Recently, several neutralizing anti-HIV antibodies have been isolated from memory B cells of HIV-infected individuals. Despite extensive evidence of B cell dysfunction in HIV disease, little is known about the cells from which these rare HIV-specific antibodies originate. Accordingly, we used HIV envelope gp140 and CD4 or coreceptor (CoR) binding site (bs) mutant probes to evaluate HIV-specific responses in peripheral blood B cells of HIV-infected individuals at various stages of infection. In contrast to non-HIV responses, HIV-specific responses against gp140 were enriched within abnormal B cells, namely activated and exhausted memory subsets, which are largely absent in the blood of uninfected individuals. Responses against the CoRbs, which is a poorly neutralizing epitope, arose early, whereas those against the well-characterized neutralizing epitope CD4bs were delayed and infrequent. Enrichment of the HIV-specific response within resting memory B cells, the predominant subset in uninfected individuals, did occur in certain infected individuals who maintained low levels of plasma viremia and immune activation with or without antiretroviral therapy. The distribution of HIV-specific responses among memory B cell subsets was corroborated by transcriptional analyses. Taken together, our findings provide valuable insight into virus-specific B cell responses in HIV infection and demonstrate that memory B cell abnormalities may contribute to the ineffectiveness of the antibody response in infected individuals.
An understanding of the antigen-specific B-cell response to the influenza virus hemagglutinin (HA) is critical to the development of universal influenza vaccines, but it has not been possible to examine these cells directly because HA binds to sialic acid (SA) on most cell types. Here, we use structure-based modification of HA to isolate HA-specific B cells by flow cytometry and characterize the features of HA stem antibodies (Abs) required for their development. Incorporation of a previously described mutation (Y98F) to the receptor binding site (RBS) causes HA to bind only those B cells that express HA-specific Abs, but it does not bind nonspecifically to B cells, and this mutation has no effect on the binding of broadly neutralizing Abs to the RBS. To test the specificity of the Y98F mutation, we first demonstrated that previously described HA nanoparticles mediate hemagglutination and then determined that the Y98F mutation eliminates this activity. Cloning of immunoglobulin genes from HA-specific B cells isolated from a single human subject demonstrates that vaccination with H5N1 influenza virus can elicit B cells expressing stem monoclonal Abs (MAbs). Although these MAbs originated mostly from the IGHV1-69 germ line, a reasonable proportion derived from other genes. Analysis of stem Abs provides insight into the maturation pathways of IGVH1-69-derived stem Abs. Furthermore, this analysis shows that multiple non-IGHV1-69 stem Abs with a similar neutralizing breadth develop after vaccination in humans, suggesting that the HA stem response can be elicited in individuals with non-stem-reactive IGHV1-69 alleles.
Needle-free delivery improves the immunogenicity of DNA vaccines but is also associated with more local reactogenicity. Here we report the first comparison of Biojector and needle administration of a candidate rAd5 HIV vaccine.
The interaction between follicular T helper cells (TFH) and B cells in the lymph nodes and spleen has a major impact on the development of antigen-specific B cell responses during infection or vaccination. Recent studies described a functional equivalent of these cells among circulating CD4 T cells, referred to as peripheral TFH cells. Here, we characterize the phenotype and in vitro B cell helper activity of peripheral TFH populations, as well as the effect of HIV infection on these populations. In co-culture experiments we confirmed CXCR5+ cells from HIV-uninfected donors provide help to B cells and more specifically, we identified a CCR7(high)CXCR5(high)CCR6(high)PD-1(high) CD4 T cell population that secretes IL-21 and enhances isotype-switched immunoglobulin production. This population is significantly decreased in treatment-naïve, HIV-infected individuals and can be recovered after anti-retroviral therapy. We found impaired immunoglobulin production in co-cultures from HIV-infected individuals and found no correlation between the frequency of peripheral TFH cells and memory B cells, or with neutralization activity in untreated HIV infection in our cohort. Furthermore, we found that within the peripheral TFH population, the expression level of TFH-associated genes more closely resembles a memory, non-TFH population, as opposed to a TFH population. Overall, our data identify a heterogeneous population of circulating CD4 T cells that provides in vitro help to B cells, and challenges the origin of these cells as memory TFH cells.
Results from recent HIV-1 vaccine studies have indicated that high serum antibody (Ab) titers may not be necessary for Ab-mediated protection, and that Abs localized to mucosal sites might be critical for preventing infection. Enzyme-linked immunosorbent assay (ELISA) has been used for decades as the gold standard for Ab measurement, though recently, highly sensitive microsphere-based assays have become available, with potential utility for improved detection of Abs. In this study, we assessed the Bio-Plex(®) Suspension Array System for the detection of simian immunodeficiency virus (SIV)-specific Abs in rhesus macaques (RMs) chronically infected with SIV, whose serum or mucosal SIV-specific Ab titers were negative by ELISA. We developed a SIVmac239-specific 4-plex bead array for the simultaneous detection of Abs binding to Env, Gag, Pol, and Nef. The 4-plex assay was used to quantify SIV-specific serum IgG and rectal swab IgA titers from control (SIV-naive) and SIVmac239-infected RMs. The Bio-Plex assay specifically detected anti-SIV Abs in specimens from SIV-infected animals for all four analytes when compared to SIV-naive control samples (p?0.04). Furthermore, in 70% of Env and 79% of Gag ELISA-negative serum samples, specific Ab was detected using the Bio-Plex assay. Similarly, 71% of Env and 48% of Gag ELISA-negative rectal swab samples were identified as positive using the Bio-Plex assay. Importantly, assay specificity (i.e., probability of true positives) was comparable to ELISA (94%-100%). The results reported here indicate that microsphere-based methods provide a substantial improvement over ELISA for the detection of Ab responses, aid in detecting specific Abs when analyzing samples containing low levels of Abs, such as during the early stages of a vaccine trial, and may be valuable in attempts to link protective efficacy of vaccines with induced Ab responses.
Here we highlight the latest advances in HIV vaccine concepts that will expand our knowledge on how to elicit effective acquisition-prevention and/or control of simian immunodeficiency virus (SIV) replication in the nonhuman primate (NHP) model.
It has recently been shown that polymorphism at the rhesus macaque TRIM5 locus can affect simian immunodeficiency virus (SIV) replication. Here we show that TRIM5 alleles can also affect acquisition of SIVsmE660. Animals coexpressing the TRIM5(TFP) and TRIM5(CypA) alleles took significantly longer to become infected with SIVsmE660, but not SIVmac239, after repeated limiting-dose intrarectal challenge than did animals expressing other TRIM5 allele combinations. Our results indicate that the TRIM5 alleles can be a barrier to productive infection and that this should be taken into account when designing acquisition studies using SIVsmE660 or related viruses.
DNA priming has previously been shown to elicit augmented immune responses when administered by electroporation (EP) or codelivered with a plasmid encoding interleukin-12 (pIL-12). We hypothesized that the efficacy of a DNA prime and recombinant adenovirus 5 boost vaccination regimen (DNA/rAd5) would be improved when incorporating these vaccination strategies into the DNA priming phase, as determined by pathogenic simian immunodeficiency virus SIVmac239 challenge outcome. The whole SIVmac239 proteome was delivered in 5 separate DNA plasmids (pDNA-SIV) by EP with or without pIL-12, followed by boosting 4 months later with corresponding rAd5-SIV vaccine vectors. Remarkably, after repeated low-dose SIVmac239 mucosal challenge, we demonstrate 2.6 and 4.4 log reductions of the median SIV peak and set point viral loads in rhesus macaques (RMs) that received pDNA-SIV by EP with pIL-12 compared to the median peak and set point viral loads in mock-immunized controls (P < 0.01). In 5 out of 6 infected RMs, strong suppression of viremia was observed, with intermittent "blips" in virus replication. In 2 RMs, we could not detect the presence of SIV RNA in tissue and lymph nodes, even after 13 viral challenges. RMs immunized without pIL-12 demonstrated a typical maximum of 1.5 log reduction in virus load. There was no significant difference in the overall magnitude of SIV-specific antibodies or CD8 T-cell responses between groups; however, pDNA delivery by EP with pIL-12 induced a greater magnitude of SIV-specific CD4 T cells that produced multiple cytokines. This vaccine strategy is relevant for existing vaccine candidates entering clinical evaluation, and this model may provide insights into control of retrovirus replication.
The goals of a T cell-based vaccine for HIV are to reduce viral peak and setpoint and prevent transmission. While it has been relatively straightforward to induce CD8(+) T cell responses against immunodominant T cell epitopes, it has been more difficult to broaden the vaccine-induced CD8(+) T cell response against subdominant T cell epitopes. Additionally, vaccine regimens to induce CD4(+) T cell responses have been studied only in limited settings. In this study, we sought to elicit CD8(+) T cells against subdominant epitopes and CD4(+) T cells using various novel and well-established vaccine strategies. We vaccinated three Mamu-A*01(+) animals with five Mamu-A*01-restricted subdominant SIV-specific CD8(+) T cell epitopes. All three vaccinated animals made high frequency responses against the Mamu-A*01-restricted Env TL9 epitope with one animal making a low frequency CD8(+) T cell response against the Pol LV10 epitope. We also induced SIV-specific CD4(+) T cells against several MHC class II DRBw*606-restricted epitopes. Electroporated DNA with pIL-12 followed by a rAd5 boost was the most immunogenic vaccine strategy. We induced responses against all three Mamu-DRB*w606-restricted CD4 epitopes in the vaccine after the DNA prime. Ad5 vaccination further boosted these responses. Although we successfully elicited several robust epitope-specific CD4(+) T cell responses, vaccination with subdominant MHC class I epitopes elicited few detectable CD8(+) T cell responses. Broadening the CD8(+) T cell response against subdominant MHC class I epitopes was, therefore, more difficult than we initially anticipated.
While the long-term goal is to develop highly effective AIDS vaccines, first generation vaccines may be only partially effective. Other HIV prevention modalities such as preexposure prophylaxis with antiretrovirals (PrEP) may have limited efficacy as well. The combined administration of vaccine and PrEP (VAXPREP), however, may have a synergistic effect leading to an overall benefit that is greater than the sum of the individual effects. We propose two test-of-concept trial designs for an AIDS vaccine plus oral or topical ARV. In one design, evidence that PrEP reduces the risk of HIV acquisition is assumed to justify offering it to all participants. A two-arm study comparing PrEP alone to VAXPREP is proposed in which 30 to 60 incident infections are observed to assess the additional benefit of vaccination on risk of infection and setpoint viral load. The demonstrated superiority of VAXPREP does not imply vaccine alone is efficacious. Similarly, the lack of superiority does not imply vaccine alone is ineffective, as antagonism could exist between vaccine and PrEP. In the other design, PrEP is assumed not to be in general use. A 2 × 2 factorial design is proposed in which high-risk individuals are randomized to one of four arms: placebo vaccine given with placebo PrEP, placebo vaccine given with PrEP, vaccine given with placebo PrEP, or VAXPREP. Between 60 and 210 infections are required to detect a benefit of vaccination with or without PrEP on risk of HIV acquisition or setpoint viral load, with fewer infections needed when synergy is present.
An effective human immunodeficiency virus (HIV) vaccine will likely need to reduce mucosal transmission and, if infection occurs, control virus replication. To determine whether our best simian immunodeficiency virus (SIV) vaccine can achieve these lofty goals, we vaccinated eight Indian rhesus macaques with SIVmac239Delta nef and challenged them intrarectally (i.r.) with repeated low doses of the pathogenic heterologous swarm isolate SIVsmE660. We detected a significant reduction in acquisition of SIVsmE660 in comparison to that for naïve controls (log rank test; P = 0.023). After 10 mucosal challenges, we detected replication of the challenge strain in only five of the eight vaccinated animals. In contrast, seven of the eight control animals became infected with SIVsmE660 after these 10 challenges. Additionally, the SIVsmE660-infected vaccinated animals controlled peak acute virus replication significantly better than did the naïve controls (Mann-Whitney U test; P = 0.038). Four of the five SIVsmE660 vaccinees rapidly brought virus replication under control by week 4 postinfection. Unfortunately, two of these four vaccinated animals lost control of virus replication during the chronic phase of infection. Bulk sequence analysis of the circulating viruses in these animals indicated that recombination had occurred between the vaccine and challenge strains and likely contributed to the increased virus replication in these animals. Overall, our results suggest that a well-designed HIV vaccine might both reduce the rate of acquisition and control viral replication.
Live attenuated simian immunodeficiency virus (SIV) vaccines (LAVs) remain the most efficacious of all vaccines in nonhuman primate models of HIV and AIDS, yet the basis of their robust protection remains poorly understood. Here we show that the degree of LAV-mediated protection against intravenous wild-type SIVmac239 challenge strongly correlates with the magnitude and function of SIV-specific, effector-differentiated T cells in the lymph node but not with the responses of such T cells in the blood or with other cellular, humoral and innate immune parameters. We found that maintenance of protective T cell responses is associated with persistent LAV replication in the lymph node, which occurs almost exclusively in follicular helper T cells. Thus, effective LAVs maintain lymphoid tissue-based, effector-differentiated, SIV-specific T cells that intercept and suppress early wild-type SIV amplification and, if present in sufficient frequencies, can completely control and perhaps clear infection, an observation that provides a rationale for the development of safe, persistent vectors that can elicit and maintain such responses.
The complex interplay between the host immune response and HIV has been the subject of intense research over the last 25 years. HIV and simian immunodeficiency virus (SIV) CD8 T cells have been of particular interest since they were demonstrated to be temporally associated with reduction in virus load shortly following transmission. Here, we briefly review the phenotypic and functional properties of HIV-specific and SIV-specific CD8 T-cell subsets during HIV infection and consider the influence of viral variation with specific responses that are associated with disease progression or control. The development of an effective HIV/AIDS vaccine combined with existing successful prevention and treatment strategies is essential for preventing new infections. In the context of previous clinical HIV/AIDS vaccine trials, we consider the challenges faced by therapeutic and vaccine strategies designed to elicit effective HIV-specific CD8 T cells.
The goal of an effective AIDS vaccine is to generate immunity that will prevent human immunodeficiency virus 1 (HIV-1) acquisition. Despite limited progress toward this goal, renewed optimism has followed the recent success of the RV144 vaccine trial in Thailand. However, the lack of complete protection in this trial suggests that breakthroughs, where infection occurs despite adequate vaccination, will be a reality for many vaccine candidates. We previously reported that neutralizing antibodies elicited by DNA prime-recombinant adenovirus serotype 5 (rAd5) boost vaccination with simian immunodeficiency virus strain mac239 (SIVmac239) Gag-Pol and Env provided protection against pathogenic SIVsmE660 acquisition after repeated mucosal challenge. Here, we report that SIV-specific CD8(+) T cells elicited by that vaccine lowered both peak and set-point viral loads in macaques that became infected despite vaccination. These SIV-specific CD8(+) T cells showed strong virus-inhibitory activity (VIA) and displayed an effector memory (EM) phenotype. VIA correlated with high levels of CD107a mobilization and perforin expression in SIV-specific CD8(+) T cells. Remarkably, both the frequency and the number of Gag CM9-specific public clonotypes were strongly correlated with VIA mediated by EM CD8(+) T cells. The ability to elicit such virus-specific EM CD8(+) T cells might contribute substantially to an efficacious HIV/AIDS vaccine, even after breakthrough infection.
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