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Background The generation of accurate and reproducible viral sequence data is necessary to understand the diversity present in populations of RNA viruses isolated from clinical samples. While various sequencing methods are available, they often require high quality templates and high viral titer to ensure reliable data. Methods We modified a multiplex PCR and sequencing approach to characterize populations of simian immunodeficiency virus (SIV) isolated from nonhuman primates. We chose this approach with the aim of reducing the number of required input templates while maintaining fidelity and sensitivity. We conducted replicate sequencing experiments using different numbers of quantified viral RNA (vRNA) or viral cDNA as input material. We performed assays with clonal SIVmac239 to detect false positives, and we mixed SIVmac239 and a variant with 24 point mutations (SIVmac239-24X) to measure variant detection sensitivity. Results We found that utilizing a starting material of quantified viral cDNA templates had a lower rate of false positives and increased reproducibility when compared to that of quantified vRNA templates. This study identifies the importance of rigorously validating deep sequencing methods and including replicate samples when using a new method to characterize low frequency variants in a population with a small number of templates. Conclusions Because the need to generate reproducible and accurate sequencing data from diverse viruses from low titer samples, we modified a multiplex PCR and sequencing approach to characterize SIV from populations from non-human primates. We found that increasing starting template numbers increased the reproducibility and decreased the number of false positives identified, and this was further seen when cDNA was used as a starting material. Ultimately, we highlight the importance of vigorously validating methods to prevent overinterpretation of low frequency variants in a sample.

We evaluated the contribution of CD8αβ(+) T cells to control of live-attenuated simian immunodeficiency virus (LASIV) replication during chronic infection and subsequent protection from pathogenic SIV challenge. Unlike previous reports with a CD8α-specific depleting monoclonal antibody (mAb), the CD8β-specific mAb CD8β255R1 selectively depleted CD8αβ(+) T cells without also depleting non-CD8(+) T cell populations that express CD8α, such as natural killer (NK) cells and γδ T cells. Following infusion with CD8β255R1, plasma viremia transiently increased coincident with declining peripheral CD8αβ(+) T cells. Interestingly, plasma viremia returned to predepletion levels even when peripheral CD8αβ(+) T cells did not. Although depletion of CD8αβ(+) T cells in the lymph node (LN) was incomplete, frequencies of these cells were 3-fold lower (P = 0.006) in animals that received CD8β255R1 than in those that received control IgG. It is possible that these residual SIV-specific CD8αβ(+) T cells may have contributed to suppression of viremia during chronic infection. We also determined whether infusion of CD8β255R1 in the LASIV-vaccinated animals increased their susceptibility to infection following intravenous challenge with pathogenic SIVmac239. We found that 7/8 animals infused with CD8β255R1, and 3/4 animals infused with the control IgG, were resistant to SIVmac239 infection. These results suggest that infusion with CD8β255R1 did not eliminate the protection afforded to LASIV vaccination. This provides a comprehensive description of the impact of CD8β255R1 infusion on the immunological composition in cynomolgus macaques, compared to an isotype-matched control IgG, while showing that the control of LASIV viremia and protection from challenge can occur even after CD8β255R1 administration. IMPORTANCE Studies of SIV-infected macaques that deplete CD8(+) T cells in vivo with monoclonal antibodies have provided compelling evidence for their direct antiviral role. These studies utilized CD8α-specific mAbs that target both the major (CD8αβ(+)) and minor (CD8αα(+)) populations of CD8(+) T cells but additionally deplete non-CD8(+) T cell populations that express CD8α, such as NK cells and γδ T cells. In the current study, we administered the CD8β-specific depleting mAb CD8β255R1 to cynomolgus macaques chronically infected with a LASIV to selectively deplete CD8αβ(+) T cells without removing CD8αα(+) lymphocytes. We evaluated the impact on control of virus replication and protection from pathogenic SIVmac239 challenge. These results underscore the utility of CD8β255R1 for studying the direct contribution of CD8αβ(+) T cells in various disease states.

We evaluated the contribution of CD8αβ+ T cells on control of live-attenuated simian immunodeficiency virus (LASIV) replication during chronic infection and subsequent protection from pathogenic SIV challenge. Unlike previous reports with a CD8α-specific depleting monoclonal antibody (mAb), the CD8β-specific mAb CD8β255R1 selectively depleted CD8αβ+ T cells without also depleting non-CD8+ T cell populations that express CD8α, such as natural killer (NK) cells and γδ T cells. Following infusion with CD8β255R1, plasma viremia transiently increased coincident with declining peripheral CD8αβ+ T cells. Interestingly, plasma viremia returned to pre-depletion levels even when peripheral CD8αβ+ T cells did not. Although depletion of CD8αβ+ T cells in the lymph node (LN) was incomplete, frequencies of these cells were three-fold lower (p=0.006) in animals that received CD8β255R1 compared to control IgG. It is possible that these residual SIV-specific CD8αβ+ T cells may have contributed to suppression of viremia during chronic infection. We also determined whether infusion of CD8β255R1 in the LASIV-vaccinated animals increased their susceptibility to infection following intravenous challenge with pathogenic SIVmac239. We found that 7/8 animals infused with CD8β255R1, and 3/4 animals infused with the control IgG, were resistant to SIVmac239 infection. These results suggest that infusion with CD8β255R1 did not eliminate the protection afforded to LASIV vaccination. This provides a comprehensive description of the impact of CD8β255R1 infusion on the immunological composition of the host, when compared to an isotype matched control IgG, while showing that the control of LASIV viremia and protection from challenge can occur even after CD8β255R1 administration.ImportanceStudies of SIV-infected macaques that deplete CD8+ T cellsin vivowith monoclonal antibodies have provided compelling evidence for their direct antiviral role. These studies utilized CD8α-specific mAbs that target both the major (CD8αβ+) and minor (CD8αα+) populations of CD8+ T cells, but additionally deplete non-CD8+ T cell populations that express CD8α, such as NK cells and γδ T cells. In the current study, we administered the CD8β-specific depleting mAb CD8β255R1 to cynomolgus macaques chronically infected with a LASIV to selectively deplete CD8αβ+ T cells without removing CD8αα+ lymphocytes. We evaluated the impact on control of virus replication and protection from pathogenic SIVmac239 challenge. These results underscore the utility of CD8β255R1 for studying the direct contribution of CD8αβ+ T cells in various disease states.

The epitopes defined by HIV-1 broadly neutralizing antibodies (bNAbs) are valuable templates for vaccine design, and studies of the immunological development of these antibodies are providing insights for vaccination strategies. In addition, the most potent and broadly reactive of these bNAbs have potential for clinical use. We previously described a family of 12 V1V2-directed neutralizing antibodies, CAP256-VRC26, isolated from an HIV-1 clade C-infected donor at years 1, 2, and 4 of infection (N. A. Doria-Rose et al., Nature 509:55–62, 2014, http://dx.doi.org/10.1038/nature13036 ). Here, we report on the isolation and characterization of new members of the family mostly obtained at time points of peak serum neutralization breadth and potency. Thirteen antibodies were isolated from B cell culture, and eight were isolated using trimeric envelope probes for differential single B cell sorting. One of the new antibodies displayed a 10-fold greater neutralization potency than previously published lineage members. This antibody, CAP256-VRC26.25, neutralized 57% of diverse clade viral isolates and 70% of clade C isolates with remarkable potency. Among the viruses neutralized, the median 50% inhibitory concentration was 0.001 μg/ml. All 33 lineage members targeted a quaternary epitope focused on V2. While all known bNAbs targeting the V1V2 region interact with the N160 glycan, the CAP256-VRC26 antibodies showed an inverse correlation of neutralization potency with dependence on this glycan. Overall, our results highlight the ongoing evolution within a single antibody lineage and describe more potent and broadly neutralizing members with potential clinical utility, particularly in areas where clade C is prevalent. IMPORTANCE Studies of HIV-1 broadly neutralizing antibodies (bNAbs) provide valuable information for vaccine design, and the most potent and broadly reactive of these bNAbs have potential for clinical use. We previously described a family of V1V2-directed neutralizing antibodies from an HIV-1 clade C-infected donor. Here, we report on the isolation and characterization of new members of the family mostly obtained at time points of peak serum neutralization breadth and potency. One of the new antibodies, CAP256-VRC26.25, displayed a 10-fold greater neutralization potency than previously described lineage members. It neutralized 57% of diverse clade viral isolates and 70% of clade C isolates with remarkable potency: the median 50% inhibitory concentration was 0.001 μg/ml. Our results highlight the ongoing evolution within a single antibody lineage and describe more potent and broadly neutralizing members with potential clinical utility, particularly in areas where clade C is prevalent.

We manipulated SIVmac239Δnef, a model of major histocompatibility complex (MHC)-independent viral control, to evaluate characteristics of effective cellular responses mounted by Mauritian cynomolgus macaques (MCMs) that express the M3 MHC haplotype, which has been associated with poor control of pathogenic simian immunodeficiency virus (SIV). We created SIVΔnef-8x to test the hypothesis that effective SIV-specific T cell responses targeting invariant viral regions can emerge in the absence of immunodominant CD8(+) T cell responses targeting variable epitopes and that control is achievable in individuals lacking known “protective” MHC alleles. Full-proteome gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assays identified six newly targeted immunogenic regions following SIVΔnef-8x infection of M3/M3 MCMs. We deep sequenced circulating virus and found that four of the six newly targeted regions rarely accumulated mutations. Six animals infected with SIVΔnef-8x had T cell responses that targeted at least one of the four invariant regions and had a lower set point viral load than two animals that did not have T cell responses that targeted any invariant regions. We found that MHC class II molecules restricted all four of the invariant peptide regions, while the two variable regions were restricted by MHC class I molecules. Therefore, in the absence of immunodominant CD8(+) T cell responses that target variable regions during SIVmac239Δnef infection, individuals without protective MHC alleles developed predominantly CD4(+) T cell responses specific for invariant regions that may improve control of virus replication. Our results provide some evidence that antiviral CD4(+) T cells during acute SIV infection can contribute to effective viral control and should be considered in strategies to combat HIV infection. IMPORTANCE Studies defining effective cellular immune responses to human immunodeficiency virus (HIV) and SIV have largely focused on a rare population that express specific MHC class I alleles and control virus replication in the absence of antiretroviral treatment. This leaves in question whether similar effective immune responses can be achieved in the larger population. The majority of HIV-infected individuals mount CD8(+) T cell responses that target variable viral regions that accumulate high-frequency escape mutations. Limiting T cell responses to these variable regions and targeting invariant viral regions, similar to observations in rare “elite controllers,” may provide an ideal strategy for the development of effective T cell responses in individuals with diverse MHC genetics. Therefore, it is of paramount importance to determine whether T cell responses can be redirected toward invariant viral regions in individuals without protective MHC alleles and if these responses improve control of virus replication.

Increasing drug resistance and the lack of an effective vaccine are the main factors contributing to Mycobacterium tuberculosis (Mtb) being a major cause of death globally. Despite intensive research efforts, it is not well understood why some individuals control Mtb infection and some others develop active disease. HIV-1 infection is associated with an increased incidence of active tuberculosis, even in virally suppressed individuals. Mucosal-associated invariant T (MAIT) and invariant natural killer T (iNKT) cells are innate T cells that can recognize Mtb-infected cells. Contradicting results regarding the frequency of MAIT cells in latent Mtb infection have been reported. In this confirmatory study, we investigated the frequency, phenotype, and IFNγ production of MAIT and iNKT cells in subjects with latent or active Mtb infection. We found that the frequency of both cell types was increased in subjects with latent Mtb infection compared with uninfected individuals or subjects with active infection. We found no change in the expression of HLA-DR, PD-1, and CCR6, as well as the production of IFNγ by MAIT and iNKT cells, among subjects with latent Mtb infection or uninfected controls. The proportion of CD4− CD8+ MAIT cells in individuals with latent Mtb infection was, however, increased. HIV-1 infection was associated with a loss of MAIT and iNKT cells, and the residual cells had elevated expression of the exhaustion marker PD-1. Altogether, the results suggest a role for MAIT and iNKT cells in immunity against Mtb and show a deleterious impact of HIV-1 infection on those cells.

We manipulated SIVmac239Δnef, a model of MHC-independent viral control, to evaluate characteristics of effective cellular responses mounted by Mauritian cynomolgus macaques (MCMs) who express the M3 MHC haplotype that has been associated with poor control of pathogenic SIV. We created SIVΔnef-8x to test the hypothesis that effective SIV-specific T cell responses targeting invariant viral regions can emerge in the absence of immunodominant CD8+ T cell responses targeting variable epitopes, and that control is achievable in individuals lacking known protective MHC alleles. Full proteome IFNγ ELISPOT assays identified six newly targeted immunogenic regions following SIVΔnef-8x infection of M3/M3 MCMs. We deep sequenced circulating virus and found that four of the six newly targeted regions rarely accumulated mutations. Six animals infected with SIVΔnef-8x targeted at least one of the four invariant regions and had a lower set point viral load compared to two animals that did not target any invariant regions. We found that MHC class II molecules restricted all four of the invariant peptide regions, while the two variable regions were restricted by MHC class I molecules. Therefore, in the absence of immunodominant CD8+ T cell responses that target variable regions during SIVmac239Δnef infection, individuals without ‘protective’ MHC alleles developed predominantly CD4+ T cell responses specific for invariant regions that may improve control of virus replication. Our results provide some evidence that antiviral CD4+ T cells during acute SIV infection can contribute to effective viral control and should be considered in strategies to combat HIV infection.ImportanceStudies defining effective cellular immune responses to human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) have largely focused on a rare population that express specific MHC class I alleles and control virus replication in the absence of antiretroviral treatment. This leaves in question whether similar effective immune responses can be achieved in the larger population. The majority of HIV-infected individuals mount CD8+ T cell responses that target variable viral regions that accumulate high-frequency escape mutations. Limiting T cell responses to these variable regions and targeting invariant viral regions, similar to observations in rare ‘elite controllers’, may provide an ideal strategy for the development of effective T cell responses in individuals with diverse MHC genetics. Therefore, it is paramount to determine whether T cell responses can be redirected towards invariant viral regions in individuals without ‘protective’ MHC alleles and if these responses improve control of virus replication.

Increasing drug resistance and the lack of an effective vaccine are the main factors contributing to Mycobacterium tuberculosis (Mtb) being a major cause of death globally. Despite intensive research efforts, it is not well understood why some individuals control Mtb infection and some others develop active disease. HIV-1 infection is associated with an increased incidence of active tuberculosis, even in virally suppressed individuals. Mucosal-associated invariant T (MAIT) and invariant natural killer T (iNKT) cells are innate T cells that can recognize Mtb-infected cells. Contradicting results regarding the frequency of MAIT cells in latent Mtb infection have been reported. In this confirmatory study, we investigated the frequency, phenotype, and IFNγ production of MAIT and iNKT cells in subjects with latent or active Mtb infection. We found that the frequency of both cell types was increased in subjects with latent Mtb infection compared with uninfected individuals or subjects with active infection. We found no change in the expression of HLA-DR, PD-1, and CCR6, as well as the production of IFNγ by MAIT and iNKT cells, among subjects with latent Mtb infection or uninfected controls. The proportion of CD4− CD8+ MAIT cells in individuals with latent Mtb infection was, however, increased. HIV-1 infection was associated with a loss of MAIT and iNKT cells, and the residual cells had elevated expression of the exhaustion marker PD-1. Altogether, the results suggest a role for MAIT and iNKT cells in immunity against Mtb and show a deleterious impact of HIV-1 infection on those cells.

Computational neutralization fingerprinting, NFP, is an efficient and accurate method for predicting the epitope specificities of polyclonal antibody responses to HIV-1 infection. Here, we present next-generation NFP algorithms that substantially improve prediction accuracy for individual donors and enable serologic analysis for entire cohorts. Specifically, we developed algorithms for: (a) selection of optimized virus neutralization panels for NFP analysis, (b) estimation of NFP prediction confidence for each serum sample, and (c) identification of sera with potentially novel epitope specificities. At the individual donor level, the next-generation NFP algorithms particularly improved the ability to detect multiple epitope specificities in a sample, as confirmed both for computationally simulated polyclonal sera and for samples from HIV-infected donors. Specifically, the next-generation NFP algorithms detected multiple specificities in twice as many samples of simulated sera. Further, unlike the first-generation NFP, the new algorithms were able to detect both of the previously confirmed antibody specificities, VRC01-like and PG9-like, in donor CHAVI 0219. At the cohort level, analysis of ~150 broadly neutralizing HIV-infected donor samples suggested a potential connection between clade of infection and types of elicited epitope specificities. Most notably, while 10E8-like antibodies were observed in infections from different clades, an enrichment of such antibodies was predicted for clade B samples. Ultimately, such large-scale analyses of antibody responses to HIV-1 infection can help guide the design of epitope-specific vaccines that are tailored to take into account the prevalence of infecting clades within a specific geographic region. Overall, the next-generation NFP technology will be an important tool for the analysis of broadly neutralizing polyclonal antibody responses against HIV-1., Author Summary HIV-1 remains a significant global health threat, with no effective vaccine against the virus currently available. Since traditional vaccine design efforts have had limited success, much effort in recent years has focused on gaining a better understanding of the ways select individuals are able to effectively neutralize the virus upon natural infection, and to utilize that knowledge for the design of optimized vaccine candidates. Primary emphasis has been placed on characterizing the antibody arm of the immune system, and specifically on antibodies capable of neutralizing the majority of circulating HIV-1 strains. Various experimental techniques can be applied to map the epitope targets of these antibodies, but more recently, the development of computational methods has provided an efficient and accurate alternative for understanding the complex antibody responses to HIV-1 in a given individual. Here, we present the next generation of this computational technology, and show that these new methods have significantly improved accuracy and confidence, and that they enable the interrogation of biologically important questions that can lead to new insights for the design of an effective vaccine against HIV-1.

One of the goals of HIV-1 vaccine development is the elicitation of neutralizing antibodies against vulnerable regions on the envelope glycoprotein (Env) viral spike. Broadly neutralizing antibodies targeting the Env glycan-V3 region (also called the N332 glycan supersite) have been described previously, with several single lineages each derived from different individual donors. We used a high-throughput B-cell culture method to isolate neutralizing antibodies from an HIV-1-infected donor with high serum neutralization breadth. Clonal relatives from three distinct antibody lineages were isolated. Each of these antibody lineages displayed modest breadth and potency but shared several characteristics with the well-characterized glycan-V3 antibodies, including dependence on glycans N332 and N301, VH4 family gene utilization, a heavy chain complementarity-determining region 2 (CDRH2) insertion, and a longer-than-average CDRH3. In contrast to previously described glycan-V3 antibodies, these antibodies preferentially recognized the native Env trimer compared to monomeric gp120. These data indicate the diversity of antibody specificities that target the glycan-V3 site. The quaternary binding preference of these antibodies suggests that that their elicitation likely requires the presentation of a native-like trimeric Env immunogen. IMPORTANCE Broadly neutralizing antibodies targeting the HIV-1 glycan-V3 region with single lineages from individual donors have been described previously. Here we describe three lineages from a single donor, each of which targets glycan-V3. Unlike previously described glycan-V3 antibodies, these mature antibodies bind preferentially to the native Env trimer and weakly to the gp120 monomer. These data extend our knowledge of the immune response recognition of the N332 supersite region and suggest that the mode of epitope recognition is more complex than previously anticipated.

Few studies have evaluated the impact of the viral challenge route on protection against a heterologous simian immunodeficiency virus (SIV) challenge. We vaccinated seven macaques with a live attenuated SIV that differed from SIVmac239Δnef by 24 amino acids, called m3KOΔnef. All animals were protected from an intrarectal SIVmac239 challenge, whereas only four animals were protected from subsequent intravenous SIVmac239 challenge. These data suggest that immune responses elicited by vaccination with live attenuated SIV in an individual animal can confer protection from intrarectal challenge while remaining insufficient for protection from intravenous challenge. IMPORTANCE Our study is important because we show that vaccinated animals can be protected from a mucosal challenge with a heterologous SIV, but the same animals are not necessarily protected from intravenous challenge with the same virus. This is unique because in most studies, either vaccinated animals are challenged multiple times by the same route or only a single challenge is performed. An individually vaccinated animal is rarely challenged multiple times by different routes, so protection from different challenge routes cannot be measured in the same animal. Our data imply that vaccine-elicited responses in an individual animal may be insufficient for protection from intravenous challenge but may be suitable for protection from a mucosal challenge that better approximates human immunodeficiency virus (HIV) exposure.