable, produced higher levels of antigen and grew faster than

those that expressed the unmodified Gag (BCG[pWB105]). The re-

combinant BCG that expressed the modified HIV-1 Gag induced 2 to

3 fold higher levels of Gag-specific CD4 T cells than those expressing

the unmodified Gag (BCG[pWB105]). Mice primed with 107 CFU BCG

[pWB206] and then boosted with MVA-Gag developed Gag-specific

CD8 T cells with a frequency of 1343±17 SFU/106 splenocytes, 16

fold greater than the response induced with MVA-Gag alone. Levels of

Gag-specific CD4 T cells were approximately 5 fold higher in mice

primed with BCG[pWB206] and boosted with MVA-Gag than in those

receiving the MVA-Gag boost alone. In addition mice vaccinated with

BCG[pWB206] were protected from a surrogate vaccinia virus chal-

lenge.

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Mariba Lebeko

V I R O L O G Y

M A T T E R S


Mlisana K, Werner l, Garrett NJ, McKinnon LR, van Loggerenberg F,

Passmore J-AS, Gray CM, Morris L, Williamson C, Abdool Karim SS

and the CAPRISA 002 Study Team.

Rapid disease progression in HIV-1 subtype C infected South African wom-

en: Antiretroviral therapy initiation within 12 months of infection.

Clinical Infectious Diseases. 2014. Nov 1;59(9):1322-31

Abstract

BACKGROUNDS:

While HIV subtype-B infected individuals generally progress to AIDS within

8-10 years, limited data exist for other clades, especially from Africa. We in-

vestigated rates of HIV disease progression of clade C-infected South Afri-

can women.

RESULTS:

Sixty-two women were identified at median 42 days post-infection (IQR 34-

59), contributing 282 person-years of follow-up. Mean CD4 count dropped

by 39.6% at 3 months and 46.7% at 6 months post-infection in women with

pre-infection measurements. CD4 decline to <350 cells/µl occurred in 31%,

44%, and 55% at 1, 2, and 3 years post-infection, respectively, and to <500

cells/µl in 69%, 79% and 81% at equivalent time-points. Predictors of rapid

progression were CD4 count at 3 months post-infection [hazard ratio (HR)

2.07, 95% confidence interval 1.31-3.28, p=0.002], set-point viral load [HR

3.82 (1.51-9.67), p=0.012] and hepatitis B co-infection [HR 4.54 (1.31-

15.69), p=0.017]. Conversely, presence of any of HLAB*1302, B*27, B*57,

B*5801 or B*8101 alleles predicted non-rapid progression [HR 0.19 (0.05-

0.74), p=0.016].

CONCLUSION:

Nearly half of subtype C infected women progressed to CD4<350 cells/µl

within two years of infection. Implementing 2013 WHO treatment guidelines

(CD4<500) would require most individuals to start antiretroviral therapy with-

in one year of HIV infection

Page 14

Mariba Lebeko

V I R O L O G Y

M A T T E R S


Kharsany AB, Buthelezi TJ, Frohlich JA, Yende-Zuma N, Sam-

sunder N, Mahlase G, Williamson C, Travers S, Marais JC, Dellar

RC, Karim QA, Karim SS.

HIV infection in rural South African high schools: Role of transmis-

sions among students.

AIDS Res Hum Retroviruses. 2014 Oct;30(10):956-65

In South Africa, adolescents constitute a key population at high risk

of HIV acquisition. However, little is known about HIV transmission

among students within schools. This study was undertaken to as-

sess the risk factors for HIV infection and the extent of transmission

among rural high school students. Between February and May 2012,

consenting students from five randomly selected public sector high

schools in rural KwaZulu-Natal participated in an anonymized cross-

sectional survey. Dried blood spot samples were collected and test-

ed for HIV. βHCG levels were measured in females for pregnancy.

Family circumstances, socio-demographic and behavioral factors

were assessed as potential risk factors. A subset (106/148, 72%) of

HIV positive samples underwent gag p17p24 sequencing for phylo-

genetic analysis. A total of 3242 students (81.7% of enrolled stu-

dents) participated. HIV prevalence was 6.8% [95% Confidence In-

terval (CI) 3.9-9.8%] in girls and 2.7% (CI 1.6-3.8%) in boys

(aOR=3.0, CI 2.4-3.8; p<0.001). HIV prevalence increased from

4.6% (95% CI 1.9-7.3) in the 12-15 year old girls to 23.1% (95% CI

7.7-38.5) in girls over 20 years, whilst in boys HIV prevalence in-

creased from 2.7% (95% CI 0.6-4.9) in the 12-15 year olds to 11.1%

(95% CI 2.7-19.4) in those over 20 years. Sequencing of samples

obtained from students revealed only 2 clusters suggesting within-

school transmission and 3 inter-school clusters, while the remainder

was most likely acquired from sources other than those currently at-

tending the school concerned. HIV prevalence in both girls

(aOR=3.6, CI 2.9-4.5; p<0.001) and boys (aOR=2.8, CI 1.2-6.2;

p=0.01) was higher in those without a living biological mother. The

high burden of HIV infection among students was not associated

with intra-school transmission in this rural setting. Lack of a living

parent is an important factor defining high risk in this group of ado-

lescents

Page 15


Hraber P, Korber BT, Lapedes AS, Bailer RT, Seaman MS, Gao H, Greene KM,

McCutchan F, Williamson C, Kim JH, Tovanabutra S, Hahn BH, Swanstrom R, Thomson

MM, Gao F, Harris L, Giorgi E, Hengartner N, Bhattacharya T, Mascola JR, Montefiori DC.

Impact of clade, geography and age of the epidemic on HIV-1 neutralization by antibodies.

J Virol. 2014 Nov 1;88(21):12623-43

Abstract Neutralizing antibodies (nAbs) are a high priority for vaccines that aim to prevent the ac-quisition of HIV-1 infection. Vaccine effectiveness will depend on the extent to which in-duced antibodies neutralize the global diversity of circulating HIV-1 variants. Using large panels of genetically and geographically diverse HIV-1 Env-pseudotyped viruses and chronic infection plasma samples, we unambiguously show that cross-clade nAb respons-es are commonly induced in response to infection by any virus clade. Nonetheless, neu-tralization was significantly greater when the plasma clade matched the clade of the virus being tested. This within-clade advantage was diminished in older, more diverse epidem-ics in southern Africa, the US and Europe compared to more recent epidemics in Asia. It was most pronounced for circulating recombinant form (CRF) 07_BC, which is common in China and is the least divergent lineage studied; this was followed by the slightly more di-verse Asian CRF01_AE. We found no evidence that transmitted/founder viruses are gen-erally more susceptible to neutralization and are therefore easier targets for vaccination than chronic viruses. Features of the gp120 V1V2 loop, in particular length, net charge and number of N-linked glycans, were associated with Env susceptibility and plasma neutrali-zation potency in a manner consistent with neutralization-escape being a force that drives viral diversification and plasma neutralization breadth. The overall susceptibility of Envs and potencies of plasmas were highly predictive of the neutralization outcome of any sin-gle virus/plasma combination. These findings highlight important considerations for the de-sign and testing of candidate HIV-1 vaccines that aim to elicit effective nAbs.

IMPORTANCE: An effective HIV-1 vaccine will need to overcome the extraordinary variability of the virus, which is most pronounced in the envelope glycoproteins (Env) that are the sole targets for neutralizing antibodies (nAbs). Distinct genetic lineages, or clades, of HIV-1 occur in differ-ent locales that may require special consideration when designing and testing vaccines candidates. We show that nAb responses to HIV-1 infection are generally active across clades but are most potent within clades. Because effective vaccine-induced nAbs are like-ly to share these properties, optimal coverage of a particular clade or combination of clades may require clade-matched immunogens. Optimal within-clade coverage might be easier to achieve in regions such as China and Thailand, where the epidemic is more re-cent and the virus less diverse than in southern Africa, the US and Europe. Finally, fea-tures of the first and second hypervariable regions of gp120 (V1V2) may be critical for opti-mal vaccine design.

Page 16


Mann JK, Chopera D, Omarjee S, Kuang XT, Le AQ, Anmole G,

Danroth R, Mwimanzi P, Reddy T, Carlson J, Radebe M, Goulder

PJ, Walker BD, Abdool Karim S, Novitsky V, Williamson C,

Brockman MA, Brumme ZL, Ndung'u T.

Nef-mediated down-regulation of CD4 and HLA class I in HIV-1 sub-

type C infection: Association with disease progression and influence

of immune pressure.

Virology. 2014 Sep 2;468-470C:214-225

Nef plays a major role in HIV-1 pathogenicity. We studied HIV-1 sub-type C infected individuals in acute/early (n=120) or chronic (n=207) infection to investigate the relationship between Nef-mediated CD4/HLA-I down-regulation activities and disease progression, and the influence of immune-driven sequence variation on these Nef func-tions. A single Nef sequence per individual was cloned into an ex-pression plasmid, followed by transfection of a T cell line and meas-urement of CD4 and HLA-I expression. In early infection, a trend of higher CD4 down-regulation ability correlating with higher viral load set point was observed (r=0.19, p=0.05), and higher HLA-I down-regulation activity was significantly associated with faster rate of CD4 decline (p=0.02). HLA-I down-regulation function correlated in-versely with the number HLA-associated polymorphisms previously associated with reversion in the absence of the selecting HLA allele (r=-0.21, p=0.0002). These data support consideration of certain Nef regions in HIV-1 vaccine strategies designed to attenuate the infec-tion course

Page 17


Kleppa E, Ramsuran V, Zulu S, Karlsen GH, Bere A, Passmore JA, Ndhlovu P, Lillebø K, Holmen SD, Onsrud M, Gundersen SG, Taylor M, Kjetland EF, Ndung'u T. Efect of female genital schistosomiasis and antischistosomal treatment on monocytes, CD4+ T-cells and CCR5 expression in the female genital tract. Plos One 2014 Jun 4;9(6):e98593 BACKGROUND: Schistosoma haematobium is a waterborne para-site that may cause female genital schistosomiasis (FGS), charac-terized by genitalmucosal lesions. There is clinical and epidemiologi-cal evidence for a relationship between FGS and HIV. We investigat-ed the impact of FGS on HIV target cell density and expression of the HIV co-receptor CCR5 in blood and cervical cytobrush samples. Furthermore we evaluated the effect of anti-schistosomal treatment on these cell populations. DESIGN: The study followed a case-control design with post treatment follow-up, nested in an on-going field study on FGS. METHODS: Blood and cervical cytobrush samples were collected from FGS neg-ative and positive women for flow cytometry analyses. Urine sam-ples were investigated for schistosome ova by microscopy and poly-merase chain reaction (PCR). RESULTS: FGS was associated with a higher frequency of CD14+ cells (monocytes) in blood (11.5% in FGS+ vs. 2.2% in FGS-, p = 0.042). Frequencies of CD4+ cells expressing CCR5 were higher in blood samples from FGS+ than from FGS- women (4.7% vs. 1.5%, p = 0.018). The CD14+ cell population decreased significantly in both compartments after anti-schistosomal treatment (p = 0.043). Alt-hough the frequency of CD4+ cells did not change after treatment, frequencies of CCR5 expression by CD4+ cells decreased signifi-cantly in both compartments (from 3.4% to 0.5% in blood, p = 0.036; and from 42.4% to 5.6% in genital samples, p = 0.025). CONCLUSIONS: The results support the hypothesis that FGS may increase the risk of HIV acquisition, not only through damage of the mucosal epithelial barrier, but also by affecting HIV target cell populations, and that anti-schistosomal treatment can modify this.

Page 18

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International Papillomavirus Conference

Prof Anna-Lise Williamson and Dr Zizipho Mbulawa attended the 29th Interna-tional Papillomavirus Conference and Public Health and Clinical Workshop that was held at the Washington State Convention Center, Seattle, Washington, USA, 21-25 August 2014. Prof Anna-Lise’s proposal to host the 31st International Papillomavirus Conference and Public Health and Clinical Workshop in Cape Town, South Africa, at the beginning of 2017 was accepted. Dr Zizipho Mbulawa gave an oral presentation during the conference on the 24th August 2014; her presentation was entitled “Risk factors for oral Human Papillomavirus in heterosexual couples in an African set-tings”. After the presentation she received a good response from the audience and suggestions from international experts in the field.

2014 International Conference on Poxvirus, Asfarvirus &

Iridovirus

Kristy Offerman, Niki Douglass and Anna-Lise Williamson attended the 2014 International Conference on Poxvirus, Asfarvirus & Iridovirus in Victoria, Canada. Anna-Lise and Niki each presented a poster. Kristy was one of the only students to get an oral presentation which was entitled “Poxviruses induce different host gene expression profiles in a model system’ with authors - Kristy Offerman, Armin Deffur, Olivia Carulei, Robert Wilkinson, Nicola Douglass and Anna-Lise Williamson. The conference also allowed time for sight-seeing.

Orca off the coat of Victoria photographed by Anna-Lise Williamson

Niki and Kristy after a long day at the

conference

Anna-Lise with Chris Upton a long time poxvirus researcher who visited UCT in 1989

Conferences

Page 19

.


Workshop on HIV

Transmission Various members of Virology

attended the 9th International

workshop on HIV transmission in

Cape Town in October. Carolyn

Williamson was the co-chair of the

workshop.

HIV R4P The HIV R4P conference at the CTICC was well attended by staff and students

from Virology in October. Jo-Ann Passmore gave a

plenary lecture and Shameem

Jaumdally

and Smritee

Dabee also

presented

Page 20

.


Events

Ruwayhida Thebus

Now that I have completed my MSc , I

have time to enjoy the sun, outdoors

and the road. While I am deciding what

to study next, I completed my first 21km

half marathon ( Atlantis marathon ) in

August and hope to make a debut at

the Old Mutual Two Oceans half mara-

thon, 2015

Murray Logan competing in a CrossFit winter games in July. He did a 150kg deadlift — amazing!

Ziyaad Valley-Omar com-pleted the Impi Challenge in October. This is trail running with an adventurous obstacle course.

Lerato Majara and Shera-zaan Ismail took part in the 10km Gun Run on 12 Octo-ber

Page 21


Congratulations

Zizi Mbulawa got engaged to

Ntuthuko Mbatha from KwaZulu

Natal (Ulundi)

Natalie Strickland married

Mathew Jewell in April

Compiled and edited by Debbie Stewart ([email protected])

Dieter Mielke and Rudo Gunda

got married in Harare in July

Documents

SPECIAL POINTS OF INTEREST: VIROLOGY MATTERS NRF RCA …