Scientific Publications

We have demonstrated that a liposomal array of well-ordered trimers enhances B cell activation, germinal center formation, and the elicitation of tier-2 autologous neutralizing antibodies. Previously, we coupled well-ordered cleavage-independent NFL trimers via their C-terminal polyhistidine tails to nickel lipids integrated into the lipid bilayer. Despite favorable effects, concern remained over the potentially longer-term instability of noncovalent linkage of the trimers to the liposomes. Accordingly, we tested both cobalt coupling and covalent linkage of the trimers to the liposomes by reengineering the polyhistidine tail to include a free cysteine on each protomer of model BG505 NFL trimers to allow covalent linkage. Both cobalt and cysteine coupling resulted in a high-density array of NFL trimers that was stable in both 20% mouse serum and 100 mM EDTA, whereas the nickel-conjugated trimers were not stable under these conditions. Binding analysis and calcium flux with anti-Env-specific B cells confirmed that the trimers maintained conformational integrity following coupling. Following immunization of mice, serologic analysis demonstrated that the covalently coupled trimers elicited Env-directed antibodies in a manner statistically significantly improved compared to soluble trimers and nickel-conjugated trimers. Importantly, the covalent coupling not only enhanced gp120-directed responses compared to soluble trimers, it also completely eliminated antibodies directed to the C-terminal His tag located at the 'bottom' of the spike. In contrast, soluble and noncovalent formats efficiently elicited anti-His tag antibodies. These data indicate that covalent linkage of well-ordered trimers to liposomes in high-density array displays multiple advantages and Enveloped viruses typically encode a surface-bound glycoprotein that mediates viral entry into host cells and is a primary target for vaccine design. Liposomes with modified lipid head groups have a unique feature of capturing and displaying antigens on their surfaces, mimicking the native pathogens. Our first-generation nickel-based liposomes captured HIV-1 Env glycoprotein trimers via a noncovalent linkage with improved efficacy over soluble glycoprotein in activating germinal center B cells and eliciting tier-2 autologous neutralizing antibodies. In this study, we report the development of second-generation cobalt- and maleimide-based liposomes that have improved stability over nickel-based liposomes. In particular, the maleimide liposomes captured HIV-1 Env trimers via a more stable covalent bond, resulting in enhanced germinal center B cell responses that generated higher antibody titers than the soluble trimers and liposome-bearing trimers via noncovalent linkages. We further demonstrate that covalent coupling prevents release of the trimers prior to recognition by B cells and masks a nonneutralizing determinant located at the bottom of the trimer.

Cytomegalovirus (CMV)-based vaccines have shown remarkable efficacy in the rhesus macaque model of acquired immune deficiency syndrome, enabling 50% of vaccinated monkeys to clear a subsequent virulent simian immunodeficiency virus challenge. The protective vaccine elicited unconventional CD8 T cell responses that were entirely restricted by MHC II or the nonclassical MHC I molecule, MHC-E. These unconventional responses were only elicited by a fibroblast-adapted rhesus CMV vector with limited tissue tropism; a repaired vector with normal tropism elicited conventional responses. Testing whether these unusual protective CD8 T responses could be elicited in humans requires vaccinating human subjects with a fibroblast-adapted mutant of human CMV (HCMV). In this study, we describe the CD8 T cell responses of human subjects vaccinated with two fibroblast-adapted HCMV vaccines. Most responses were identified as conventional classically MHC I restricted, and we found no evidence for MHC II or HLA-E restriction. These results indicate that fibroblast adaptation alone is unlikely to explain the unconventional responses observed in macaques.

Home-based HIV counselling and testing (HBHCT) has the potential to increase HIV testing uptake in sub-Saharan Africa (SSA), but data on linkage to HIV care after HBHCT are scarce. We conducted a systematic review of linkage to care after HBHCT in SSA.

In this paper we evaluate the effects of heavy alcohol consumption on sexual behavior, HIV acquisition, and antiretroviral treatment (ART) initiation in a longitudinal open cohort of 1929 serodiscordant couples in Lusaka, Zambia from 2002 to 2012. We evaluated factors associated with baseline heavy alcohol consumption and its association with condomless sex with the study partner, sex outside of the partnership, and ART initiation using multivariable logistic regression. We estimated the effect of alcohol consumption on HIV acquisition using multivariable Cox models. Baseline factors significantly associated with women's heavy drinking (drunk weekly or more in 12-months before enrollment) included woman's older age (adjusted prevalence odds ratio [aPOR] = 1.04), partner heavy drinking (aPOR = 3.93), and being HIV-infected (aPOR = 2.03). Heavy drinking among men was associated with less age disparity with partner (aPOR per year disparity = 0.97) and partner heavy drinking (aPOR = 1.63). Men's being drunk daily (aOR = 1.18), women's being drunk less than monthly (aOR = 1.39) vs. never drunk and being in a male HIV-negative and female HIV-positive union (aOR = 1.45) were associated with condomless sex. Heavy alcohol use was associated with having 1 or more outside sex partners among men (aOR drunk daily = 1.91, drunk weekly = 1.32, drunk monthly = 2.03 vs. never), and women (aOR drunk monthly = 2.75 vs. never). Being drunk weekly or more increased men's risk of HIV acquisition (adjusted hazard ratio [aHR] = 1.72). Men and women being drunk weekly or more was associated (p < 0.1) with women's seroconversion (aHR = 1.42 and aHR = 3.71 respectively). HIV-positive women who were drunk monthly or more had lower odds of initiating ART (aOR = 0.83; 95% CI = 0.70-0.99) adjusting for age, months since baseline and previous pregnancies. Individuals in HIV-serodiscordant couples who reported heavy drinking had more outside sex partnerships and condomless sex with their study partner and were more likely to acquire HIV. HIV-positive women had lower odds of initiating ART if they were heavy drinkers.

The unchanged global HIV incidence may be related to ignoring acute HIV infection (AHI). This scoping review examines diagnostic, clinical, and public health implications of identifying and treating persons with AHI.

Analyzing surface epitopes of single HIV particles holds great potential for the development of vaccine candidates. However, existing technologies do not allow corresponding screens at high throughput. We present here a single-virus droplet-based microfluidics platform enabling sorting of millions of HIV-1 particles with >99% efficiency, based on the expression of epitopes recognized by broadly neutralizing antibodies. We show that virus particles displaying these epitopes can be identified, sorted, and analyzed by next-generation sequencing: an approximately 1,900-fold enrichment of viral particles displaying neutralizing epitopes could be obtained in a single sort, thus opening the way for screening diverse virus libraries with optimal antigenic features for HIV vaccine candidates.

Early treatment of acute HIV-1 infection (AHI) is beneficial for patients and could reduce onward transmission. However, guidelines on whom to test for AHI with HIV-1 RNA testing are lacking.

Native-like trimers of the SOSIP design are being developed as immunogens in human immunodeficiency virus type 1 (HIV-1) vaccine development programs. These trimers display the epitopes for multiple broadly neutralizing antibodies (bNAbs) but can also expose binding sites for some types of nonneutralizing antibodies (non-NAbs). Among the latter are epitopes in the gp120 V3 region that are highly immunogenic when SOSIP trimers are evaluated in animal models. It is presently uncertain whether antibodies against V3 can interfere with the induction of NAbs, but there are good arguments in favor of suppressing such 'off-target' immune responses. Accordingly, we have assessed how to minimize the exposure of V3 non-NAb epitopes and thereby reduce their immunogenicity by introducing -glycans within the V3 region of BG505 SOSIP trimers. We found that inserting glycans at positions 306 and 314 (termed M1 and M7) markedly reduced V3 antigenicity while improving the presentation of trimer apex bNAb epitopes. Both added glycans were shown to be predominantly of the ManGlcNAc form. The additional introduction of the E64K ground-state stabilizing substitution markedly reduced or ablated soluble CD4 (sCD4) induction of non-NAb epitopes in V3 and/or associated with the coreceptor binding site. When a V3 glycan- and E64K-modified trimer variant, BG505 SOSIP.664-E64K.M1M7, was tested in rabbits, V3 immunogenicity was eliminated while the autologous NAb response was unchanged. Trimeric proteins are being developed for future HIV-1 vaccine trials in humans, with the goal of eliciting broadly active neutralizing antibodies (NAbs) that are active against a wide variety of circulating strains. In animal models, the present generation of native-like trimer immunogens, exemplified by the BG505 SOSIP.664 construct, induces narrow-specificity antibodies against the neutralization-resistant (tier-2), sequence-matched virus and more broadly active antibodies against sequence-divergent atypically neutralization-sensitive (tier-1) viruses. A concern in the trimer immunogen design field has been whether the latter off-target antibodies might interfere with the induction of the more desired responses to tier-2 epitopes. Here, we have inserted two glycans into the dominant site for tier-1 NAbs, the gp120 V3 region, to block the induction of off-target antibodies. We characterized the new trimers, tested them as immunogens in rabbits, and found that the blocking glycans eliminated the induction of tier-1 NAbs to V3-epitopes.

We present temporal trends in self-reported and biological markers of unprotected sex and sex with concurrent partners in discordant couples receiving couples' voluntary HIV counselling and testing (CVCT).

: Understanding HIV-1 transmission dynamics is relevant to both screening and intervention strategies of HIV-1 infection. Commonly, HIV-1 transmission chains are determined based on sequence similarity assessed either directly from a sequence alignment or by inferring a phylogenetic tree. This review is aimed at both nonexperts interested in understanding and interpreting studies of HIV-1 transmission, and experts interested in finding the most appropriate cluster definition for a specific dataset and research question. We start by introducing the concepts and methodologies of how HIV-1 transmission clusters usually have been defined. We then present the results of a systematic review of 105 HIV-1 molecular epidemiology studies summarizing the most common methods and definitions in the literature. Finally, we offer our perspectives on how HIV-1 transmission clusters can be defined and provide some guidance based on examples from real life datasets.

Kenya is a generally homophobic country where homosexuality is criminalised and people who engage in same sex sexuality face stigma and discrimination. In 2013, we developed a 16 min documentary entitled '' that aimed at sharing information on how and why men who have sex with men (MSM) are involved in on-going KEMRI HIV prevention research, and associated community engagement. To consider the film's usefulness as a communication tool, and its perceived security risks in case the film was publicly released, we conducted nine facilitated viewings with 122 individuals representing seven different stakeholder groups. The documentary was seen as a strong visual communication tool with potential to reduce stigma related to homosexuality, and facilitated film viewings were identified as platforms with potential to support open dialogue about HIV research involving MSM. Despite the potential, there were concerns over possible risks to LGBT communities and those working with them following public release. We opted-giving emphasis to the 'do no harm' principle-to use the film only in facilitated settings where audience knowledge and attitudes can be carefully considered and discussed. The results highlight the importance of carefully assessing the range of possible impacts when using visuals in community engagement.

Soluble, recombinant native-like envelope glycoprotein (Env) trimers of various human immunodeficiency virus type 1 (HIV-1) genotypes are being developed for structural studies and as vaccine candidates aimed at the induction of broadly neutralizing antibodies (bNAbs). The prototypic design is designated SOSIP.664, but many HIV-1 genes do not yield fully native-like trimers efficiently. One such gene is CZA97.012 from a neutralization-resistant (tier 2) clade C virus. As appropriately purified, native-like CZA97.012 SOSIP.664 trimers induce autologous neutralizing antibodies (NAbs) efficiently in immunized rabbits, we sought to improve the efficiency with which they can be produced and to better understand the limitations to the original design. By using structure- and antigenicity-guided mutagenesis strategies focused on the V2 and V3 regions and the gp120-gp41 interface, we developed the CZA97 SOSIP.v4.2-M6.IT construct. Fully native-like, stable trimers that display multiple bNAb epitopes could be expressed from this construct in a stable CHO cell line and purified at an acceptable yield using either a PGT145 or a 2G12 bNAb affinity column. We also show that similar mutagenesis strategies can be used to improve the yields and properties of SOSIP.664 trimers of the DU422, 426c, and 92UG037 genotypes. Recombinant trimeric proteins based on HIV-1 genes are being developed for future vaccine trials in humans. A feature of these proteins is their mimicry of the envelope glycoprotein (Env) structure on virus particles that is targeted by neutralizing antibodies, i.e., antibodies that prevent cells from becoming infected. The vaccine concept under exploration is that recombinant trimers may be able to elicit virus-neutralizing antibodies when delivered as immunogens. Because HIV-1 is extremely variable, a practical vaccine may need to incorporate Env trimers derived from multiple different virus sequences. Accordingly, we need to understand how to make recombinant trimers from many different genes. Here, we show how to produce trimers from a clade C virus, CZA97.012, by using an array of protein engineering techniques to improve a prototypic construct. We also show that the methods may have wider utility for other genes, thereby further guiding immunogen design.

A key aspect to finding an efficacious human immunodeficiency virus (HIV) vaccine is the optimization of vaccine schedules that can mediate the efficient maturation of protective immune responses. In the present study, we investigated the effect of alternate booster regimens on the immune responses to a candidate HIV-1 clade C CN54gp140 envelope protein, which was coadministered with the TLR4-agonist glucopyranosyl lipid A-aqueous formulation. Twelve study participants received a common three-dose intramuscular priming series followed by a final booster at either 6 or 12 months. The two homologous prime-boost regimens were well tolerated and induced CN54gp140-specific responses that were observed in both the systemic and mucosal compartments. Levels of vaccine-induced IgG-subclass antibodies correlated significantly with FcγR engagement, and both vaccine regimens were associated with strikingly similar patterns in antibody titer and FcγR-binding profiles. In both groups, identical changes in the antigen (Ag)-specific IgG-subclass fingerprint, leading to a decrease in IgG1 and an increase in IgG4 levels, were modulated by booster injections. Here, the dissection of immune profiles further supports the notion that prime-boost strategies are essential for the induction of diverse Ag-specific HIV-1 responses. The results reported here clearly demonstrate that identical responses were effectively and safely induced by both vaccine regimens, indicating that an accelerated 6-month regimen could be employed for the rapid induction of immune responses against CN54gp140 with no apparent impact on the overall quality of the induced immune response. (This study has been registered at http://ClinicalTrials.gov under registration no. NCT01966900.).