Search

Abstract Understanding the nature and extent of non-canonical human leukocyte antigen (HLA) presentation in tumour cells is a priority for target antigen discovery for the development of next generation immunotherapies in cancer. We here employ a de novo mass spectrometric sequencing approach with a refined, MHC-centric analysis strategy to detect non-canonical MHC-associated peptides specific to cancer without any prior knowledge of the target sequence from genomic or RNA sequencing data. Our strategy integrates MHC binding rank, Average local confidence scores, and peptide Retention time prediction for improved de novo candidate Selection; culminating in the machine learning model MARS. We benchmark our model on a large synthetic peptide library dataset and reanalysis of a published dataset of high-quality non-canonical MHC-associated peptide identifications in human cancer. We achieve almost 2-fold improvement for high quality spectral assignments in comparison to de novo sequencing alone with an estimated accuracy of above 85.7% when integrated with a stepwise peptide sequence mapping strategy. Finally, we utilize MARS to detect and validate lncRNA-derived peptides in human cervical tumour resections, demonstrating its suitability to discover novel, immunogenic, non-canonical peptide sequences in primary tumour tissue.

The commonly used antibodies 3D12 and 4D12 recognise the human leukocyte antigen E (HLA-E) protein. These antibodies bind distinct epitopes on HLA-E and differ in their ability to bind alleles of the major histocompatibility complex E (MHC-E) proteins of rhesus and cynomolgus macaques. We confirmed that neither antibody cross-reacts with classical HLA alleles, and used hybrids of different MHC-E alleles to map the regions that are critical for their binding. 3D12 recognises a region on the alpha 3 domain, with its specificity for HLA-E resulting from the amino acids present at three key positions (219, 223 and 224) that are unique to HLA-E, while 4D12 binds to the start of the alpha 2 domain, adjacent to the C terminus of the presented peptide. 3D12 staining is increased by incubation of cells at 27°C, and by addition of the canonical signal sequence peptide presented by HLA-E peptide (VL9, VMAPRTLVL). This suggests that 3D12 may bind peptide-free forms of HLA-E, which would be expected to accumulate at the cell surface when cells are incubated at lower temperatures, as well as HLA-E with peptide. Therefore, additional studies are required to determine exactly what forms of HLA-E can be recognised by 3D12. In contrast, while staining with 4D12 was also increased when cells were incubated at 27°C, it was decreased when the VL9 peptide was added. We conclude that 4D12 preferentially binds to peptide-free HLA-E, and, although not suitable for measuring the total cell surface levels of MHC-E, may putatively identify peptide-receptive forms.

Summary: Human immunodeficiency virus 1 (HIV-1) causes major health burdens worldwide and still lacks curative therapies and vaccines. Circadian rhythms are endogenous daily oscillations that coordinate an organism’s response to its environment and invading pathogens. Peripheral viral loads of HIV-1 infected patients show diurnal variation; however, the underlying mechanisms remain unknown. Here, we demonstrate a role for the cell-intrinsic clock to regulate rhythmic HIV-1 replication in circadian-synchronized systems. Silencing the circadian activator Bmal1 abolishes this phenotype, and we observe BMAL1 binding to the HIV-1 promoter. Importantly, we show differential binding of the nuclear receptors REV-ERB and ROR to the HIV-long terminal repeat at different circadian times, demonstrating a dynamic interplay in time-of-day regulation of HIV-1 transcription. Bioinformatic analysis shows circadian regulation of host factors that control HIV-1 replication, providing an additional mechanism for rhythmic viral replication. This study increases our understanding of the circadian regulation of HIV-1, which can ultimately inform new therapies.

The identification and structural analysis of HLA-E-VL9-targeting antibodies that block a natural killer (NK) cell receptor pathway and regulate NK function in vitro.

Human leukocyte antigen (HLA) genes are the most polymorphic loci in the human genome and code for proteins that play a key role in guiding adaptive immune responses by presenting foreign and self peptides (ligands) to T cells. Each person carries up to 6 HLA class I variants (maternal and paternal copies of HLA-A, HLA-B and HLA-C genes) and also multiple HLA class II variants, which cumulatively define the landscape of peptides presented to T cells. Each HLA variant has its own repertoire of presented peptides with a certain sequence motif which is mainly defined by peptide anchor residues (typically the second and the last positions for HLA class I ligands) forming key interactions with the peptide-binding groove of HLA. In this study, we aimed to characterize HLA binding preferences in terms of molecular functions of presented proteins. To focus on the ligand presentation bias introduced specifically by HLA-peptide interaction we performed large-scale in silico predictions of binding of all peptides from human proteome for a wide range of HLA variants and established which functions are characteristic for proteins that are more or less preferentially presented by different HLA variants using statistical calculations and gene ontology (GO) analysis. We demonstrated marked distinctions between HLA variants in molecular functions of preferentially presented proteins (e.g. some HLA variants preferentially present membrane and receptor proteins, while others – ribosomal and DNA-binding proteins) and reduced presentation of extracellular matrix and collagen proteins by the majority of HLA variants. To explain these observations we demonstrated that HLA preferentially presents proteins enriched in amino acids which are required as anchor residues for the particular HLA variant. Our observations can be extrapolated to explain the protective effect of certain HLA alleles in infectious diseases, and we hypothesize that they can also explain susceptibility to certain autoimmune diseases and cancers. We demonstrate that these differences lead to differential presentation of HIV, influenza virus, SARS-CoV-1 and SARS-CoV-2 proteins by various HLA alleles. Taking into consideration that HLA alleles are inherited in haplotypes, we hypothesized that haplotypes composed of a combination of HLA variants with different presentation preferences should be more advantageous as they allow presenting a larger repertoire of peptides and avoiding holes in immunopeptidome. Indeed, we demonstrated that HLA-A/HLA-B and HLA-A/HLA-C haplotypes which have a high frequency in the human population are comprised of HLA variants that are more distinct in terms of functions of preferentially presented proteins than the control pairs.

Elucidation of broadly neutralizing antibodies (bnAb) is a goal in HIV vaccine development. Here, Bradley et al. show that administration of CTLA-4 blocking antibody with vaccine antigens increases HIV-1 envelope antibody responses in macaques and a bnAb precursor mouse model.

Human immunodeficiency virus type 1 (HIV-1) infection triggers rapid induction of multiple innate cytokines including type I interferons, which play important roles in viral control and disease pathogenesis. The transforming growth factor (TGF)-β superfamily is a pleiotropic innate cytokine family, some members of which (activins and bone morphogenetic proteins (BMPs)) were recently demonstrated to exert antiviral activity against Zika and hepatitis B and C viruses but are poorly studied in HIV-1 infection. Here, we show that TGF-β1 is systemically induced with very rapid kinetics (as early as 1–4 days after viremic spread begins) in acute HIV-1 infection, likely due to release from platelets, and remains upregulated throughout infection. Contrastingly, no substantial systemic upregulation of activins A and B or BMP-2 was observed during acute infection, although plasma activin levels trended to be elevated during chronic infection. HIV-1 triggered production of type I interferons but not TGF-β superfamily cytokines from plasmacytoid dendritic cells (DCs) in vitro, putatively explaining their differing in vivo induction; whilst lipopolysaccharide (but not HIV-1) elicited activin A production from myeloid DCs. These findings underscore the need for better definition of the protective and pathogenic capacity of TGF-β superfamily cytokines, to enable appropriate modulation for therapeutic purposes.

Proteasomes catalyze the degradation of endogenous proteins into oligopeptides, but can concurrently create spliced oligopeptides through ligation of previously non-contiguous peptide fragments. Recent studies have uncovered a formerly unappreciated role for proteasome-catalyzed peptide splicing (PCPS) in the generation of non-genomically templated human leukocyte antigen class I (HLA-I)-bound cis-spliced peptides that can be targeted by CD8+ T cells in cancer and infection. However, the mechanisms defining PCPS reactions are poorly understood. Here, we experimentally define the biochemical constraints of proteasome-catalyzed cis-splicing reactions by examination of in vitro proteasomal digests of a panel of viral- and self-derived polypeptide substrates using a tailored mass-spectrometry-based de novo sequencing workflow. We show that forward and reverse PCPS reactions display unique splicing signatures, defined by preferential fusion of distinct amino acid residues with stringent peptide length distributions, suggesting sequence- and size-dependent accessibility of splice reactants for proteasomal substrate binding pockets. Our data provide the basis for a more informed mechanistic understanding of PCPS that will facilitate future prediction of spliced peptides from protein sequences.

Natural Killer (NK) cells play an important role in antiviral defense and their potent effector function identifies them as key candidates for immunotherapeutic interventions in chronic viral infections. Their remarkable functional agility is achieved by virtue of a wide array of germline-encoded inhibitory and activating receptors ensuring a self-tolerant and tunable repertoire. NK cell diversity is generated by a combination of factors including genetic determinants and infections/environmental factors, which together shape the NK cell pool and functional potential. Recently a genetic polymorphism at position -21 of HLA-B, which influences the supply of HLA-E binding peptides and availability of HLA-E for recognition by the inhibitory NK cell receptor NKG2A, was shown to have a marked influence on NK cell functionality in healthy human cytomegalovirus (HCMV) seronegative Caucasian individuals. In this study, -21 methionine (M)-expressing alleles supplying HLA-E binding peptides were largely poor ligands for inhibitory killer immunoglobulin-like receptors (KIRs), and a bias to NKG2A-mediated education of functionally-potent NK cells was observed. Here, we investigated the effect of this polymorphism on the phenotype and functional capacity of peripheral blood NK cells in a cohort of 36 African individuals with human immunodeficiency virus type 1 (HIV-1)/HCMV co-infection. A similarly profound influence of dimorphism at position -21 of HLA-B on NK cells was not evident in these subjects. They predominantly expressed African specific HLA-B and -C alleles that contribute a distinct supply of NKG2A and KIR ligands, and these genetic differences were compounded by the marked effect of HIV-1/HCMV co-infection on NK cell differentiation. Together, these factors resulted in a lack of correlation of the HLA-B -21 polymorphism with surface abundance of HLA-E and loss of the NK cell functional advantage in subjects with -21M HLA-B alleles. Instead, our data suggest that during HIV/HCMV co-infection exposure of NK cells to an environment that displays altered HLA-E ligands drives adaptive NKG2C+ NK cell expansions influencing effector responses. Increased efforts to understand how NK cells are functionally calibrated to self-HLA during chronic viral infections will pave the way to developing targeted therapeutic interventions to overcome the current barriers to enhancing immune-based antiviral control.

Summary: T follicular helper (Tfh) cells are fundamental for B cell selection and antibody maturation in germinal centers. Circulating Tfh (cTfh) cells constitute a minor proportion of the CD4+ T cells in peripheral blood, but their clonotypic relationship to Tfh populations resident in lymph nodes and the extent to which they differ from non-Tfh CD4+ cells have been unclear. Using donor-matched blood and tonsil samples, we investigate T cell receptor (TCR) sharing between tonsillar Tfh cells and peripheral Tfh and non-Tfh cell populations. TCR transcript sequencing reveals considerable clonal overlap between peripheral and tonsillar Tfh cell subsets as well as a clear distinction between Tfh and non-Tfh cells. Furthermore, influenza-specific cTfh cell clones derived from blood can be found in the repertoire of tonsillar Tfh cells. Therefore, human blood samples can be used to gain insight into the specificity of Tfh responses occurring in lymphoid tissues, provided that cTfh subsets are studied. : CD4+ T follicular helper (Tfh) cells are fundamental for antibody production. Brenna et al. demonstrate extensive repertoire overlap between Tfh populations in human blood and tonsils, whereas non-Tfh repertoires differ profoundly. Therefore, analysis of Tfh but not of total circulating CD4+ T cells can reflect the specificity of lymphoid tissue Tfh cells. Keywords: T follicular helper cells, TCR repertoire, influenza, tonsil, blood

Human leucocyte antigen E (HLA-E) directly engages NK cells but also presents antigen to CD8+ T cells. Here the authors show crystal structures of HLA-E in complex with peptides derived from HIV and Mycobacterium tuberculosis, and describe binding conformations, the positional impact of residues involved and discuss implications for functional presentation to CD8+ T cells.

Anti-retroviral therapy successfully suppresses HIV-1 infection, but fails to provide a cure. During infection Type 1 IFNs normally play an essential role in viral clearance, but in vivo IFN-α only has a modest impact on HIV-1 infection, suggesting its possible targeting by HIV. Here, we report that the HIV protein, Vif, inhibits effective IFN-α signalling via degradation of essential JAK/STAT pathway components. We found that STAT1 and STAT3 are specifically reduced in HEK293T cells expressing Vif and that full length, infectious HIV-1 IIIB strain promotes their degradation in a Vif-dependent manner. HIV-1 IIIB infection of myeloid ThP-1 cells also reduced the IFN-α-mediated induction of the anti-viral gene, ISG15, but not MxA, revealing a functional consequence of this HIV-1-mediated immune evasion strategy. Interestingly, while total STAT levels were not reduced upon in vitro IIIB infection of primary human PBMCs, IFN-α-mediated phosphorylation of STAT1 and STAT3 and ISG induction were starkly reduced, with removal of Vif (IIIBΔVif), partially restoring pSTATs, ISG15 and MxB induction. Similarly, pSTAT1 and pSTAT3 expression and IFN-α-induced ISG15 were reduced in PBMCs from HIV-infected patients, compared to healthy controls. Furthermore, IFN-α pre-treatment of a CEM T lymphoblast cells significantly inhibited HIV infection/replication (measured by cellular p24), only in the absence of Vif (IIIBΔVif), but was unable to suppress full length IIIB infection. When analysing the mechanism by which Vif might target the JAK/STAT pathway, we found Vif interacts with both STAT1 and STAT3, (but not STAT2), and its expression promotes ubiquitination and MG132-sensitive, proteosomal degradation of both proteins. Vif's Elongin-Cullin-SOCS-box binding motif enables the formation of an active E3 ligase complex, which we found to be required for Vif's degradation of STAT1 and STAT3. In fact, the E3 ligase scaffold proteins, Cul5 and Rbx2, were also found to be essential for Vif-mediated proteasomal degradation of STAT1 and STAT3. These results reveal a target for HIV-1-Vif and demonstrate how HIV-1 impairs the anti-viral activity of Type 1 IFNs, possibly explaining why both endogenous and therapeutic IFN-α fail to activate more effective control over HIV infection. Keywords: HIV-1, Type 1 IFNs, JAK/STAT, Viral immune evasion, Proteasomal degradation, ISG

A single HIV-1 variant establishes infection of the host after sexual contact. Identifying the phenotypic characteristics of these Transmitted Founder (T/F) viruses is important to understand the restriction mechanisms during transmission. Langerhans cells (LCs) are the mucosal dendritic cell subset that has been shown to have a protective role in HIV-1 transmission. Immature LCs efficiently capture and degrade HIV-1 via langerin-mediated restriction. Here we have investigated the capacity of T/F HIV-1 strains to infect mucosal Langerhans cells (LCs). Notably, most T/F variants efficiently infected immature LCs derived from skin and vaginal tissue in contrast to chronic HIV-1 laboratory strains. Next we screened a panel of T/F viruses and their matched 6-month consensus sequence viruses. Interestingly most T/F variants infected immature LCs whereas donor-matched 6-month consensus sequence viruses had lost the ability to infect LCs. However, we also identified 6-month consensus sequence viruses that had retained an ability to infect LCs similar to that of the donor-matched T/F virus. Moreover, some T/F viruses and 6-month consensus sequence viruses were unable to infect immature LCs. Further analyses indicated that T/F viruses are less sensitive to langerin-mediated restriction. These data suggest that T/F HIV-1 variants have the ability to infect immature LCs, which will facilitate transmission.

Summary: Densely arranged N-linked glycans shield the HIV-1 envelope (Env) trimer from antibody recognition. Strain-specific breaches in this shield (glycan holes) can be targets of vaccine-induced neutralizing antibodies that lack breadth. To understand the interplay between glycan holes and neutralization breadth in HIV-1 infection, we developed a sequence- and structure-based approach to identify glycan holes for individual Env sequences that are shielded in most M-group viruses. Applying this approach to 12 longitudinally followed individuals, we found that transmitted viruses with more intact glycan shields correlated with development of greater neutralization breadth. Within 2 years, glycan acquisition filled most glycan holes present at transmission, indicating escape from hole-targeting neutralizing antibodies. Glycan hole filling generally preceded the time to first detectable breadth, although time intervals varied across hosts. Thus, completely glycan-shielded viruses were associated with accelerated neutralization breadth development, suggesting that Env immunogens with intact glycan shields may be preferred components of AIDS vaccines. : Wagh et al. show that transmitted viruses with more intact glycan shields are correlated with development of neutralization breadth in HIV-1-infected individuals. This is consistent with previous findings that glycan holes in Env immunogens are targeted by strain-specific neutralizing responses, and suggests that immunogens with intact glycan shields may be advantageous. Keywords: neutralizing antibodies, glycan shield, HIV-1 envelope, transmitted founder, vaccine design

Elucidation of novel peptides presented by human leukocyte antigen (HLA) class I alleles by immunopeptidomics constitutes a powerful approach that can inform the rational design of CD8+ T cell inducing vaccines to control infection with pathogens such as human immunodeficiency virus type 1 (HIV-1) or to combat tumors. Recent advances in the sensitivity of liquid chromatography tandem mass spectrometry instrumentation have facilitated the discovery of thousands of natural HLA-restricted peptides in a single measurement. However, the extent of contamination of class I-bound peptides identified using HLA immunoprecipitation (IP)-based immunopeptidomics approaches with peptides from other sources has not previously been evaluated in depth. Here, we investigated the specificity of the IP-based immunopeptidomics methodology using HLA class I- or II-deficient cell lines and membrane protein-specific antibody IPs. We demonstrate that the 721.221 B lymphoblastoid cell line, widely regarded to be HLA class Ia-deficient, actually expresses and presents peptides on HLA-C*01:02. Using this cell line and the C8166 (HLA class I- and II-expressing) cell line, we show that some HLA class II-bound peptides were co-purified non-specifically during HLA class I and membrane protein IPs. Furthermore, IPs of “irrelevant” membrane proteins from HIV-1-infected HLA class I- and/or II-expressing cells revealed that unusually long HIV-1-derived peptides previously reported by us and other immunopeptidomics studies as potentially novel CD8+ T cell epitopes were non-specifically co-isolated, and so constitute a source of contamination in HLA class I IPs. For example, a 16-mer (FLGKIWPSYKGRPGNF), which was detected in all samples studied represents the full p1 segment of the abundant intracellular or virion-associated proteolytically-processed HIV-1 Gag protein. This result is of importance, as these long co-purified HIV-1 Gag peptides may not elicit CD8+ T cell responses when incorporated into candidate vaccines. These results have wider implications for HLA epitope discovery from abundant or membrane-associated antigens by immunopeptidomics in the context of infectious diseases, cancer, and autoimmunity.

Human cytomegalovirus (HCMV) co-infection is highly prevalent within HIV-1 cohorts and is an important cofactor in driving ongoing immune activation, even during effective antiretroviral treatment. HCMV infection has recently been associated with expansion of adaptive-like natural killer (NK) cells, which harbor epigenetic alterations that impact on their cellular function and phenotype. The influence of HCMV co-infection on the considerable heterogeneity among NK cells and their functional responses to different stimuli was assessed in a cohort of HIV-1-infected individuals sampled during different stages of infection, compared with healthy subjects stratified according to HCMV serostatus. Our data demonstrate a reshaping of the NK cell pool in HIV-1 infection of HCMV-seropositive individuals, with an accentuated peripheral transition of CD56dim NK cells toward a mature CD57+ CD85j+ NKG2C+ NKG2A− phenotype. Lack of PLZF further distinguishes adaptive NK cells from other NK cells expressing CD57 or NKG2C. PLZF− NK cells from HIV-infected individuals had high expression of CD2, were Siglec-7 negative and exhibited downregulation of key signaling molecules, SYK and FcεRI-γ, overwhelmingly displaying features of adaptive NK cells that correlated with HCMV serum Ab levels. Notably this adaptive-like signature was detected during early HIV-1 infection and persisted during treatment. Adaptive-like NK cell subsets in HIV-1-infected individuals displayed enhanced IFN-γ production following Fc receptor triggering compared with their conventional NK cell counterparts, and their ability to produce TNF-α and degranulate was preserved. Together, these data suggest that HMCV infection/reactivation, a hallmark of HIV-1 infection, plays a role in driving a relative expansion of NK cells with adaptive features during HIV-1 infection. The identification of selective NK subsets with retained effector activity in HIV-1-infected subjects raises the possibility of developing therapeutic strategies that exploit specific NK subpopulations to achieve better HIV-1 control.

The original version of this Article contained an error in the spelling of the author Jonah B Sacha, which was incorrectly given as Jonah Sacha. These errors have now been corrected in both the PDF and HTML versions of the Article.

Arenaviruses merit interest both as tractable experimental model systems to study acute and persistent viral infections, and as clinically-important human pathogens. Several arenaviruses cause hemorrhagic fever (HF) disease in humans. In addition, evidence indicates that the globally-distributed prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is a human pathogen of clinical significance in congenital infections, and also poses a great danger to immunosuppressed individuals. Arenavirus persistence and pathogenesis are facilitated by their ability to overcome the host innate immune response. Mammalian hosts have developed both membrane toll-like receptors (TLR) and cytoplasmic pattern recognition receptors (PRRs) that recognize specific pathogen-associated molecular patterns (PAMPs), resulting in activation of the transcription factors IRF3 or IRF7, or both, which together with NF-κB and ATF-2/c-JUN induce production of type I interferon (IFN-I). IFN-I plays a key role in host anti-microbial defense by mediating direct antiviral effects via up-regulation of IFN-I stimulated genes (ISGs), activating dendritic cells (DCs) and natural killer (NK) cells, and promoting the induction of adaptive responses. Accordingly, viruses have developed a plethora of strategies to disrupt the IFN-I mediated antiviral defenses of the host, and the viral gene products responsible for these disruptions are often major virulence determinants.IRF3- and IRF7-dependent induction of host innate immune responses is frequently targeted by viruses. Thus, the arenavirus nucleoprotein (NP) was shown to inhibit the IFN‑I response by interfering with the activation of IRF3. This NP anti-IFN activity, together with alterations in the number and function of DCs observed in mice chronically infected with LCMV, likely play an important role in LCMV persistence in its murine host. In this review we will discuss current knowledge about the cellular and molecular mechanisms by which arenaviruses can subvert the host innate immune response and their implications for understanding HF arenaviral disease as well as arenavirus persistence in their natural hosts.

Heterosexual transmission of HIV-1 is characterized by a genetic bottleneck that selects a single viral variant, the transmitted/founder (TF), during most transmission events. To assess viral characteristics influencing HIV-1 transmission, we sequenced 167 near full-length viral genomes and generated 40 infectious molecular clones (IMC) including TF variants and multiple non-transmitted (NT) HIV-1 subtype C variants from six linked heterosexual transmission pairs near the time of transmission. Consensus-like genomes sensitive to donor antibodies were selected for during transmission in these six transmission pairs. However, TF variants did not demonstrate increased viral fitness in terms of particle infectivity or viral replicative capacity in activated peripheral blood mononuclear cells (PBMC) and monocyte-derived dendritic cells (MDDC). In addition, resistance of the TF variant to the antiviral effects of interferon-α (IFN-α) was not significantly different from that of non-transmitted variants from the same transmission pair. Thus neither in vitro viral replicative capacity nor IFN-α resistance discriminated the transmission potential of viruses in the quasispecies of these chronically infected individuals. However, our findings support the hypothesis that within-host evolution of HIV-1 in response to adaptive immune responses reduces viral transmission potential.

HIV-1 can disseminate between susceptible cells by two mechanisms: cell-free infection following fluid-phase diffusion of virions and by highly-efficient direct cell-to-cell transmission at immune cell contacts. The contribution of this hybrid spreading mechanism, which is also a characteristic of some important computer worm outbreaks, to HIV-1 progression in vivo remains unknown. Here we present a new mathematical model that explicitly incorporates the ability of HIV-1 to use hybrid spreading mechanisms and evaluate the consequences for HIV-1 pathogenenesis. The model captures the major phases of the HIV-1 infection course of a cohort of treatment naive patients and also accurately predicts the results of the Short Pulse Anti-Retroviral Therapy at Seroconversion (SPARTAC) trial. Using this model we find that hybrid spreading is critical to seed and establish infection, and that cell-to-cell spread and increased CD4+ T cell activation are important for HIV-1 progression. Notably, the model predicts that cell-to-cell spread becomes increasingly effective as infection progresses and thus may present a considerable treatment barrier. Deriving predictions of various treatments' influence on HIV-1 progression highlights the importance of earlier intervention and suggests that treatments effectively targeting cell-to-cell HIV-1 spread can delay progression to AIDS. This study suggests that hybrid spreading is a fundamental feature of HIV infection, and provides the mathematical framework incorporating this feature with which to evaluate future therapeutic strategies.

Control of virus replication in HIV-1 infection is critical to delaying disease progression. While cellular immune responses are a key determinant of control, relatively little is known about the contribution of the infecting virus to this process. To gain insight into this interplay between virus and host in viral control, we conducted a detailed analysis of two heterosexual HIV-1 subtype A transmission pairs in which female recipients sharing three HLA class I alleles exhibited contrasting clinical outcomes: R880F controlled virus replication while R463F experienced high viral loads and rapid disease progression. Near full-length single genome amplification defined the infecting transmitted/founder (T/F) virus proteome and subsequent sequence evolution over the first year of infection for both acutely infected recipients. T/F virus replicative capacities were compared in vitro, while the development of the earliest cellular immune response was defined using autologous virus sequence-based peptides. The R880F T/F virus replicated significantly slower in vitro than that transmitted to R463F. While neutralizing antibody responses were similar in both subjects, during acute infection R880F mounted a broad T cell response, the most dominant components of which targeted epitopes from which escape was limited. In contrast, the primary HIV-specific T cell response in R463F was focused on just two epitopes, one of which rapidly escaped. This comprehensive study highlights both the importance of the contribution of the lower replication capacity of the transmitted/founder virus and an associated induction of a broad primary HIV-specific T cell response, which was not undermined by rapid epitope escape, to long-term viral control in HIV-1 infection. It underscores the importance of the earliest CD8 T cell response targeting regions of the virus proteome that cannot mutate without a high fitness cost, further emphasizing the need for vaccines that elicit a breadth of T cell responses to conserved viral epitopes.

Antibodies that neutralize (nAbs) genetically diverse HIV-1 strains have been recovered from a subset of HIV-1 infected subjects during chronic infection. Exact mechanisms that expand the otherwise narrow neutralization capacity observed during early infection are, however, currently undefined. Here we characterized the earliest nAb responses in a subtype A HIV-1 infected Rwandan seroconverter who later developed moderate cross-clade nAb breadth, using (i) envelope (Env) glycoproteins from the transmitted/founder virus and twenty longitudinal nAb escape variants, (ii) longitudinal autologous plasma, and (iii) autologous monoclonal antibodies (mAbs). Initially, nAbs targeted a single region of gp120, which flanked the V3 domain and involved the alpha2 helix. A single amino acid change at one of three positions in this region conferred early escape. One immunoglobulin heavy chain and two light chains recovered from autologous B cells comprised two mAbs, 19.3H-L1 and 19.3H-L3, which neutralized the founder Env along with one or three of the early escape variants carrying these mutations, respectively. Neither mAb neutralized later nAb escape or heterologous Envs. Crystal structures of the antigen-binding fragments (Fabs) revealed flat epitope contact surfaces, where minimal light chain mutation in 19.3H-L3 allowed for additional antigenic interactions. Resistance to mAb neutralization arose in later Envs through alteration of two glycans spatially adjacent to the initial escape signatures. The cross-neutralizing nAbs that ultimately developed failed to target any of the defined V3-proximal changes generated during the first year of infection in this subject. Our data demonstrate that this subject's first recognized nAb epitope elicited strain-specific mAbs, which incrementally acquired autologous breadth, and directed later B cell responses to target distinct portions of Env. This immune re-focusing could have triggered the evolution of cross-clade antibodies and suggests that exposure to a specific sequence of immune escape variants might promote broad humoral responses during HIV-1 infection.

A genome-wide screen for large structural variants showed that a copy number variant (CNV) in the region encoding killer cell immunoglobulin-like receptors (KIR) associates with HIV-1 control as measured by plasma viral load at set point in individuals of European ancestry. This CNV encompasses the KIR3DL1-KIR3DS1 locus, encoding receptors that interact with specific HLA-Bw4 molecules to regulate the activation of lymphocyte subsets including natural killer (NK) cells. We quantified the number of copies of KIR3DS1 and KIR3DL1 in a large HIV-1 positive cohort, and showed that an increase in KIR3DS1 count associates with a lower viral set point if its putative ligand is present (p = 0.00028), as does an increase in KIR3DL1 count in the presence of KIR3DS1 and appropriate ligands for both receptors (p = 0.0015). We further provide functional data that demonstrate that NK cells from individuals with multiple copies of KIR3DL1, in the presence of KIR3DS1 and the appropriate ligands, inhibit HIV-1 replication more robustly, and associated with a significant expansion in the frequency of KIR3DS1+, but not KIR3DL1+, NK cells in their peripheral blood. Our results suggest that the relative amounts of these activating and inhibitory KIR play a role in regulating the peripheral expansion of highly antiviral KIR3DS1+ NK cells, which may determine differences in HIV-1 control following infection.

Acute HIV infection (AHI) is a critical phase of infection when irreparable damage to the immune system occurs and subjects are very infectious. We studied subjects with AHI prospectively to develop better treatment and public health interventions.Cross-sectional screening was employed to detect HIV RNA positive, antibody negative subjects. Date of HIV acquisition was estimated from clinical history and correlated with sequence diversity assessed by single genome amplification (SGA). Twenty-two cytokines/chemokines were measured from enrollment through week 24.Thirty-seven AHI subjects were studied. In 7 participants with limited exposure windows, the median exposure to HIV occurred 14 days before symptom onset. Lack of viral sequence diversification confirmed the short duration of infection. Transmission dates estimated by SGA/sequencing using molecular clock models correlated with transmission dates estimated by symptom onset in individuals infected with single HIV variants (mean of 28 versus 33 days). Only 10 of 22 cytokines/chemokines were significantly elevated among AHI participants at enrollment compared to uninfected controls, and only 4 participants remained seronegative at enrollment.The results emphasize the difficulty in recruiting subjects early in AHI. Viral sequence diversity proved accurate in estimating time of infection. Regardless of aggressive screening, peak viremia and inflammation occurred before enrollment and potential intervention. Given the personal and public health importance, improved AHI detection is urgently needed.

In the present study, we analyzed the functional profile of CD8+ T-cell responses directed against autologous transmitted/founder HIV-1 isolates during acute and early infection, and examined whether multifunctionality is required for selection of virus escape mutations. Seven anti-retroviral therapy-naïve subjects were studied in detail between 1 and 87 weeks following onset of symptoms of acute HIV-1 infection. Synthetic peptides representing the autologous transmitted/founder HIV-1 sequences were used in multiparameter flow cytometry assays to determine the functionality of HIV-1-specific CD8+ T memory cells. In all seven patients, the earliest T cell responses were predominantly oligofunctional, although the relative contribution of multifunctional cell responses increased significantly with time from infection. Interestingly, only the magnitude of the total and not of the poly-functional T-cell responses was significantly associated with the selection of escape mutants. However, the high contribution of MIP-1β-producing CD8+ T-cells to the total response suggests that mechanisms not limited to cytotoxicity could be exerting immune pressure during acute infection. Lastly, we show that epitope entropy, reflecting the capacity of the epitope to tolerate mutational change and defined as the diversity of epitope sequences at the population level, was also correlated with rate of emergence of escape mutants.

The risk of postnatal HIV transmission is associated with the magnitude of the milk virus load. While HIV-specific cellular immune responses control systemic virus load and are detectable in milk, the contribution of these responses to the control of virus load in milk is unknown.We assessed the magnitude of the immunodominant GagRY11 and subdominant EnvKY9-specific CD8+ T lymphocyte response in blood and milk of 10 A*3002+, HIV-infected Malawian women throughout the period of lactation and correlated this response to milk virus RNA load and markers of breast inflammation.The magnitude and kinetics of the HIV-specific CD8+ T lymphocyte responses were discordant in blood and milk of the right and left breast, indicating independent regulation of these responses in each breast. However, there was no correlation between the magnitude of the HIV-specific CD8+ T lymphocyte response and the milk virus RNA load. Further, there was no correlation between the magnitude of this response and markers of breast inflammation.The magnitude of the HIV-specific CD8+ T lymphocyte response in milk does not appear to be solely determined by the milk virus RNA load and is likely only one of the factors contributing to maintenance of low virus load in milk.

HIV-1 is present in anatomical compartments and bodily fluids. Most transmissions occur through sexual acts, making virus in semen the proximal source in male donors. We find three distinct relationships in comparing viral RNA populations between blood and semen in men with chronic HIV-1 infection, and we propose that the viral populations in semen arise by multiple mechanisms including: direct import of virus, oligoclonal amplification within the seminal tract, or compartmentalization. In addition, we find significant enrichment of six out of nineteen cytokines and chemokines in semen of both HIV-infected and uninfected men, and another seven further enriched in infected individuals. The enrichment of cytokines involved in innate immunity in the seminal tract, complemented with chemokines in infected men, creates an environment conducive to T cell activation and viral replication. These studies define different relationships between virus in blood and semen that can significantly alter the composition of the viral population at the source that is most proximal to the transmitted virus.

We used ultra-deep sequencing to obtain tens of thousands of HIV-1 sequences from regions targeted by CD8+ T lymphocytes from longitudinal samples from three acutely infected subjects, and modeled viral evolution during the critical first weeks of infection. Previous studies suggested that a single virus established productive infection, but these conclusions were tempered because of limited sampling; now, we have greatly increased our confidence in this observation through modeling the observed earliest sample diversity based on vastly more extensive sampling. Conventional sequencing of HIV-1 from acute/early infection has shown different patterns of escape at different epitopes; we investigated the earliest escapes in exquisite detail. Over 3-6 weeks, ultradeep sequencing revealed that the virus explored an extraordinary array of potential escape routes in the process of evading the earliest CD8 T-lymphocyte responses--using 454 sequencing, we identified over 50 variant forms of each targeted epitope during early immune escape, while only 2-7 variants were detected in the same samples via conventional sequencing. In contrast to the diversity seen within epitopes, non-epitope regions, including the Envelope V3 region, which was sequenced as a control in each subject, displayed very low levels of variation. In early infection, in the regions sequenced, the consensus forms did not have a fitness advantage large enough to trigger reversion to consensus amino acids in the absence of immune pressure. In one subject, a genetic bottleneck was observed, with extensive diversity at the second time point narrowing to two dominant escape forms by the third time point, all within two months of infection. Traces of immune escape were observed in the earliest samples, suggesting that immune pressure is present and effective earlier than previously reported; quantifying the loss rate of the founder virus suggests a direct role for CD8 T-lymphocyte responses in viral containment after peak viremia. Dramatic shifts in the frequencies of epitope variants during the first weeks of infection revealed a complex interplay between viral fitness and immune escape.

The earliest immune responses activated in acute human immunodeficiency virus type 1 infection (AHI) exert a critical influence on subsequent virus spread or containment. During this time frame, components of the innate immune system such as macrophages and DCs, NK cells, beta-defensins, complement and other anti-microbial factors, which have all been implicated in modulating HIV infection, may play particularly important roles. A proteomics-based screen was performed on a cohort from whom samples were available at time points prior to the earliest positive HIV detection. The ability of selected factors found to be elevated in the plasma during AHI to inhibit HIV-1 replication was analyzed using in vitro PBMC and DC infection models. Analysis of unique plasma donor panels spanning the eclipse and viral expansion phases revealed very early alterations in plasma proteins in AHI. Induction of acute phase protein serum amyloid A (A-SAA) occurred as early as 5-7 days prior to the first detection of plasma viral RNA, considerably prior to any elevation in systemic cytokine levels. Furthermore, a proteolytic fragment of alpha-1-antitrypsin (AAT), termed virus inhibitory peptide (VIRIP), was observed in plasma coincident with viremia. Both A-SAA and VIRIP have anti-viral activity in vitro and quantitation of their plasma levels indicated that circulating concentrations are likely to be within the range of their inhibitory activity. Our results provide evidence for a first wave of host anti-viral defense occurring in the eclipse phase of AHI prior to systemic activation of other immune responses. Insights gained into the mechanism of action of acute-phase reactants and other innate molecules against HIV and how they are induced could be exploited for the future development of more efficient prophylactic vaccine strategies.

Predictive models of peptide-Major Histocompatibility Complex (MHC) binding affinity are important components of modern computational immunovaccinology. Here, we describe the development and deployment of a reliable peptide-binding prediction method for a previously poorly-characterized human MHC class I allele, HLA-Cw*0102.Using an in-house, flow cytometry-based MHC stabilization assay we generated novel peptide binding data, from which we derived a precise two-dimensional quantitative structure-activity relationship (2D-QSAR) binding model. This allowed us to explore the peptide specificity of HLA-Cw*0102 molecule in detail. We used this model to design peptides optimized for HLA-Cw*0102-binding. Experimental analysis showed these peptides to have high binding affinities for the HLA-Cw*0102 molecule. As a functional validation of our approach, we also predicted HLA-Cw*0102-binding peptides within the HIV-1 genome, identifying a set of potent binding peptides. The most affine of these binding peptides was subsequently determined to be an epitope recognized in a subset of HLA-Cw*0102-positive individuals chronically infected with HIV-1.A functionally-validated in silico-in vitro approach to the reliable and efficient prediction of peptide binding to a previously uncharacterized human MHC allele HLA-Cw*0102 was developed. This technique is generally applicable to all T cell epitope identification problems in immunology and vaccinology.