Jill Gilmour

Induction of Multifunctional Human Immunodeficiency Virus Type 1 (HIV-1)-Specific T Cells Capable of Proliferation in Healthy Subjects by Using a Prime-Boost Regimen of DNA- and Modified Vaccinia Virus Ankara-Vectored Vaccines Expressing HIV-1 Gag Coupled to CD8+ T-Cell Epitopes

ABSTRACT A double-blind randomized phase I trial was conducted in human immunodeficiency virus type 1 (HIV-1)-negative subjects receiving vaccines vectored by plasmid DNA and modified vaccinia virus Ankara (MVA) expressing HIV-1 p24/p17 gag linked to a string of CD8+ T-cell epitopes. The trial had two groups. One group received either two doses of MVA.HIVA (2× MVA.HIVA) (n = 8) or two doses of placebo (2× placebo) (n = 4). The second group received 2× pTHr.HIVA followed by one dose of MVA.HIVA (n = 8) or 3× placebo (n = 4). In the pTHr.HIVA-MVA.HIVA group, HIV-1-specific T-cell responses peaked 1 week after MVA.HIVA vaccination in both ex vivo gamma interferon (IFN-γ) ELISPOT (group mean, 210 spot-forming cells/106 cells) and proliferation (group mean stimulation index, 37), with assays detecting positive responses in four out of eight and five out of eight subjects, respectively. No HIV-1-specific T-cell responses were detected in either assay in the 2× MVA.HIVA group or subjects receiving placebo. Using a highly sensitive and reproducible cultured IFN-γ ELISPOT assay, positive responses mainly mediated by CD4+ T cells were detected in eight out of eight vaccinees in the pTHr.HIVA-MVA.HIVA group and four out of eight vaccinees in the 2× MVA.HIVA group. Importantly, no false-positive responses were detected in the eight subjects receiving placebo. Of the 12 responders, 11 developed responses to previously identified immunodominant CD4+ T-cell epitopes, with 6 volunteers having responses to more than one epitope. Five out of 12 responders also developed CD8+ T-cell responses to the epitope string. Induced T cells produced a variety of anti-viral cytokines, including tumor necrosis factor alpha and macrophage inflammatory protein 1β. These data demonstrate that prime-boost vaccination with recombinant DNA and MVA vectors can induce multifunctional HIV-1-specific T cells in the majority of vaccinees.

Plasmacytoid Dendritic Cells Are Highly Susceptible to Human Immunodeficiency Virus Type 1 Infection and Release Infectious Virus

ABSTRACT Plasmacytoid dendritic cells (pcDC) and myeloid dendritic cells (myDC) are shown to express CD4 and low levels of CCR5 and CXCR4, but only myDC express DC SIGN, a C-type lectin that binds human immunodeficiency virus but does not mediate virus entry. Both DC types were more susceptible to infection with a macrophage than a lymphotropic strain of human immunodeficiency virus type 1, but pcDC were more readily infected than myDC.

Standardization of cytokine flow cytometry assays

BackgroundCytokine flow cytometry (CFC) or intracellular cytokine staining (ICS) can quantitate antigen-specific T cell responses in settings such as experimental vaccination. Standardization of ICS among laboratories performing vaccine studies would provide a common platform by which to compare the immunogenicity of different vaccine candidates across multiple international organizations conducting clinical trials. As such, a study was carried out among several laboratories involved in HIV clinical trials, to define the inter-lab precision of ICS using various sample types, and using a common protocol for each experiment (see additional files online).ResultsThree sample types (activated, fixed, and frozen whole blood; fresh whole blood; and cryopreserved PBMC) were shipped to various sites, where ICS assays using cytomegalovirus (CMV) pp65 peptide mix or control antigens were performed in parallel in 96-well plates. For one experiment, antigens and antibody cocktails were lyophilised into 96-well plates to simplify and standardize the assay setup. Results (CD4+cytokine+ cells and CD8+cytokine+ cells) were determined by each site. Raw data were also sent to a central site for batch analysis with a dynamic gating template.Mean inter-laboratory coefficient of variation (C.V.) ranged from 17–44% depending upon the sample type and analysis method. Cryopreserved peripheral blood mononuclear cells (PBMC) yielded lower inter-lab C.V.s than whole blood. Centralized analysis (using a dynamic gating template) reduced the inter-lab C.V. by 5–20%, depending upon the experiment. The inter-lab C.V. was lowest (18–24%) for samples with a mean of >0.5% IFNγ + T cells, and highest (57–82%) for samples with a mean of <0.1% IFNγ + cells.ConclusionICS assays can be performed by multiple laboratories using a common protocol with good inter-laboratory precision, which improves as the frequency of responding cells increases. Cryopreserved PBMC may yield slightly more consistent results than shipped whole blood. Analysis, particularly gating, is a significant source of variability, and can be reduced by centralized analysis and/or use of a standardized dynamic gating template. Use of pre-aliquoted lyophilized reagents for stimulation and staining can provide further standardization to these assays.

Evolution of HLA-B*5703 HIV-1 escape mutations in HLA-B*5703–positive individuals and their transmission recipients

HLA-B*57 is the class I allele most consistently associated with control of human immunodeficiency virus (HIV) replication, which may be linked to the specific HIV peptides that this allele presents to cytotoxic T lymphocytes (CTLs), and the resulting efficacy of these cellular immune responses. In two HIV C clade–infected populations in South Africa and Zambia, we sought to elucidate the role of HLA-B*5703 in HIV disease outcome. HLA-B*5703–restricted CTL responses select for escape mutations in three Gag p24 epitopes, in a predictable order. We show that the accumulation of these mutations sequentially reduces viral replicative capacity in vitro. Despite this, in vivo data demonstrate that there is ultimately an increase in viral load concomitant with evasion of all three HLA-B*5703–restricted CTL responses. In HLA-B*5703–mismatched recipients, the previously described early benefit of transmitted HLA-B*5703–associated escape mutations is abrogated by the increase in viral load coincident with reversion. Rapid disease progression is observed in HLA-matched recipients to whom mutated virus is transmitted. These data demonstrate that, although costly escape from CTL responses can progressively attenuate the virus, high viral loads develop in the absence of adequate, continued CTL responses. These data underline the need for a CTL vaccine against multiple conserved epitopes.

International Seroepidemiology of Adenovirus Serotypes 5, 26, 35, and 48 in Pediatric and Adult Populations

Recombinant adenovirus serotype 5 (rAd5) vaccine vectors for HIV-1 and other pathogens have been shown to be limited by high titers of Ad5 neutralizing antibodies (NAbs) in the developing world. Alternative serotype rAd vectors have therefore been constructed. Here we report Ad5, Ad26, Ad35, and Ad48 NAb titers in 4381 individuals from North America, South America, sub-Saharan Africa, and Southeast Asia. As expected, Ad5 NAb titers were both frequent and high magnitude in sub-Saharan Africa and Southeast Asia. In contrast, Ad35 NAb titers proved infrequent and low in all regions studied, and Ad48 NAbs were rare in all regions except East Africa. Ad26 NAbs were moderately common in adults in sub-Saharan Africa and Southeast Asia, but Ad26 NAb titers proved markedly lower than Ad5 NAb titers in all regions, and these relatively low Ad26 NAb titers did not detectably suppress the immunogenicity of 4×10(10)vp of a rAd26-Gag/Pol/Env/Nef vaccine in rhesus monkeys. These data inform the clinical development of alternative serotype rAd vaccine vectors in the developing world.

Selection bias at the heterosexual HIV-1 transmission bottleneck

Introduction Heterosexual HIV-1 transmission is an inefficient process with rates reported at <1% per unprotected sexual exposure. When transmission occurs, systemic infection is typically established by a single genetic variant, taken from the swarm of genetically distinct viruses circulating in the donor. Whether that founder virus represents a chance event or was systematically favored is unclear. Our work has tested a central hypothesis that founder virus selection is biased toward certain genetic characteristics. Fitter viruses (red) are favored more in woman-to-man (bottom curve) than in man-to-woman (top curve) transmission. The probability that a majority donor amino acid variant is transmitted is a function of relative fitness, here estimated by the frequency of the variant in the Zambian population. Even residues common in the population are less likely to be transmitted to healthy men than to women, indicative of higher selection bias in woman-to-man transmission. Rationale If HIV-1 transmission involves selection for viruses with certain favorable characteristics, then such advantages should emerge as statistical biases when viewed across many viral loci in many transmitting partners. We therefore identified 137 Zambian heterosexual transmission pairs, for whom plasma samples were available for both the donor and recipient partner soon after transmission, and compared the viral sequences obtained from each partner to identify features that predicted whether the majority amino acid observed at any particular position in the donor was transmitted. We focused attention on two features: viral genetic characteristics that correlate with viral fitness, and clinical factors that influence transmission. Statistical modeling indicates that the former will be favored for transmission, while the latter will nullify this relative advantage. Results We observed a highly significant selection bias that favors the transmission of amino acids associated with increased fitness. These features included the frequency of the amino acid in the study cohort, the relative advantage of the amino acid with respect to the stability of the protein, and features related to immune escape and compensation. This selection bias was reduced in couples with high risk of transmission. In particular, significantly less selection bias was observed in men with genital inflammation and in women (regardless of inflammation status), compared to healthy men, suggesting a more permissive environment in the female than male genital tract. Consistent with this observation, viruses transmitted to women were characterized by lower predicted fitness than those in men. The presence of amino acids favored during transmission predicted which individual virus within a donor was transmitted to their partner, while chronically infected individuals with viral populations characterized by a predominance of these amino acids were more likely to transmit to their partners. Conclusion These data highlight the clear selection biases that benefit fitter viruses during transmission in the context of a stochastic process. That such biases exist, and are tempered by certain risk factors, suggests that transmission is frequently characterized by many abortive transmission events in which some target cells are nonproductively infected. Moreover, for efficient transmission, some changes that favored survival in the transmitting partner are frequently discarded, resulting in overall slower evolution of HIV-1 in the population. Paradoxically, by increasing the selection bias at the transmission bottleneck, reduction of susceptibility may increase the expected fitness of breakthrough viruses that establish infection and may therefore worsen the prognosis for the newly infected partner. Conversely, preventive or therapeutic approaches that weaken the virus may reduce overall transmission rates via a mechanism that is independent from the quantity of circulating virus, and may therefore provide long-term benefits to the recipient if transmission does occur. HIV needs to be fit to transmit Although you might not think it, its hard to catch HIV. Less than 1% of unprotected sexual exposures result in infection. What then leads to transmission? Carlson et al. determined the amino acid sequence of viruses infecting 137 Zambian heterosexual couples in which one partner infected the other (see the Perspective by Joseph and Swanstrom). The authors then used statistical modeling and found that transmitted viruses are typically the most evolutionarily fit. That is, compared to other viral variants in the infected person, the transmitted virus most closely matches the most common viral sequence found in the Zambian population. Science, this issue 10.1126/science.1254031; see also p. 136 An analysis of discordant couples reveals that transmitted HIV-1 viruses are typically the most evolutionarily fit. [Also see Perspective by Joseph and Swanstrom] Heterosexual transmission of HIV-1 typically results in one genetic variant establishing systemic infection. We compared, for 137 linked transmission pairs, the amino acid sequences encoded by non-envelope genes of viruses in both partners and demonstrate a selection bias for transmission of residues that are predicted to confer increased in vivo fitness on viruses in the newly infected, immunologically naïve recipient. Although tempered by transmission risk factors, such as donor viral load, genital inflammation, and recipient gender, this selection bias provides an overall transmission advantage for viral quasispecies that are dominated by viruses with high in vivo fitness. Thus, preventative or therapeutic approaches that even marginally reduce viral fitness may lower the overall transmission rates and offer long-term benefits even upon successful transmission.

Vaccine-elicited Human T Cells Recognizing Conserved Protein Regions Inhibit HIV-1

Virus diversity and escape from immune responses are the biggest challenges to the development of an effective vaccine against HIV-1. We hypothesized that T-cell vaccines targeting the most conserved regions of the HIV-1 proteome, which are common to most variants and bear fitness costs when mutated, will generate effectors that efficiently recognize and kill virus-infected cells early enough after transmission to potentially impact on HIV-1 replication and will do so more efficiently than whole protein-based T-cell vaccines. Here, we describe the first-ever administration of conserved immunogen vaccines vectored using prime-boost regimens of DNA, simian adenovirus and modified vaccinia virus Ankara to uninfected UK volunteers. The vaccine induced high levels of effector T cells that recognized virus-infected autologous CD4(+) cells and inhibited HIV-1 replication by up to 5.79 log10. The virus inhibition was mediated by both Gag- and Pol- specific effector CD8(+) T cells targeting epitopes that are typically subdominant in natural infection. These results provide proof of concept for using a vaccine to target T cells at conserved epitopes, showing that these T cells can control HIV-1 replication in vitro.

Broadly Neutralizing Antibody Responses in a Large Longitudinal Sub-Saharan HIV Primary Infection Cohort.

Broadly neutralizing antibodies (bnAbs) are thought to be a critical component of a protective HIV vaccine. However, designing vaccines immunogens able to elicit bnAbs has proven unsuccessful to date. Understanding the correlates and immunological mechanisms leading to the development of bnAb responses during natural HIV infection is thus critical to the design of a protective vaccine. The IAVI Protocol C program investigates a large longitudinal cohort of primary HIV-1 infection in Eastern and South Africa. Development of neutralization was evaluated in 439 donors using a 6 cross-clade pseudo-virus panel predictive of neutralization breadth on larger panels. About 15% of individuals developed bnAb responses, essentially between year 2 and year 4 of infection. Statistical analyses revealed no influence of gender, age or geographical origin on the development of neutralization breadth. However, cross-clade neutralization strongly correlated with high viral load as well as with low CD4 T cell counts, subtype-C infection and HLA-A*03(-) genotype. A correlation with high overall plasma IgG levels and anti-Env IgG binding titers was also found. The latter appeared not associated with higher affinity, suggesting a greater diversity of the anti-Env responses in broad neutralizers. Broadly neutralizing activity targeting glycan-dependent epitopes, largely the N332-glycan epitope region, was detected in nearly half of the broad neutralizers while CD4bs and gp41-MPER bnAb responses were only detected in very few individuals. Together the findings suggest that both viral and host factors are critical for the development of bnAbs and that the HIV Env N332-glycan supersite may be a favorable target for vaccine design.

Viral Inhibition Assay: A CD8 T Cell Neutralization Assay for Use in Clinical Trials of HIV-1 Vaccine Candidates

We have characterized an assay measuring CD8 T cell-mediated inhibition of human immunodeficiency virus (HIV) type 1 replication, demonstrating specificity and reproducibility and employing a panel of primary HIV-1 isolates. The assay uses relatively simple autologous cell culture and enzyme-linked immunosorbent assay, avoids generation of T cell clones, and can be performed with <2 million peripheral blood mononuclear cells. Efficient CD8 T cell-mediated cross-clade inhibition of HIV-1 replication in vitro was demonstrated in antiretroviral therapy-naive HIV-1-infected subjects with controlled viral replication in vivo but not in viremic subjects. An HIV-1 vaccine candidate, consisting of DNA and recombinant adenovirus 5 vectors tested in a phase I clinical trial, induced CD8 T cells that efficiently inhibited HIV-1 in a HLA-I-dependent manner. Assessment of direct antiviral T cell function by this assay provides additional information to guide vaccine design and the prioritizing of candidates for further clinical trials.

Role of Transmitted Gag CTL Polymorphisms in Defining Replicative Capacity and Early HIV-1 Pathogenesis

Initial studies of 88 transmission pairs in the Zambia Emory HIV Research Project cohort demonstrated that the number of transmitted HLA-B associated polymorphisms in Gag, but not Nef, was negatively correlated to set point viral load (VL) in the newly infected partners. These results suggested that accumulation of CTL escape mutations in Gag might attenuate viral replication and provide a clinical benefit during early stages of infection. Using a novel approach, we have cloned gag sequences isolated from the earliest seroconversion plasma sample from the acutely infected recipient of 149 epidemiologically linked Zambian transmission pairs into a primary isolate, subtype C proviral vector, MJ4. We determined the replicative capacity (RC) of these Gag-MJ4 chimeras by infecting the GXR25 cell line and quantifying virion production in supernatants via a radiolabeled reverse transcriptase assay. We observed a statistically significant positive correlation between RC conferred by the transmitted Gag sequence and set point VL in newly infected individuals (p = 0.02). Furthermore, the RC of Gag-MJ4 chimeras also correlated with the VL of chronically infected donors near the estimated date of infection (p = 0.01), demonstrating that virus replication contributes to VL in both acute and chronic infection. These studies also allowed for the elucidation of novel sites in Gag associated with changes in RC, where rare mutations had the greatest effect on fitness. Although we observed both advantageous and deleterious rare mutations, the latter could point to vulnerable targets in the HIV-1 genome. Importantly, RC correlated significantly (p = 0.029) with the rate of CD4+ T cell decline over the first 3 years of infection in a manner that is partially independent of VL, suggesting that the replication capacity of HIV-1 during the earliest stages of infection is a determinant of pathogenesis beyond what might be expected based on set point VL alone.