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1.  Lack of Protection following Passive Transfer of Polyclonal Highly Functional Low-Dose Non-Neutralizing Antibodies 
PLoS ONE  2014;9(5):e97229.
Recent immune correlates analysis from the RV144 vaccine trial has renewed interest in the role of non-neutralizing antibodies in mediating protection from infection. While neutralizing antibodies have proven difficult to induce through vaccination, extra-neutralizing antibodies, such as those that mediate antibody-dependent cellular cytotoxicity (ADCC), are associated with long-term control of infection. However, while several non-neutralizing monoclonal antibodies have been tested for their protective efficacy in vivo, no studies to date have tested the protective activity of naturally produced polyclonal antibodies from individuals harboring potent ADCC activity. Because ADCC-inducing antibodies are highly enriched in elite controllers (EC), we passively transferred highly functional non-neutralizing polyclonal antibodies, purified from an EC, to assess the potential impact of polyclonal non-neutralizing antibodies on a stringent SHIV-SF162P3 challenge in rhesus monkeys. Passive transfer of a low-dose of ADCC inducing antibodies did not protect from infection following SHIV-SF162P3 challenge. Passively administered antibody titers and gp120-specific, but not gp41-specific, ADCC and antibody induced phagocytosis (ADCP) were detected in the majority of the monkeys, but did not correlate with post infection viral control. Thus these data raise the possibility that gp120-specific ADCC activity alone may not be sufficient to control viremia post infection but that other specificities or Fc-effector profiles, alone or in combination, may have an impact on viral control and should be tested in future passive transfer experiments.
doi:10.1371/journal.pone.0097229
PMCID: PMC4018276  PMID: 24820481
2.  Association of HLA-DRB1-restricted CD4+ T cell responses with HIV immune control 
Nature medicine  2013;19(7):930-933.
The contribution of HLA class II-restricted CD4+ T cell responses to HIV immune control is poorly defined. Here, we delineated novel peptide-DRB1 restrictions in functional assays and analyzed the host genetic effects of HLA-DRB1 alleles on HIV viremia in a large cohort of HIV controllers and progressors (n=1085). We found distinct stratifications in the effect of HLA-DRB1 alleles on HIV viremia, with DRB1*15:02 significantly associated with low viremia (P=0.003, q=0.04) and DRB1*03:01 significantly associated with high viremia (P=0.004, q=0.04). Interestingly, a sub-group of HLA-DRB1 alleles linked with low viremia showed the ability to promiscuously present a larger breadth of peptides with lower functional avidity when compared to HLA-DRB1 alleles linked with high viremia (p=0.018). Our data provide systematic evidence that HLA-DRB1 allele expression significantly impacts the durable control of HIV replication, an effect that appears to be mediated primarily by the protein-specificity of HIV-specific CD4+ T cell responses to Gag and Nef.
doi:10.1038/nm.3229
PMCID: PMC3974408  PMID: 23793098
3.  HIV-specific cytolytic CD4 T cell responses during acute HIV infection predict disease outcome 
Science translational medicine  2012;4(123):123ra25.
Early immunological events during acute HIV infection are thought to fundamentally influence long-term disease outcome. Whereas the contribution of HIV-specific CD8 T cell responses to early viral control is well established, the role of HIV-specific CD4 T cell responses in the control of viral replication following acute infection is unknown. A growing body of evidence suggests that CD4 T cells - besides their helper function - have the capacity to directly recognize and kill virally infected cells. In a longitudinal study of a cohort of individuals acutely infected with HIV, we observed that subjects able to spontaneously control HIV replication in the absence of antiretroviral therapy showed a significant expansion of HIV-specific CD4 T cell responses—but not CD8 T cell responses–compared to subjects who progressed to a high viral set point (p=0.038). Strikingly, this expansion occurred prior to differences in viral load or CD4 T cell count and was characterized by robust cytolytic activity and expression of a distinct profile of perforin and granzymes at the earliest time point. Kaplan-Meier analysis revealed that the emergence of Granzyme A+ HIV-specific CD4 T cell responses at baseline was highly predictive of slower disease progression and clinical outcome (average days to CD4 T cell count <350/μl was 575 versus 306, p=0.001). These data demonstrate that HIV-specific CD4 T cell responses can be used during the earliest phase of HIV infection as an immunological predictor of subsequent viral set point and disease outcome. Moreover, these data suggest that expansion of Granzyme A+ HIV-specific cytolytic CD4 T cell responses early during acute HIV infection contributes substantially to the control of viral replication.
doi:10.1126/scitranslmed.3003165
PMCID: PMC3918726  PMID: 22378925
4.  Temporal effect of HLA-B*57 on viral control during primary HIV-1 infection 
Retrovirology  2013;10:139.
Background
HLA-B alleles are associated with viral control in chronic HIV-1 infection, however, their role in primary HIV-1 disease is unclear. This study sought to determine the role of HLA-B alleles in viral control during the acute phase of HIV-1 infection and establishment of the early viral load set point (VLSP).
Findings
Individuals identified during primary HIV-1 infection were HLA class I typed and followed longitudinally. Associations between HLA-B alleles and HIV-1 viral replication during acute infection and VLSP were analyzed in untreated subjects. The results showed that neither HLA-B*57 nor HLA-B*27 were significantly associated with viral control during acute HIV-1 infection (Fiebig stage I-IV, n=171). HLA-B*57 was however significantly associated with a subsequent lower VLSP (p<0.001, n=135) with nearly 1 log10 less median viral load. Analysis of a known polymorphism at position 97 of HLA-B showed significant associations with both lower initial viral load (p<0.01) and lower VLSP (p<0.05). However, this association was dependent on different amino acids at this position for each endpoint.
Conclusions
The effect of HLA-B*57 on viral control is more pronounced during the later stages of primary HIV-1 infection, which suggests the underlying mechanism of control occurs at a critical period in the first several months after HIV-1 acquisition. The risk profile of polymorphisms at position 97 of HLA-B are more broadly associated with HIV-1 viral load during primary infection and may serve as a focal point in further studies of HLA-B function.
doi:10.1186/1742-4690-10-139
PMCID: PMC3874665  PMID: 24245727
HLA-B*57; HLA-B; Acute HIV-1 infection; Primary HIV-1 infection; Viral load set point; MHC class I
5.  A BLUEPRINT FOR HIV VACCINE DISCOVERY 
Cell host & microbe  2012;12(4):396-407.
Despite numerous attempts over many years to develop an HIV vaccine based on classical strategies, none has convincingly succeeded to date. A number of approaches are being pursued in the field, including building upon possible efficacy indicated by the recent RV144 clinical trial, which combined two HIV vaccines. Here, we argue for an approach based, in part, on understanding the HIV envelope spike and its interaction with broadly neutralizing antibodies (bnAbs) at the molecular level and using this understanding to design immunogens as possible vaccines. BnAbs can protect against virus challenge in animal models and many such antibodies have been isolated recently. We further propose that studies focused on how best to provide T cell help to B cells that produce bnAbs are crucial for optimal immunization strategies. The synthesis of rational immunogen design and immunization strategies, together with iterative improvements, offers great promise for advancing toward an HIV vaccine.
doi:10.1016/j.chom.2012.09.008
PMCID: PMC3513329  PMID: 23084910
7.  Preserved Function of Regulatory T Cells in Chronic HIV-1 Infection Despite Decreased Numbers in Blood and Tissue 
The Journal of Infectious Diseases  2012;205(10):1495-1500.
Regulatory T cells (Tregs) are potent immune modulators, but their role in human immunodeficiency virus type 1 (HIV-1) pathogenesis remains poorly understood. We performed a detailed analysis of the frequency and function of Tregs in a large cohort of HIV-1–infected individuals and HIV-1 negative controls. While HIV “elite controllers” and uninfected individuals had similar Treg numbers and frequencies, the absolute numbers of Tregs declined in blood and gut-associated lymphoid tissue in patients with chronic progressive HIV-1 infection. Despite quantitative changes in Tregs, HIV-1 infection was not associated with an impairment of ex vivo suppressive function of flow-sorted Tregs in both HIV controllers and untreated chronic progressors.
doi:10.1093/infdis/jis236
PMCID: PMC3415814  PMID: 22427677
8.  Harnessing CD4+ T cell responses in HIV vaccine development 
Nature medicine  2013;19(2):143-149.
CD4+ T cells can perform a panoply of tasks to shape an effective response against a pathogen. Limited attention has been paid to the potential importance of functional CD4+ T cell responses in the context of the development of next-generation vaccines, including HIV vaccines. Many CD4+ T cell functions are newly appreciated and only partially understood. A workshop was held as a forum to bring together a small group of experts to exchange ideas on the role of CD4+ T cells in developing durable functional antibody responses, via follicular helper T cells, as well as on the roles of CD4+ T cells in other aspects of protective immunity. Here we discuss whether CD4+ T cell responses may represent a beneficial component of an efficacious HIV vaccine.
doi:10.1038/nm.3054
PMCID: PMC3626561  PMID: 23389614
9.  Induction of Strong HIV-1-specific CD4+ T Cell Responses using an HIV-1 gp120/NefTat Vaccine adjuvanted with AS02A in ARV Treated HIV-1-Infected Individuals 
Background
Induction of HIV-1-specific CD4+ T cell responses by therapeutic vaccination represents an attractive intervention to potentially increase immune control of HIV-1.
Methods
We performed a double-blinded, randomized, placebo-controlled clinical trial to determine the safety and immunogenicity of GSK Biologicals' HIV-1 gp120/NefTat subunit protein vaccine formulated with the AS02A adjuvant in subjects with well controlled chronic HIV-1 infection on HAART. Ten individuals received the vaccine; while adjuvant alone or placebo was given to five subjects each. Immunogenicity was monitored by intracellular cytokine flow cytometry and CFSE-based proliferation assays.
Results
The vaccine was well tolerated with no related SAEs. Vaccine recipients had significantly stronger gp120-specific CD4+ T cell responses which persisted until week 48 and greater gp120-specific CD4+ T cell proliferation activity as compared to controls. In the vaccine group, the number of participants that demonstrated positive responses for both gp120-specific CD4+ T cell IL-2 production and gp120-specific CD8+ T cell proliferation was significantly higher at week 6.
Conclusions
The gp120/NefTat/AS02A vaccine induced strong gp120-specific CD4+ T cell responses, and a higher number of vaccinees developed both HIV-1-specific CD4+ T cell responses and CD8+ T cell proliferation. The induction of these responses may be important in enhancing immune-mediated viral control.
doi:10.1097/QAI.0b013e3182373b77
PMCID: PMC3241906  PMID: 21963936
HIV-1; Vaccination; Therapeutic Vaccination; HIV-1-specific CD4+ cells
10.  CD4+ CD25+ Regulatory T Cells Impair HIV-1-Specific CD4 T Cell Responses by Upregulating Interleukin-10 Production in Monocytes 
Journal of Virology  2012;86(12):6586-6594.
T cell dysfunction in the presence of ongoing antigen exposure is a cardinal feature of chronic viral infections with persistent high viremia, including HIV-1. Although interleukin-10 (IL-10) has been implicated as an important mediator of this T cell dysfunction, the regulation of IL-10 production in chronic HIV-1 infection remains poorly understood. We demonstrated that IL-10 is elevated in the plasma of individuals with chronic HIV-1 infection and that blockade of IL-10 signaling results in a restoration of HIV-1-specific CD4 T cell proliferation, gamma interferon (IFN-γ) secretion, and, to a lesser extent, IL-2 production. Whereas IL-10 blockade leads to restoration of IFN-γ secretion by HIV-1-specific CD4 T cells in all categories of subjects investigated, significant enhancement of IL-2 production and improved proliferation of CD4 T helper cells are restricted to viremic individuals. In peripheral blood mononuclear cells (PBMCs), this IL-10 is produced primarily by CD14+ monocytes, but its production is tightly controlled by regulatory T cells (Tregs), which produce little IL-10 directly. When Tregs are depleted from PBMCs of viremic individuals, the effect of the IL-10 signaling blockade is abolished and IL-10 production by monocytes decreases, while the production of proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-α), increases. The regulation of IL-10 by Tregs appears to be mediated primarily by contact or paracrine-dependent mechanisms which involve IL-27. This work describes a novel mechanism by which regulatory T cells control IL-10 production and contribute to dysfunctional HIV-1-specific CD4 T cell help in chronic HIV-1 infection and provides a unique mechanistic insight into the role of regulatory T cells in immune exhaustion.
doi:10.1128/JVI.06251-11
PMCID: PMC3393541  PMID: 22496237
11.  Short Communication: CD8+ T Cell Polyfunctionality Profiles in Progressive and Nonprogressive Pediatric HIV Type 1 Infection 
AIDS Research and Human Retroviruses  2011;27(9):1005-1012.
Abstract
Pediatric HIV-1 infection is characterized by rapid disease progression and without antiretroviral therapy (ART), more than 50% of infected children die by the age of 2 years. However, a small subset of infected children progresses slowly to disease in the absence of ART. This study aimed to identify functional characteristics of HIV-1-specific T cell responses that distinguish children with rapid and slow disease progression. Fifteen perinatally HIV-infected children (eight rapid and seven slow progressors) were longitudinally studied to monitor T cell polyfunctionality. HIV-1-specific interferon (IFN)-γ+ CD8+ T cell responses gradually increased over time but did not differ between slow and rapid progressors. However, polyfunctional HIV-1-specific CD8+ T cell responses, as assessed by the expression of four functions (IFN-γ, CD107a, TNF-α, MIP-1β), were higher in slow compared to rapid progressors (p=0.05) early in infection, and was associated with slower subsequent disease progression. These data suggest that the quality of the HIV-specific CD8+ T cell response is associated with the control of disease in children as has been shown in adult infection.
doi:10.1089/aid.2010.0227
PMCID: PMC3332389  PMID: 21288139
12.  Expansion of HIV-specific T follicular helper cells in chronic HIV infection 
The Journal of Clinical Investigation  2012;122(9):3271-3280.
HIV targets CD4 T cells, which are required for the induction of high-affinity antibody responses and the formation of long-lived B cell memory. The depletion of antigen-specific CD4 T cells during HIV infection is therefore believed to impede the development of protective B cell immunity. Although several different HIV-related B cell dysfunctions have been described, the role of CD4 T follicular helper (TFH) cells in HIV infection remains unknown. Here, we assessed HIV-specific TFH responses in the lymph nodes of treatment-naive and antiretroviral-treated HIV-infected individuals. Strikingly, both the bulk TFH and HIV-specific TFH cell populations were significantly expanded in chronic HIV infection and were highly associated with viremia. In particular, GAG-specific TFH cells were detected at significantly higher levels in the lymph nodes compared with those of GP120-specific TFH cells and showed preferential secretion of the helper cytokine IL-21. In addition, TFH cell expansion was associated with an increase of germinal center B cells and plasma cells as well as IgG1 hypersecretion. Thus, our study suggests that high levels of HIV viremia drive the expansion of TFH cells, which in turn leads to perturbations of B cell differentiation, resulting in dysregulated antibody production.
doi:10.1172/JCI64314
PMCID: PMC3428098  PMID: 22922259
13.  Broadly directed virus-specific CD4+ T cell responses are primed during acute hepatitis C infection, but rapidly disappear from human blood with viral persistence 
Early after symptom onset, HCV-specific CD4+ T cell responses are primed and detectable in patients regardless of clinical outcome, but without early antiviral therapy these T cells become exhausted or deleted in chronically infected patients.
Vigorous proliferative CD4+ T cell responses are the hallmark of spontaneous clearance of acute hepatitis C virus (HCV) infection, whereas comparable responses are absent in chronically evolving infection. Here, we comprehensively characterized the breadth, specificity, and quality of the HCV-specific CD4+ T cell response in 31 patients with acute HCV infection and varying clinical outcomes. We analyzed in vitro T cell expansion in the presence of interleukin-2, and ex vivo staining with HCV peptide-loaded MHC class II tetramers. Surprisingly, broadly directed HCV-specific CD4+ T cell responses were universally detectable at early stages of infection, regardless of the clinical outcome. However, persistent viremia was associated with early proliferative defects of the HCV-specific CD4+ T cells, followed by rapid deletion of the HCV-specific response. Only early initiation of antiviral therapy was able to preserve CD4+ T cell responses in acute, chronically evolving infection. Our results challenge the paradigm that HCV persistence is the result of a failure to prime HCV-specific CD4+ T cells. Instead, broadly directed HCV-specific CD4+ T cell responses are usually generated, but rapid exhaustion and deletion of these cells occurs in the majority of patients. The data further suggest a short window of opportunity to prevent the loss of CD4+ T cell responses through antiviral therapy.
doi:10.1084/jem.20100388
PMCID: PMC3260872  PMID: 22213804
14.  HIV-Specific CD4 T Cell Responses to Different Viral Proteins Have Discordant Associations with Viral Load and Clinical Outcome 
Journal of Virology  2012;86(1):277-283.
A successful prophylactic vaccine is characterized by long-lived immunity, which is critically dependent on CD4 T cell-mediated helper signals. Indeed, most licensed vaccines induce antigen-specific CD4 T cell responses, in addition to high-affinity antibodies. However, despite the important role of CD4 T cells in vaccine design and natural infection, few studies have characterized HIV-specific CD4 T cells due to their preferential susceptibility to HIV infection. To establish at the population level the impact of HIV-specific CD4 T cells on viral control and define the specificity of HIV-specific CD4 T cell peptide targeting, we conducted a comprehensive analysis of these responses to the entire HIV proteome in 93 subjects at different stages of HIV infection. We show that HIV-specific CD4 T cell responses were detectable in 92% of individuals and that the breadth of these responses showed a significant inverse correlation with the viral load (P = 0.009, R = −0.31). In particular, CD4 T cell responses targeting Gag were robustly associated with lower levels of viremia (P = 0.0002, R = −0.45). Importantly, differences in the immunodominance profile of HIV-specific CD4 T cell responses distinguished HIV controllers from progressors. Furthermore, Gag/Env ratios were a potent marker of viral control, with a high frequency and magnitude of Gag responses and low proportion of Env responses associated with effective immune control. At the epitope level, targeting of three distinct Gag peptides was linked to spontaneous HIV control (P = 0.60 to 0.85). Inclusion of these immunogenic proteins and peptides in future HIV vaccines may act as a critical cornerstone for enhancing protective T cell responses.
doi:10.1128/JVI.05577-11
PMCID: PMC3255877  PMID: 22031937
15.  Whole Genome Deep Sequencing of HIV-1 Reveals the Impact of Early Minor Variants Upon Immune Recognition During Acute Infection 
PLoS Pathogens  2012;8(3):e1002529.
Deep sequencing technologies have the potential to transform the study of highly variable viral pathogens by providing a rapid and cost-effective approach to sensitively characterize rapidly evolving viral quasispecies. Here, we report on a high-throughput whole HIV-1 genome deep sequencing platform that combines 454 pyrosequencing with novel assembly and variant detection algorithms. In one subject we combined these genetic data with detailed immunological analyses to comprehensively evaluate viral evolution and immune escape during the acute phase of HIV-1 infection. The majority of early, low frequency mutations represented viral adaptation to host CD8+ T cell responses, evidence of strong immune selection pressure occurring during the early decline from peak viremia. CD8+ T cell responses capable of recognizing these low frequency escape variants coincided with the selection and evolution of more effective secondary HLA-anchor escape mutations. Frequent, and in some cases rapid, reversion of transmitted mutations was also observed across the viral genome. When located within restricted CD8 epitopes these low frequency reverting mutations were sufficient to prime de novo responses to these epitopes, again illustrating the capacity of the immune response to recognize and respond to low frequency variants. More importantly, rapid viral escape from the most immunodominant CD8+ T cell responses coincided with plateauing of the initial viral load decline in this subject, suggestive of a potential link between maintenance of effective, dominant CD8 responses and the degree of early viremia reduction. We conclude that the early control of HIV-1 replication by immunodominant CD8+ T cell responses may be substantially influenced by rapid, low frequency viral adaptations not detected by conventional sequencing approaches, which warrants further investigation. These data support the critical need for vaccine-induced CD8+ T cell responses to target more highly constrained regions of the virus in order to ensure the maintenance of immunodominant CD8 responses and the sustained decline of early viremia.
Author Summary
The ability of HIV-1 and other highly variable pathogens to rapidly mutate to escape vaccine-induced immune responses represents a major hurdle to the development of effective vaccines to these highly persistent pathogens. Application of next-generation or deep sequencing technologies to the study of host pathogens could significantly improve our understanding of the mechanisms by which these pathogens subvert host immunity, and aid in the development of novel vaccines and therapeutics. Here, we developed a 454 deep sequencing approach to enable the sensitive detection of low-frequency viral variants across the entire HIV-1 genome. When applied to the acute phase of HIV-1 infection we observed that the majority of early, low frequency mutations represented viral adaptations to host cellular immune responses, evidence of strong host immunity developing during the early decline of peak viral load. Rapid viral escape from the most dominant immune responses however correlated with loss of this initial viral control, suggestive of the importance of mounting immune responses against more conserved regions of the virus. These data provide a greater understanding of the early evolutionary events subverting the ability of host immune responses to control early HIV-1 replication, yielding important insight into the design of more effective vaccine strategies.
doi:10.1371/journal.ppat.1002529
PMCID: PMC3297584  PMID: 22412369
16.  T cell immunity in acute HIV-1 infection 
The Journal of infectious diseases  2010;202(Suppl 2):S302-S308.
Exceedingly high viral loads and rapid loss of CD4+ T cells in all tissue compartments is a hallmark of acute HIV-1 infection, which is often accompanied by clinical symptoms, such as fever, maculopapular rash and/or lymphadenopathy. The resolution of the clinical symptoms, and the subsequent decrease of plasma viremia are associated with the emergence of HIV-1-specific CD4+ and CD8+ T cell responses. The remarkable early inhibition of viremia by CD8+ T cells appears to be precipitated by only a limited number of specific CD8+ T cell responses, and the plasma viremia is reduced to a “set point” level. Over time the breadth and magnitude of CD8+ T cell responses increase, but without a change in the control of viral replication or further reduction in the viral set point. Moreover, the early viral set point, consequent upon the first CD8+ T cell responses, is highly predictive for the later course of disease progression. Thus, HIV-1-specific CD8+ T cell responses in acute HIV-1 infection appear to be uniquely able to efficiently suppress viral replication, while CD8+ T cell responses generated in the chronic phase of the infection appear often impaired.
doi:10.1086/655652
PMCID: PMC2954287  PMID: 20846037
17.  Cytolytic CD4+ T cells in viral immunity 
Expert review of vaccines  2010;9(12):1453-1463.
It is generally believed that the role of CD4+ T cells is to coordinate the different arms of the adaptive immune system to shape an effective response against a pathogen and regulate nonessential or deleterious activities. However, a growing body of evidence suggests that effector CD4+ T cells can directly display potent antiviral activity themselves. The presence of cytolytic CD4+ T cells has been demonstrated in the immune response to numerous viral infections in both humans and in animal models and it is likely that they play a critical role in the control of viral replication in vivo. This article describes the current research on virus-specific cytolytic CD4+ T cells, with a focus on HIV-1 infection and the implications that this immune response has for vaccine design.
doi:10.1586/erv.10.132
PMCID: PMC3033049  PMID: 21105780
cytolytic CD4+ Tcells; HIV; vaccines; viral infection
18.  HIV-1-Specific Interleukin-21+ CD4+ T Cell Responses Contribute to Durable Viral Control through the Modulation of HIV-Specific CD8+ T Cell Function ▿ †  
Journal of Virology  2010;85(2):733-741.
Functional defects in cytotoxic CD8+ T cell responses arise in chronic human viral infections, but the mechanisms involved are not well understood. In mice, CD4 cell-mediated interleukin-21 (IL-21) production is necessary for the maintenance of CD8+ T cell function and control of persistent viral infections. To investigate the potential role of IL-21 in a chronic human viral infection, we studied the rare subset of HIV-1 controllers, who are able to spontaneously control HIV-1 replication without treatment. HIV-specific triggering of IL-21 by CD4+ T cells was significantly enriched in these persons (P = 0.0007), while isolated loss of IL-21-secreting CD4+ T cells was characteristic for subjects with persistent viremia and progressive disease. IL-21 responses were mediated by recognition of discrete epitopes largely in the Gag protein, and expansion of IL-21+ CD4+ T cells in acute infection resulted in lower viral set points (P = 0.002). Moreover, IL-21 production by CD4+ T cells of HIV controllers enhanced perforin production by HIV-1-specific CD8+ T cells from chronic progressors even in late stages of disease, and HIV-1-specific effector CD8+ T cells showed an enhanced ability to efficiently inhibit viral replication in vitro after IL-21 binding. These data suggest that HIV-1-specific IL-21+ CD4+ T cell responses might contribute to the control of viral replication in humans and are likely to be of great importance for vaccine design.
doi:10.1128/JVI.02030-10
PMCID: PMC3020027  PMID: 21047960
19.  Early Selection in Gag by Protective HLA Alleles Contributes to Reduced HIV-1 Replication Capacity That May Be Largely Compensated for in Chronic Infection▿ †  
Journal of Virology  2010;84(22):11937-11949.
Mutations that allow escape from CD8 T-cell responses are common in HIV-1 and may attenuate pathogenesis by reducing viral fitness. While this has been demonstrated for individual cases, a systematic investigation of the consequence of HLA class I-mediated selection on HIV-1 in vitro replication capacity (RC) has not been undertaken. We examined this question by generating recombinant viruses expressing plasma HIV-1 RNA-derived Gag-Protease sequences from 66 acute/early and 803 chronic untreated subtype B-infected individuals in an NL4-3 background and measuring their RCs using a green fluorescent protein (GFP) reporter CD4 T-cell assay. In acute/early infection, viruses derived from individuals expressing the protective alleles HLA-B*57, -B*5801, and/or -B*13 displayed significantly lower RCs than did viruses from individuals lacking these alleles (P < 0.05). Furthermore, acute/early RC inversely correlated with the presence of HLA-B-associated Gag polymorphisms (R = −0.27; P = 0.03), suggesting a cumulative effect of primary escape mutations on fitness during the first months of infection. At the chronic stage of infection, no strong correlations were observed between RC and protective HLA-B alleles or with the presence of HLA-B-associated polymorphisms restricted by protective alleles despite increased statistical power to detect these associations. However, RC correlated positively with the presence of known compensatory mutations in chronic viruses from B*57-expressing individuals harboring the Gag T242N mutation (n = 50; R = 0.36; P = 0.01), suggesting that the rescue of fitness defects occurred through mutations at secondary sites. Additional mutations in Gag that may modulate the impact of the T242N mutation on RC were identified. A modest inverse correlation was observed between RC and CD4 cell count in chronic infection (R = −0.17; P < 0.0001), suggesting that Gag-Protease RC could increase over the disease course. Notably, this association was stronger for individuals who expressed B*57, B*58, or B*13 (R = −0.27; P = 0.004). Taken together, these data indicate that certain protective HLA alleles contribute to early defects in HIV-1 fitness through the selection of detrimental mutations in Gag; however, these effects wane as compensatory mutations accumulate in chronic infection. The long-term control of HIV-1 in some persons who express protective alleles suggests that early fitness hits may provide lasting benefits.
doi:10.1128/JVI.01086-10
PMCID: PMC2977869  PMID: 20810731
20.  Epidemiologically-Linked Transmission of HIV-1 Illustrates the Impact of Host Genetics on Virological Outcome 
AIDS (London, England)  2009;23(2):259-262.
The diversity of HIV-1 and human genetics complicates our ability to determine the impact of treatment during primary HIV-1 infection (PHI) on disease outcome. Here we show in a small group infected with virtually identical HIV-1 strains and treated during PHI that only subjects expressing protective HLA alleles had lower viral loads following treatment discontinuation. These data suggests that genetic factors play a pivotal role in the outcome of HIV-1 infection despite early therapy.
doi:10.1097/QAD.0b013e328315a78b
PMCID: PMC3082841  PMID: 19098497
acute HIV-1 infection; HLA-B57; HLA-A11; Berlin patient; cluster
21.  Sex differences in the TLR-mediated response of pDCs to HIV-1 are associated with higher immune activation in infected women 
Nature medicine  2009;15(8):955-959.
Manifestations of viral infections can differ between women and men1, and significant sex differences have been described in the course of HIV-1 disease. HIV-1-infected women tend to have lower viral load levels early in HIV-1 infection, but progress faster to AIDS for a given viral load than men2–7. Here we demonstrate substantial sex differences in the response of plasmacytoid dendritic cells (pDCs) to HIV-1. pDCs derived from women produce significantly more interferon-α (IFN-α) in response to HIV-1-encoded TLR7 ligands than pDCs derived from men, resulting in stronger secondary activation of CD8+ T cells. In line with these in vitro studies, treatment-naïve chronically HIV-1-infected women had significantly higher levels of CD8+ T cell activation than men after adjusting for viral load. These data show that sex differences in TLR-mediated activation of pDCs can account for higher immune activation in women compared to men at a given HIV-1 viral load, and provide a mechanism by which the same level of viral replication might result in faster HIV-1 disease progression in women compared to men. Modulation of the TLR7 pathway in pDCs may therefore represent a novel approach to reduce HIV-1-associated pathology.
doi:10.1038/nm.2004
PMCID: PMC2821111  PMID: 19597505
22.  Human Immunodeficiency Virus Type 1-Specific CD8+ T-Cell Responses during Primary Infection Are Major Determinants of the Viral Set Point and Loss of CD4+ T Cells▿ †  
Journal of Virology  2009;83(15):7641-7648.
Primary HIV-1 infection (PHI) is marked by a flu-like syndrome and high levels of viremia that decrease to a viral set point with the first emergence of virus-specific CD8+ T-cell responses. Here, we investigated in a large cohort of 527 subjects the immunodominance pattern of the first virus-specific cytotoxic T-lymphocyte (CTL) responses developed during PHI in comparison to CTL responses in chronic infection and demonstrated a distinct relationship between the early virus-specific CTL responses and the viral set point, as well as the slope of CD4+ T-cell decline. CTL responses during PHI followed clear hierarchical immunodominance patterns that were lost during the transition to chronic infection. Importantly, the immunodominance patterns of human immunodeficiency virus type 1 (HIV-1)-specific CTL responses detected in primary, but not in chronic, HIV-1 infection were significantly associated with the subsequent set point of viral replication. Moreover, the preservation of the initial CD8+ T-cell immunodominance patterns from the acute into the chronic phase of infection was significantly associated with slower CD4+ T-cell decline. Taken together, these data show that the specificity of the initial CTL response to HIV is critical for the subsequent control of viremia and have important implications for the rational selection of antigens for future HIV-1 vaccines.
doi:10.1128/JVI.00182-09
PMCID: PMC2708622  PMID: 19458000
23.  Up-regulation of PD-L1 on Monocytes and Dendritic Cells by HIV-1 derived TLR Ligands 
AIDS (London, England)  2008;22(5):655-658.
Increased PD-L1 expression has been reported in HIV-1-infected individuals, but the mechanisms leading to PD-L1-up-regulation remain to be elucidated. Here we demonstrate that HIV-1-derived TLR7/8 ligands can induce MyD88-dependent up-regulation of PD-L1 on pDCs, mDCs and monocytes. These data suggest a mechanism through which HIV-1-derived TLR ligands might contribute to the functional impairment of virus-specific PD-1+ T cells by inducing the up-regulation of PD-L1 on antigen-presenting cells.
doi:10.1097/QAD.0b013e3282f4de23
PMCID: PMC2810187  PMID: 18317010
24.  Rapid Loss of Dendritic Cell and Monocyte Responses to TLR Ligands following Venipuncture 
Journal of immunological methods  2008;339(2):132-140.
Blood samples from multiple sites are collected in multicenter trials, and frequently shipped to centralized laboratories for processing and comparable experimental evaluation. It is therefore of crucial interest to assess the preservation of immune cell functions after overnight shipment of whole blood. Here we evaluated the ability of pDCs, mDCs and monocytes to respond to TLR ligands at multiple timepoints following venipuncture as compared to immediate processing. Our results demonstrate a profound impairment of APC function, in particular of IFN-α production of pDCs, if whole blood was processed later than 6hours after venipuncture. Overnight shipment or extended rest of whole blood before processing therefore severely compromises the ability of APCs to respond to TLR ligands, and this has to be taken into consideration when designing multicenter trials.
doi:10.1016/j.jim.2008.09.007
PMCID: PMC2613681  PMID: 18848564
Innate Immunity; Antigen Presenting Cells; Dendritic Cells; Monocytes; Toll Like Receptors; Vaccine Trials
25.  Protective HLA Class I Alleles That Restrict Acute-Phase CD8+ T-Cell Responses Are Associated with Viral Escape Mutations Located in Highly Conserved Regions of Human Immunodeficiency Virus Type 1▿ ‡ 
Journal of Virology  2008;83(4):1845-1855.
The control of human immunodeficiency virus type 1 (HIV-1) associated with particular HLA class I alleles suggests that some CD8+ T-cell responses may be more effective than others at containing HIV-1. Unfortunately, substantial diversities in the breadth, magnitude, and function of these responses have impaired our ability to identify responses most critical to this control. It has been proposed that CD8 responses targeting conserved regions of the virus may be particularly effective, since the development of cytotoxic T-lymphocyte (CTL) escape mutations in these regions may significantly impair viral replication. To address this hypothesis at the population level, we derived near-full-length viral genomes from 98 chronically infected individuals and identified a total of 76 HLA class I-associated mutations across the genome, reflective of CD8 responses capable of selecting for sequence evolution. The majority of HLA-associated mutations were found in p24 Gag, Pol, and Nef. Reversion of HLA-associated mutations in the absence of the selecting HLA allele was also commonly observed, suggesting an impact of most CTL escape mutations on viral replication. Although no correlations were observed between the number or location of HLA-associated mutations and protective HLA alleles, limiting the analysis to mutations selected by acute-phase immunodominant responses revealed a strong positive correlation between mutations at conserved residues and protective HLA alleles. These data suggest that control of HIV-1 may be associated with acute-phase CD8 responses capable of selecting for viral escape mutations in highly conserved regions of the virus, supporting the inclusion of these regions in the design of an effective vaccine.
doi:10.1128/JVI.01061-08
PMCID: PMC2643763  PMID: 19036810

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