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Although 15–20% of COVID‐19 patients experience hyper‐inflammation induced by massive cytokine production, cellular triggers of this process and strategies to target them remain poorly understood. Here, we show that the N‐terminal domain (NTD) of the SARS‐CoV‐2 spike protein substantially induces multiple inflammatory molecules in myeloid cells and human PBMCs. Using a combination of phenotypic screening with machine learning‐based modeling, we identified and experimentally validated several protein kinases, including JAK1, EPHA7, IRAK1, MAPK12, and MAP3K8, as essential downstream mediators of NTD‐induced cytokine production, implicating the role of multiple signaling pathways in cytokine release. Further, we found several FDA‐approved drugs, including ponatinib, and cobimetinib as potent inhibitors of the NTD‐mediated cytokine release. Treatment with ponatinib outperforms other drugs, including dexamethasone and baricitinib, inhibiting all cytokines in response to the NTD from SARS‐CoV‐2 and emerging variants. Finally, ponatinib treatment inhibits lipopolysaccharide‐mediated cytokine release in myeloid cells in vitro and lung inflammation mouse model. Together, we propose that agents targeting multiple kinases required for SARS‐CoV‐2‐mediated cytokine release, such as ponatinib, may represent an attractive therapeutic option for treating moderate to severe COVID‐19., Massive cytokine production and hyper‐inflammation have been associated with fatal SARS‐CoV‐2 infection outcomes. Phenotypic screening combined with machine learning‐based modeling identifies regulators and therapeutics for targeting SARS‐CoV‐2‐induced cytokine release.

Animal model studies highlight the role of innate-like lymphocyte populations in the early inflammatory response and subsequent parasite control following Plasmodium infection. IFN-γ production by these lymphocytes likely plays a key role in the early control of the parasite and disease severity. Analyzing human innate-like T cell and NK cell responses following infection with Plasmodium has been challenging because the early stages of infection are clinically silent. To overcome this limitation, we examined blood samples from a controlled human malaria infection (CHMI) study in a Tanzanian cohort, in which volunteers underwent CHMI with a low or high dose of Plasmodium falciparum sporozoites. The CHMI differentially affected NK, NKT (invariant NKT), and mucosal-associated invariant T cell populations in a dose-dependent manner, resulting in an altered composition of this innate-like lymphocyte compartment. Although these innate-like responses are typically thought of as short-lived, we found that changes persisted for months after the infection was cleared, leading to significantly increased frequencies of mucosal-associated invariant T cells 6 mo postinfection. We used single-cell RNA sequencing and TCR αβ-chain usage analysis to define potential mechanisms for this expansion. These single-cell data suggest that this increase was mediated by homeostatic expansion-like mechanisms. Together, these data demonstrate that CHMI leads to previously unappreciated long-lasting alterations in the human innate-like lymphocyte compartment. We discuss the consequences of these changes for recurrent parasite infection and infection-associated pathologies and highlight the importance of considering host immunity and infection history for vaccine design.

Conventional memory CD8+ T cells and mucosal-associated invariant T cells (MAIT cells) are found in blood, liver, and mucosal tissues and have similar effector potential following activation, specifically expression of IFN-γ and granzyme B. To better understand each subset’s unique contributions to immunity and pathology, we interrogated inflammation- and TCR-driven activation requirements using human memory CD8+ T and MAIT cells isolated from blood and mucosal tissue biopsies in ex vivo functional assays and single cell gene expression experiments. We found that MAIT cells had a robust IFN-γ and granzyme B response to inflammatory signals but limited responsiveness when stimulated directly via their TCR. Importantly, this is not due to an overall hyporesponsiveness to TCR signals. When delivered together, TCR and inflammatory signals synergize to elicit potent effector function in MAIT cells. This unique control of effector function allows MAIT cells to respond to the same TCR signal in a dichotomous and situation-specific manner. We propose that this could serve to prevent responses to antigen in noninflamed healthy mucosal tissue, while maintaining responsiveness and great sensitivity to inflammation-eliciting infections. We discuss the implications of these findings in context of inflammation-inducing damage to tissues such as BM transplant conditioning or HIV infection.

HIV has evolved sophisticated mechanisms to avoid restriction by intracellular innate immune defenses that otherwise serve to control acute viral infection and virus dissemination. Innate defenses are triggered when pattern recognition receptor (PRR) proteins of the host cell engage pathogen-associated molecule patterns (PAMPs) present in viral products. Interferon regulatory factor 3 (IRF3) plays a central role in PRR signaling of innate immunity to drive the expression of type I interferon (IFN) and interferon-stimulated genes (ISGs), including a variety of HIV restriction factors, that serve to limit viral replication directly and/or program adaptive immunity. Productive infection of T cells by HIV is dependent upon the targeted proteolysis of IRF3 that occurs through a virus-directed mechanism that results in suppression of innate immune defenses. However, the mechanisms by which HIV controls innate immune signaling and IRF3 function are not defined. Here, we examined the innate immune response induced by HIV strains identified through their differential control of PRR signaling. We identified viruses that, unlike typical circulating HIV strains, lack the ability to degrade IRF3. Our studies show that IRF3 regulation maps specifically to the HIV accessory protein Vpu. We define a molecular interaction between Vpu and IRF3 that redirects IRF3 to the endolysosome for proteolytic degradation, thus allowing HIV to avoid the innate antiviral immune response. Our studies reveal that Vpu is an important IRF3 regulator that supports acute HIV infection through innate immune suppression. These observations define the Vpu-IRF3 interface as a novel target for therapeutic strategies aimed at enhancing the immune response to HIV.

The RNA helicase DDX6 promotes HIV-1 assembly in a co-opted cellular complex containing P body proteins and ABCE1., To produce progeny virus, human immunodeficiency virus type I (HIV-1) Gag assembles into capsids that package the viral genome and bud from the infected cell. During assembly of immature capsids, Gag traffics through a pathway of assembly intermediates (AIs) that contain the cellular adenosine triphosphatase ABCE1 (ATP-binding cassette protein E1). In this paper, we showed by coimmunoprecipitation and immunoelectron microscopy (IEM) that these Gag-containing AIs also contain endogenous processing body (PB)–related proteins, including AGO2 and the ribonucleic acid (RNA) helicase DDX6. Moreover, we found a similar complex containing ABCE1 and PB proteins in uninfected cells. Additionally, knockdown and rescue studies demonstrated that the RNA helicase DDX6 acts enzymatically to facilitate capsid assembly independent of RNA packaging. Using IEM, we localized the defect in DDX6-depleted cells to Gag multimerization at the plasma membrane. We also confirmed that DDX6 depletion reduces production of infectious HIV-1 from primary human T cells. Thus, we propose that assembling HIV-1 co-opts a preexisting host complex containing cellular facilitators such as DDX6, which the virus uses to catalyze capsid assembly.

In HIV-1-infected individuals, G-to-A hypermutation is found in HIV-1 DNA isolated from peripheral blood mononuclear cells (PBMCs). These mutations are thought to result from editing by one or more host enzymes in the APOBEC3 (A3) family of cytidine deaminases, which act on CC (APOBEC3G) and TC (other A3 proteins) dinucleotide motifs in DNA (edited cytidine underlined). Although many A3 proteins display high levels of deaminase activity in model systems, only low levels of A3 deaminase activity have been found in primary cells examined to date. In contrast, here we report high levels of deaminase activity at TC motifs when whole PBMCs or isolated primary monocyte-derived cells were treated with interferon-alpha (IFNalpha) or IFNalpha-inducing toll-like receptor ligands. Induction of TC-specific deaminase activity required new transcription and translation and correlated with the appearance of two APOBEC3A (A3A) isoforms. Knockdown of A3A in monocytes with siRNA abolished TC-specific deaminase activity, confirming that A3A isoforms are responsible for all TC-specific deaminase activity observed. Both A3A isoforms appear to be enzymatically active; moreover, our mutational studies raise the possibility that the smaller isoform results from internal translational initiation. In contrast to the high levels of TC-specific activity observed in IFNalpha-treated monocytes, CC-specific activity remained low in PBMCs, suggesting that A3G deaminase activity is relatively inhibited, unlike that of A3A. Together, these findings suggest that deaminase activity of A3A isoforms in monocytes and macrophages may play an important role in host defense against viruses.

Interferon regulatory factor 3 (IRF-3) is essential for innate intracellular immune defenses that limit virus replication, but these defenses fail to suppress human immunodeficiency virus (HIV) infection, which can ultimately associate with opportunistic coinfections and the progression to AIDS. Here, we examined antiviral defenses in CD4+cells during virus infection and coinfection, revealing that HIV type 1 (HIV-1) directs a global disruption of innate immune signaling and supports a coinfection model through suppression of IRF-3. T cells responded to paramyxovirus infection to activate IRF-3 and interferon-stimulated gene expression, but they failed to mount a response against HIV-1. The lack of response associated with a marked depletion of IRF-3 but not IRF-7 in HIV-1-infected cells, which supported robust viral replication, whereas ectopic expression of active IRF-3 suppressed HIV-1 infection. IRF-3 depletion was dependent on a productive HIV-1 replication cycle and caused the specific disruption of Toll-like receptor and RIG-I-like receptor innate immune signaling that rendered cells permissive to secondary virus infection. IRF-3 levels were reduced in vivo within CD4+T cells from patients with acute HIV-1 infection but not from long-term nonprogressors. Our results indicate that viral suppression of IRF-3 promotes HIV-1 infection by disrupting IRF-3-dependent signaling pathways and innate antiviral defenses of the host cell. IRF-3 may direct an innate antiviral response that regulates HIV-1 replication and viral set point while governing susceptibility to opportunistic virus coinfections.

CD8+cytotoxic T lymphocytes (CTL) are strong mediators of human immunodeficiency virus type 1 (HIV-1) control, yet HIV-1 frequently mutates to escape CTL recognition. In an analysis of sequences in the Los Alamos HIV-1 database, we show that emerging CTL escape mutations were more often present at lower frequencies than the amino acid(s) that they replaced. Furthermore, epitopes that underwent escape contained amino acid sites of high variability, whereas epitopes persisting at high frequencies lacked highly variable sites. We therefore infer that escape mutations are likely to be associated with weak functional constraints on the viral protein. This was supported by an extensive analysis of one subject for whom all escape mutations within defined CTL epitopes were studied and by an analysis of all reported escape mutations of defined CTL epitopes in the HIV Immunology Database. In one of these defined epitopes, escape mutations involving the substitution of amino acids with lower database frequencies occurred, and the epitope soon reverted back to the sensitive form. We further show that this escape mutation substantially diminished viral fitness in in vitro competition assays. Coincident with the reversion in vivo, we observed the fixation of a mutation 3 amino acids C terminal to the epitope, coincident with the ablation of the corresponding CTL response. The C-terminal mutation did not restore replication fitness reduced by the escape mutation in the epitope and by itself had little effect on replication fitness. Therefore, this C-terminal mutation presumably impaired the processing and presentation of the epitope. Finally, for one persistent epitope, CTL cross-reactivity to a mutant form may have suppressed the mutant to undetected levels, whereas for two other persistent epitopes, each of two mutants showed poor cross-reactivity and appeared in the subject at later time points. Thus, a viral dynamic exists between the advantage of immune escape, peptide cross-reactivity, and the disadvantage of lost replication fitness, with the balance playing an important role in determining whether a CTL epitope will persist or decline during infection.

Human immunodeficiency virus type 1 (HIV-1)-specific CD8+T cells provide an important defense in controlling HIV-1 replication, particularly following acquisition of infection. To delineate the breadth and potency of these responses in patients upon initial presentation and before treatment, we determined the fine specificities and frequencies of gamma interferon (IFN-γ)-secreting CD8+T cells recognizing all HIV-1 proteins in patients with primary infection. In these subjects, the earliest detected responses were directed predominantly against Nef, Tat, Vpr, and Env. Tat- and Vpr-specific CD8+T cells accounted for the greatest frequencies of mean IFN-γ spot-forming cells (SFC). Nef-specific responses (10 of 21) were more commonly detected. A mean of 2.3 epitopes were recognized with various avidities per subject, and the number increased with the duration of infection (R= 0.47,P= 0.031). The mean frequency of CD8+T cells (985 SFC/106peripheral blood mononuclear cells) correlated with the number of epitopes recognized (R= 0.84,P< 0.0001) and the number of HLA-restricting alleles (R= 0.79,P< 0.0001). Neither the total SFC frequencies nor the number of epitopes recognized correlated with the concurrent plasma viral load. Seventeen novel epitopes were identified, four of which were restricted to HLA alleles (A23 and B72) that are common among African descendents. Thus, primary HIV-1 infection induces strong CD8+-T-cell immunity whose specificities broaden over time, but their frequencies and breadth do not correlate with HIV-1 containment when examined concurrently. Many novel epitopes, particularly directed to Nef, Tat, and Env, and frequently with unique HLA restrictions, merit further consideration in vaccine design.

HLA-F is expressed as a protein independent of bound peptide or β2-microglobulin and surface expression is upregulated upon activation in dendritic cells, monocytes and most lymphocyte subsets. MHC class I (MHCI) is also expressed on proliferating lymphoid cells as open conformers (OCs), in addition to the ubiquitously expressed complexed form. Previous studies showed that HLA-F binds most MHCI proteins as open conformers without peptide but not as peptide bound complex. These studies were extended to show both HLA-F and MHCI OC are ligands for a subset of killer Ig-like receptors (KIRs), defining a new paradigm for MHCI function in the innate and acquired immune responses. The HLA-F/MHCI physical interaction was further implicated in the function of HLA-F and MHCI OCs in a general mode of exogenous MHCI antigen uptake and antigen presentation by activated immune cells that differs from the canonical MHCI endogenous antigen presentation. We are currently testing the hypothesis that antigen entry is governed by a synergy between specific structural characteristics of the exogenous antigen and the MHC-I allele types of target cells. New evidence is presented using HIV-1 p24 gag derived long polypeptides suggesting a requirement for HLA class I peptide epitope specificity in antigen uptake, upstream of antigen presentation, which subsequently can be presented by either or both of MHCI and MHCII. These experiments suggested that the physical proximity of HIV-1 class I and class II peptide epitopes within a p24 polypeptide can influence antigen presentation of epitopes through either MHCI or MHCII or both. A goal is to manipulate these features in designing effective immunogens for directed stimulation of antigen-specific host responses.

The RV144 HIV-1 vaccine trial demonstrated partial efficacy of 31% against HIV-1 infection. Studies into possible correlates of protection found that antibodies specific to the V1 and V2 (V1/V2) region of envelope correlated inversely with infection risk and that viruses isolated from trial participants contained genetic signatures of vaccine-induced pressure in the V1/V2 region. We explored the hypothesis that the genetic signatures in V1 and V2 could be partly attributed to selection by vaccine-primed T cells. We performed a T-cell-based sieve analysis of breakthrough viruses in the RV144 trial and found evidence of predicted HLA binding escape that was greater in vaccine versus placebo recipients. The predicted escape depended on class I HLA A*02- and A*11-restricted epitopes in the MN strain rgp120 vaccine immunogen. Though we hypothesized that this was indicative of postacquisition selection pressure, we also found that vaccine efficacy (VE) was greater in A*02-positive (A*02 + ) participants than in A*02 − participants (VE = 54% versus 3%, P = 0.05). Vaccine efficacy against viruses with a lysine residue at site 169, important to antibody binding and implicated in vaccine-induced immune pressure, was also greater in A*02 + participants (VE = 74% versus 15%, P = 0.02). Additionally, a reanalysis of vaccine-induced immune responses that focused on those that were shown to correlate with infection risk suggested that the humoral responses may have differed in A*02 + participants. These exploratory and hypothesis-generating analyses indicate there may be an association between a class I HLA allele and vaccine efficacy, highlighting the importance of considering HLA alleles and host immune genetics in HIV vaccine trials. IMPORTANCE The RV144 trial was the first to show efficacy against HIV-1 infection. Subsequently, much effort has been directed toward understanding the mechanisms of protection. Here, we conducted a T-cell-based sieve analysis, which compared the genetic sequences of viruses isolated from infected vaccine and placebo recipients. Though we hypothesized that the observed sieve effect indicated postacquisition T-cell selection, we also found that vaccine efficacy was greater for participants who expressed HLA A*02, an allele implicated in the sieve analysis. Though HLA alleles have been associated with disease progression and viral load in HIV-1 infection, these data are the first to suggest the association of a class I HLA allele and vaccine efficacy. While these statistical analyses do not provide mechanistic evidence of protection in RV144, they generate testable hypotheses for the HIV vaccine community and they highlight the importance of assessing the impact of host immune genetics in vaccine-induced immunity and protection. (This study has been registered at ClinicalTrials.gov under registration no. NCT00223080.)

Mucosal-associated invariant T (MAIT) cells recognize bacterial metabolites presented on MR1 via their semi-invariant T cell receptor (TCR). MAIT cells are a potent effector population located in tissues that are constantly exposed to the microbiome. This raises the question how MAIT cell activation is regulated to avoid unwanted responses to microbiome-derived metabolites. To address this we dissected the MAIT cell response to TCR- and cytokine-mediated signals. We found that stimulating the MAIT TCR in the absence of inflammatory cytokines was not sufficient to induce effector function such as granzyme B and interferon-g expression. Inflammatory signals acting directly on MAIT cells were absolutely necessary to allow for TCR-mediated acquisition of effector function. Importantly, MAIT cells are not generally hyporesponsive to TCR-mediated signals as TCR stimulation in the absence of inflammation elicited a distinct chemokine response. Single-cell gene expression analysis of MAIT cells isolated from healthy human mucosal tissue revealed increased expression of inflammatory genes compared to their counterparts in the blood, indicating that provided the proper signals mucosal MAIT cells are poised to respond rapidly to infection. Importantly, MAIT cells in the mucosa do not produce inflammatory cytokines directly ex-vivo, despite expressing higher levels of inflammatory transcript. We propose a novel model in which the strict dependence of TCR-mediated signals on pro-inflammatory cytokines to induce effector function represents a MAIT cell-intrinsic mechanism to prevent unwanted responses to commensal bacteria-derived bacterial metabolites, while allowing potent effector responses following infection.

HLA-F is expressed as a protein independent of bound peptide or b2-microglobulin and surface expression is upregulated in dendritic cells, monocytes and most lymphocyte subsets upon activation. Classical MHC class I (MHCI) is also expressed on proliferating lymphoid cells as so-called ‘open conformers (OCs)’, in addition to the ubiquitously expressed form complexed with peptide and b2-microglobulin. Previous studies showed that HLA-F binds most MHCI proteins as open conformers without peptide but not as peptide bound complex. These studies were extended to show that both HLA-F and MHCI OC are ligands for a specific subset of killer Ig-like receptors (KIRs). The HLA-F/MHCI physical interaction was further implicated in the function of HLA-F and MHCI open conformers in a general mode of exogenous MHCI antigen uptake and antigen presentation by activated immune cells that differs from the canonical MHCI endogenous antigen presentation. We are currently testing the hypothesis that antigen entry is governed by a synergy between specific structural characteristics of the exogenous antigen and the MHC-I allele types of target cells. New evidence is presented using HIV-1 p24 gag derived long polypeptides suggesting a requirement for HLA class I peptide epitope specificity in antigen uptake, upstream of antigen presentation, which subsequently can be presented by either or both of MHCI and MHCII. These experiments suggested that the physical proximity of HIV-1 class I and class II peptide epitopes within a p24 polypeptide can govern antigen presentation of epitopes through either MHCI or MHCII or both. A goal is to manipulate these features in designing effective immunogens for directed stimulation of antigen-specific host responses.

Acute virus infection induces a cell-intrinsic innate immune response comprising our first line of immunity to limit virus replication and spread, but viruses have developed strategies to overcome these defenses. HIV-1 is a major public health problem; however, the virus-host interactions that regulate innate immune defenses against HIV-1 are not fully defined. We have recently identified the viral protein Vpu to be a key determinant responsible for HIV-1 targeting and degradation of interferon regulatory factor 3 (IRF3), a central transcription factor driving host cell innate immunity. IRF3 plays a major role in pathogen recognition receptor (PRR) signaling of innate immunity to drive the expression of type I interferon (IFN) and interferon-stimulated genes (ISGs), including a variety of HIV restriction factors, that serve to limit viral replication directly and/or program adaptive immunity. Here we interrogate the cellular responses to target cell infection with Vpu-deficient HIV-1 strains. Remarkably, in the absence of Vpu, HIV-1 triggers a potent intracellular innate immune response that suppresses infection. Thus, HIV-1 can be recognized by PRRs within the host cell to trigger an innate immune response, and this response is unmasked only in the absence of Vpu. Vpu modulation of IRF3 therefore prevents virus induction of specific innate defense programs that could otherwise limit infection. These observations show that HIV-1 can indeed be recognized as a pathogen in infected cells and provide a novel and effective platform for defining the native innate immune programs of target cells of HIV-1 infection.

HIV-1 transmission and viral evolution in the first year of infection were studied in 11 individuals representing four transmitter-recipient pairs and three independent seroconverters. Nine of these individuals were enrolled during acute infection; all were men who have sex with men (MSM) infected with HIV-1 subtype B. A total of 475 nearly full-length HIV-1 genome sequences were generated, representing on average 10 genomes per specimen at 2 to 12 visits over the first year of infection. Single founding variants with nearly homogeneous viral populations were detected in eight of the nine individuals who were enrolled during acute HIV-1 infection. Restriction to a single founder variant was not due to a lack of diversity in the transmitter as homogeneous populations were found in recipients from transmitters with chronic infection. Mutational patterns indicative of rapid viral population growth dominated during the first 5 weeks of infection and included a slight contraction of viral genetic diversity over the first 20 to 40 days. Subsequently, selection dominated, most markedly in env and nef . Mutants were detected in the first week and became consensus as early as day 21 after the onset of symptoms of primary HIV infection. We found multiple indications of cytotoxic T lymphocyte (CTL) escape mutations while reversions appeared limited. Putative escape mutations were often rapidly replaced with mutually exclusive mutations nearby, indicating the existence of a maturational escape process, possibly in adaptation to viral fitness constraints or to immune responses against new variants. We showed that establishment of HIV-1 infection is likely due to a biological mechanism that restricts transmission rather than to early adaptive evolution during acute infection. Furthermore, the diversity of HIV strains coupled with complex and individual-specific patterns of CTL escape did not reveal shared sequence characteristics of acute infection that could be harnessed for vaccine design.

Improved understanding of the dynamics of host immune responses and viral evolution is critical for effective HIV-1 vaccine design. We comprehensively analyzed Cytotoxic T-lymphocyte (CTL)-viral epitope dynamics in an antiretroviral therapy-naive subject over the first four years of HIV-1 infection. We found that CTL responses developed sequentially and required constant antigenic stimulation for maintenance. CTL responses exerting strong selective pressure emerged early and led to rapid escape, proliferated rapidly and were predominant during acute/early infection. Although CTL responses to a few persistent epitopes developed over the first two months of infection, they proliferated slowly. As CTL epitopes were replaced by mutational variants, the corresponding responses immediately declined, most rapidly in the cases of strongly selected epitopes. CTL recognition of epitope variants, via cross-reactivity and de novo responses, was common throughout the period of study. Our data demonstrate that HIV-specific CTL responses, especially in the critical acute/early stage, were focused on regions that are prone to escape. Failure of CTL responses to strongly target functional or structurally critical regions of the virus, as well as the sequential cascade of CTL responses, followed closely by viral escape and decline of the corresponding responses, likely contribute to a lack of sustainable viral suppression. Focusing early and rapidly proliferating CTL on persistent epitopes may be essential for durable viral control in HIV-1 infection.

The principal surface lipids of Arabidopsis are n -nonacosane, 14- and 15-nonacosanol, 15-nonacosanone, C 16 –C 30 free fatty acids, C 26 –C 30 primary alcohols and C 26 –C 30 aldehydes. We have analysed the chemical composition of the epicuticular wax of 10 Arabidopsis thaliana eceriferum ( cer ) mutants. One of the mutants ( cer 2) is blocked in the elongation of octacosanoic acid and accumulates large amounts of primary alcohols and fatty acids in its epicuticular wax. The surface lipid composition of another mutant ( cer 4) appears to be defective in the production of primary alcohols and accumulates elevated levels of epicuticular alkanes. We have also identified a mutant ( cer 1) which accumulates epicuticular aldehydes and is severely deficient in alkanes on its surface. Seven other mutants had only slightly different epicuticular wax compositions compared to those of wild type plants.

Understanding the mechanisms underlying potential altered susceptibility to human immunodeficiency virus type 1 (HIV-1) infection in highly exposed seronegative (ES) individuals and the later clinical consequences of breakthrough infection can provide insight into strategies to control HIV-1 with an effective vaccine. From our Seattle ES cohort, we identified one individual (LSC63) who seroconverted after over 2 years of repeated unprotected sexual contact with his HIV-1-infected partner (P63) and other sexual partners of unknown HIV-1 serostatus. The HIV-1 variants infecting LSC63 were genetically unrelated to those sequenced from P63. This may not be surprising, since viral load measurements in P63 were repeatedly below 50 copies/ml, making him an unlikely transmitter. However, broad HIV-1-specific cytotoxic T-lymphocyte (CTL) responses were detected in LSC63 before seroconversion. Compared to those detected after seroconversion, these responses were of lower magnitude and half of them targeted different regions of the viral proteome. Strong HLA-B27-restricted CTLs, which have been associated with disease control, were detected in LSC63 after but not before seroconversion. Furthermore, for the majority of the protein-coding regions of the HIV-1 variants in LSC63 (except gp41 , nef , and the 3′ half of pol ), the genetic distances between the infecting viruses and the viruses to which he was exposed through P63 (termed the exposed virus) were comparable to the distances between random subtype B HIV-1 sequences and the exposed viruses. These results suggest that broad preinfection immune responses were not able to prevent the acquisition of HIV-1 infection in LSC63, even though the infecting viruses were not particularly distant from the viruses that may have elicited these responses.

Human lymphocyte antigen (HLA)-restricted CD8+ cytotoxic T lymphocytes (CTL) target and kill HIV-infected cells expressing cognate viral epitopes. This response selects for escape mutations within CTL epitopes that can diminish viral replication fitness. Here, we assess the fitness impact of escape mutations emerging in seven CTL epitopes in the gp120 Env and p24 Gag coding regions of an individual followed longitudinally from the time of acute HIV-1 infection, as well as some of these same epitopes recognized in other HIV-1-infected individuals. Nine dominant mutations appeared in five gp120 epitopes within the first year of infection, whereas all four mutations found in two p24 epitopes emerged after nearly two years of infection. These mutations were introduced individually into the autologous gene found in acute infection and then placed into a full-length, infectious viral genome. When competed against virus expressing the parental protein, fitness loss was observed with only one of the nine gp120 mutations, whereas four had no effect and three conferred a slight increase in fitness. In contrast, mutations conferring CTL escape in the p24 epitopes significantly decreased viral fitness. One particular escape mutation within a p24 epitope was associated with reduced peptide recognition and high viral fitness costs but was replaced by a fitness-neutral mutation. This mutation appeared to alter epitope processing concomitant with a reduced CTL response. In conclusion, CTL escape mutations in HIV-1 Gag p24 were associated with significant fitness costs, whereas most escape mutations in the Env gene were fitness neutral, suggesting a balance between immunologic escape and replicative fitness costs., Author Summary Upon infection with a pathogen, the host mounts an immune response of specific killing by recognizing infected cells presenting the foreign entity via host cell surface proteins (aka MHC) encoded by specific HLA genes. The pathogen's proteins are chopped up into peptides (short protein sequences) by cellular enzymes. Some of these peptides are bound to MHC class I proteins and then presented on the cell surface, signifying a pathogen intrusion to the killer T cells (CTLs). In order to survive, HIV-1 evolves to mutate the specific peptides (aka epitopes) and escape recognition by MHC I and CTLs. These “forced” or selected mutations are thought to come at a cost, e.g., result in slower pathogen growth/replication. Here, we show that the escape mutations in HIV-1 genes showing slow evolution result in dramatic losses in HIV-1 replication (or fitness). In contrast, CTL escape mutations in HIV-1 genes with rapid evolution do not have a negative impact on pathogen fitness. These findings could suggest that HIV-1 (and possibly other rapidly evolving pathogens) could avoid the host's immune system with minimal detriment to its own survival and growth within the host.

This study tested whether donor-derived HIV-specific immune responses could be detected when viral replication was completely suppressed by the continuous administration of highly active antiretroviral therapy (HAART). A regimen of fludarabine and 200 cGy total body irradiation was followed by infusion of allogeneic donor peripheral blood cells and posttransplantation cyclosporine and mycophenolate mofetil. Viral load, lymphocyte counts, and HIV-1–specific CD8+ cell immune responses were compared before and after hematopoietic cell transplantation (HCT). Uninterrupted administration of HAART was feasible during nonmyeloablative conditioning and after HCT. The HIV RNA remained undetectable and no HIV-associated infections were observed. CD8+ T-cell responses targeting multiple epitopes were detected before HCT. After HCT a different pattern of donor-derived HIV-specific CTL responses emerged by day +80, presumably primed in vivo. We conclude that allogeneic HCT offers the unique ability to characterize de novo HIV-1–specific immune responses. This clinical trial was registered at ClinicalTrials.gov (identifier: NCT00112593).

Human immunodeficiency virus type 1 (HIV-1) undergoes remarkable sequence evolution in humans during the course of infection. Viral evolution is driven largely by selective immune evasion (1, 16, 24, 33) and drug resistance mutations in combination with reversions (2, 12, 22, 23, 43) to restore replication fitness (25). HIV-specific CD8+ cytotoxic T lymphocytes (CTL) are major contributors of immune effector activities against HIV-1. Typically, CTL are induced during acute HIV-1 infection and broaden in specificity over time (7, 8, 47). To date, hundreds of major histocompatibility complex (MHC) class I-restricted CTL epitopes have been identified, and each of the major HIV-1 proteins contains known epitopes (www.hiv.lanl.gov). The antiviral activities of CD8+ T cells can exert selective immune pressure, which can lead to viral escape mutations within the epitopes recognized during acute infection (6, 8, 47) and over the course of chronic infection (20, 37). Most often, this occurs by substitution of an amino acid at a position in the epitope critical for interaction with its cognate MHC or T-cell receptor, but it can also involve changes in flanking residues (11, 46). The known epitopes may differ in their abilities to stimulate highly avid CTL among HIV-positive individuals, gain immunodominance, and acquire escape mutations (11, 14, 22, 24, 32, 47). The properties of the epitopes that account for these differences merit further understanding, since they potentially govern viral selection and replication fitness within the host and among populations at risk. Reverse transcriptase and protease inhibitors have been the cornerstone of antiretroviral therapy over the past decade. As resistance to these agents has developed, distinct viral genotypes conferring resistance have been identified, particularly with advances in resistance monitoring (reviewed in reference 42). Parallel investigations of the host immune response to HIV-1 infection indicate that HIV-specific CD8+ CTL can recognize epitopes within the active sites of HIV-1 reverse transcriptase and protease targeted by these therapeutic agents, suggesting that these alterations may influence viral replication capacity (38). Recent studies have characterized CTL epitopes directed to drug resistance mutations (26), and some drug resistance mutations have been shown to sustain or enhance CTL recognition (31). To gain insight into immune selection of HIV-1 and its impact on viral replication and diversity, we assessed and reported detailed T-cell responses for 21 Seattle, WA, patients with newly diagnosed primary infection (7). Forty-one unique HLA class I-restricted epitopes were identified (7), and the epitope specificities broadened over the course of infection. The concomitant immune and viral sequence analyses of patient PIC1362, who enrolled during acute infection and elected not to receive antiretroviral therapy during the first 4 years of infection, provided particular insight (8, 24, 25). Escape mutations in the initially recognized HIV-1 Tat, Vpr, and Env epitopes occurred in PIC1362 within weeks of infection (8, 24, 25). Subsequently, as we report here, an unusual escape mutation emerged, corresponding to a 3-amino-acid insertion in the p6Pol protein. The same insertion had previously been shown to occur during the use of nucleoside analog reverse transcriptase inhibitors (NRTI). Thus, HIV-1 may be selected by the same mechanism through antiretroviral therapy or immune pressure.

Typically during human immunodeficiency virus type 1 (HIV-1) infection, a nearly homogeneous viral population first emerges and then diversifies over time due to selective forces that are poorly understood. To identify these forces, we conducted an intensive longitudinal study of viral genetic changes and T-cell immunity in one subject at ≤17 time points during his first 3 years of infection, and in his infecting partner near the time of transmission. Autologous peptides covering amino acid sites inferred to be under positive selection were powerful for identifying HIV-1-specific cytotoxic-T-lymphocyte (CTL) epitopes. Positive selection and mutations resulting in escape from CTLs occurred across the viral proteome. We detected 25 CTL epitopes, including 14 previously unreported. Seven new epitopes mapped to the viral Env protein, emphasizing Env as a major target of CTLs. One-third of the selected sites were associated with epitopic mutational escapes from CTLs. Most of these resulted from replacement with amino acids found at low database frequency. Another one-third represented acquisition of amino acids found at high database frequency, suggesting potential reversions of CTL epitopic sites recognized by the immune system of the transmitting partner and mutation toward improved viral fitness in the absence of immune targeting within the recipient. A majority of the remaining selected sites occurred in the envelope protein and may have been subjected to humoral immune selection. Hence, a majority of the amino acids undergoing selection in this subject appeared to result from fitness-balanced CTL selection, confirming CTLs as a dominant selective force in HIV-1 infection.

Induction of HIV-1-specific CD8+ T cells during acute infection is associated with a decline in viremia. The role CD8+ effectors play in subsequently establishing viral set point remains unclear. To address this, we focused on two acutely infected patients with the same initial Tat-specific CD8+ response, analyzing their CD8+ T cell responses longitudinally in conjunction with viral load and sequence evolution. In one patient initiating treatment during acute infection, the frequencies of Tat-specific CD8+ T cells gradually diminished but persisted, and the Tat epitope sequence was unaltered. By contrast, in the second patient who declined treatment, the Tat-specific CD8+ T cells disappeared below detection, in conjunction with Gag-specific CD4+ T cell loss, as plasma viremia reached a set point. This coincided with the emergence of an escape variant within the Tat epitope and an additional Vpr epitope. New CD8+ T cell responses emerged but with no further associated decline in viremia. These findings indicate that, in the absence of treatment, the initial CD8+ T cell responses have the greatest impact on reducing viremia, and that later, continuously evolving responses are less efficient in further reducing viral load. The results also suggest that T cell help may contribute to the antiviral efficiency of the acute CD8+ T cell response.

The human immunodeficiency virus (HIV) pandemic is of staggering proportions, with nearly 40 million individuals infected (1). This has created an urgent need to develop a safe, efficacious vaccine as the single prevention strategy to benefit the largest number of persons. In the absence of established correlates of protection, most experts predict that both humoral and cellular immunity will be required for sustained immunity against HIV type 1 (HIV-1). Successful vaccines against other viral infections rely on induction of protective levels of antiviral antibodies (10). Likewise, the first generation of HIV vaccine candidates, notably, recombinant monomeric envelope protein subunit vaccines, was designed to elicit antibody responses. Studies of individuals who participated in these trials but subsequently became infected with HIV (breakthrough infections), therefore, have focused on characterization of their HIV-specific neutralizing antibody responses prior to infection and on the ability of these antibodies to neutralize viruses infecting vaccine recipients (5, 12, 17, 23). Unfortunately, these studies revealed that nearly all vaccine-induced antibodies by this approach lack potency and breadth of reactivity against transmitting strains (discussed in reference 8) and ultimately fail to provide HIV protection. While new strategies for the design of vaccines which induce effective anti-HIV-l antibodies are still being identified, the emphasis has shifted to the design of vaccines that will induce antiviral CD8+ T-cell immunity. Although HIV-specific CD8+ T cells alone are unlikely to prevent HIV infection, they may have an important impact in rapidly controlling viral replication following infection. A large effort has centered upon the development of recombinant attenuated poxviruses as a safe approach in humans to induce HIV-1-specific T-cell immunity. Recombinant canarypox HIV vaccines, the most extensively tested to date, induce CD8+ T-cell responses at various frequencies (9 to 70%) depending upon the construct tested and the method of detection (4, 11, 15, 18). Here we investigate the characteristics of the T cells present during early infection in individuals previously enrolled in HIV vaccine trials evaluating recombinant canarypox candidate vaccines, contrasting their responses with those of nonvaccinated subjects who have recently acquired HIV-1 infection. The vaccine constructs contained full-length gag (HIV-1 LAI) and env gp120 (HIV-1 MN) coding regions, with some constructs also containing parts of pol and nef. Several individuals also received a recombinant gp120 SF-2 protein boost (Chiron) or a recombinant gp120 MN protein boost (Vaxgen Inc.). Vaccinees with breakthrough infections described in this report took part in phase I/II HIV vaccine trials where the net cumulative cytotoxic T-lymphocyte (CTL) response rates were between 33 and 50%, as assessed by the chromium release assay (4, 18). We determined whether any of the T-cell responses detected during early infection in these individuals represented anamnestic responses induced by immunization prior to infection. Furthermore, we characterized longitudinally the function of HIV-specific T cells postinfection in the only volunteer controlling infection in the absence of antiretroviral therapy (ART). These are the first comprehensive data describing HIV-specific T-cell responses pre- and postinfection in participants who became HIV-infected in HIV vaccine trials of vaccine candidates designed to elicit CD8+ T-cell immunity.

HIV-1-infected persons with HLA-B27 and -B57 alleles commonly remain healthy for decades without antiretroviral therapy. Properties of CD8+ T cells restricted by these alleles considered to confer disease protection in these individuals are elusive but important to understand and potentially elicit by vaccination. To address this, we compared CD8+ T cell function induced by HIV-1 immunogens and natural infection using polychromatic flow cytometry. HIV-1-specific CD8+ T cells from all four uninfected immunized and 21 infected subjects secreted IFN-γ and TNF-α. However, CD8+ T cells induced by vaccination and primary infection, but not chronic infection, proliferated to their cognate epitopes. Notably, B27- and B57-restricted CD8+ T cells from nonprogressors exhibited greater expansion than those restricted by other alleles. Hence, CD8+ T cells restricted by certain protective alleles can resist replicative defects, which permits expansion and antiviral effector activities. Our findings suggest that the capacity to maintain CD8+ T cell proliferation, regardless of MHC-restriction, may serve as an important correlate of disease protection in the event of infection following vaccination.