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Numerous studies have demonstrated that CD8 + T lymphocytes suppress virus replication during human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) infection. However, the mechanisms underlying this activity of T cells remain incompletely understood. Here, we conducted CD8 + T lymphocyte depletion in 15 rhesus macaques (RMs) infected intravenously (i.v.) with SIV mac239 . At day 70 postinfection, the animals (10 progressors with high viremia and 5 controllers with low viremia) were CD8 depleted by i.v. administration of the antibody M-T807R1. As expected, CD8 depletion resulted in increased virus replication, more prominently in controllers than progressors, which correlated inversely with predepletion viremia. Of note, the feature of CD8 + T lymphocyte predepletion that correlated best with the increase in viremia postdepletion was the level of CD8 + T-bet + lymphocytes. We next found that CD8 depletion resulted in a homogenous increase of SIV RNA in superficial and mesenteric lymph nodes, spleen, and the gastrointestinal tract of both controllers and progressors. Interestingly, the level of SIV DNA increased postdepletion in both CD4 + central memory T lymphocytes (T CM ) and CD4 + effector memory T lymphocytes (T EM ) in progressor RMs but decreased in the CD4 + T CM of 4 out of 5 controllers. Finally, we found that CD8 depletion is associated with a greater increase in CD4 + T lymphocyte activation (measured by Ki-67 expression) in controllers than in progressors. Overall, these data reveal a differential impact of CD8 + T lymphocyte depletion between controller and progressor SIV-infected RMs, emphasizing the complexity of the in vivo antiviral role of CD8 + T lymphocytes. IMPORTANCE In this study, we further dissect the impact of CD8 + T lymphocytes on HIV/SIV replication during SIV infection. CD8 + T lymphocyte depletion leads to a relatively homogenous increase in viral replication in peripheral blood and tissues. CD8 + T lymphocyte depletion resulted in a more prominent increase in viral loads and CD4 + T lymphocyte activation in controllers than in progressors. Interestingly, we found T-bet expression on CD8 + T lymphocytes to be the best predictor of viral load increase following depletion. The levels of SIV DNA increase postdepletion in both CD4 + T CM and T EM in progressor RMs but decrease in the CD4 + T CM of controllers. The findings described in this study provide key insights into the differential functions of CD8 + T lymphocytes in controller and progressor RMs.

The well-established safety profile of the tuberculosis vaccine strain,Mycobacterium bovisbacille Calmette-Guérin (BCG), makes it an attractive vehicle for heterologous expression of antigens from clinically relevant pathogens. However, successful generation of recombinant BCG strains possessing consistent insert expression has encountered challenges in stability. Here, we describe a method for the development of large recombinant BCG accession lots which stably express the lentiviral antigens, human immunodeficiency virus (HIV) gp120 and simian immunodeficiency virus (SIV) Gag, using selectable leucine auxotrophic complementation. Successful establishment of vaccine stability stems from stringent quality control criteria which not only screen for highly stable complemented BCG ΔleuCDtransformants but also thoroughly characterize postproduction quality. These parameters include consistent production of correctly sized antigen, retention of sequence-pure plasmid DNA, freeze-thaw recovery, enumeration of CFU, and assessment of cellular aggregates. Importantly, these quality assurance procedures were indicative of overall vaccine stability, were predictive for successful antigen expression in subsequent passaging bothin vitroandin vivo, and correlated with induction of immune responses in murine models. This study has yielded a quality-controlled BCG ΔleuCDvaccine expressing HIV gp120 that retained stable full-length expression after 1024-fold amplificationin vitroand following 60 days of growth in mice. A second vaccine lot expressed full-length SIV Gag for >1068-fold amplificationin vitroand induced potent antigen-specific T cell populations in vaccinated mice. Production of large, well-defined recombinant BCG ΔleuCDlots can allow confidence that vaccine materials for immunogenicity and protection studies are not negatively affected by instability or differences between freshly grown production batches.

Natural hosts of simian immunodeficiency virus (SIV), African green monkeys (AGMs), rarely transmit SIV via breast-feeding. In order to examine the genetic diversity of breast milk SIV variants in this limited-transmission setting, we performed phylogenetic analysis on envelope sequences of milk and plasma SIV variants of AGMs. Low-diversity milk virus populations were compartmentalized from that in plasma. However, this compartmentalization was transient, as the milk virus lineages did not persist longitudinally.

Antibodies raised in Indian rhesus macaques [Macaca mulatta (MM)] in many preclinical vaccine studies are often evaluated in vitro for titer, antigen-recognition breadth, neutralization potency, and/or effector function, and in vivo for potential associations with protection. However, despite reliance on this key animal model in translation of promising candidate vaccines for evaluation in first in man studies, little is known about the properties of MM immunoglobulin G (IgG) subclasses and how they may compare to human IgG subclasses. Here, we evaluate the binding of MM IgG1, IgG2, IgG3, and IgG4 to human Fc gamma receptors (FcγR) and their ability to elicit the effector functions of human FcγR-bearing cells, and unlike in humans, find a notable absence of subclasses with dramatically silent Fc regions. Biophysical, in vitro, and in vivo characterization revealed MM IgG1 exhibited the greatest effector function activity followed by IgG2 and then IgG3/4. These findings in rhesus are in contrast with the canonical understanding that IgG1 and IgG3 dominate effector function in humans, indicating that subclass-switching profiles observed in rhesus studies may not strictly recapitulate those observed in human vaccine studies.

Indian rhesus macaques (Macaca mulatta) are routinely used in preclinical studies to evaluate therapeutic Abs and candidate vaccines. The efficacy of these interventions in many cases is known to rely heavily on the ability of Abs to interact with a set of Ab FcγR expressed on innate immune cells. Yet, despite their presumed functional importance, M. mulatta Ab receptors are largely uncharacterized, posing a fundamental limit to ensuring accurate interpretation and translation of results from studies in this model. In this article, we describe the binding characteristics of the most prevalent allotypic variants of M. mulatta FcγR for binding to both human and M. mulatta IgG of varying subclasses. The resulting determination of the affinity, specificity, and glycan sensitivity of these receptors promises to be useful in designing and evaluating studies of candidate vaccines and therapeutic Abs in this key animal model and exposes significant evolutionary divergence between humans and macaques.

Summary Infection with HIV persists despite suppressive antiretroviral therapy (ART), and treatment interruption results in rapid viral rebound. Antibody-mediated CD8 + lymphocyte depletion in simian immunodeficiency virus (SIV)-infected rhesus macaques (RMs) shows that these cells contribute to viral control in untreated animals. However, the contribution of CD8 + lymphocytes to maintaining viral suppression under ART remains unknown. Here, we have shown that in SIV-infected RMs treated with short-term (i.e., 8–32 week) ART, depletion of CD8 + lymphocytes resulted in increased plasma viremia in all animals and that repopulation of CD8 + T cells was associated with prompt reestablishment of virus control. Although the number of SIV-DNA-positive cells remained unchanged after CD8 depletion and reconstitution, the frequency of SIV-infected CD4 + T cells before depletion positively correlated with both the peak and area under the curve of viremia after depletion. These results suggest a role for CD8 + T cells in controlling viral production during ART, thus providing a rationale for exploring immunotherapeutic approaches in ART-treated HIV-infected individuals.

Simian immunodeficiency virus (SIV) infection of natural hosts is characterized by nonpathogenic chronic viremia, maintenance of gastrointestinal epithelial barrier integrity, and low numbers of target cells. Assessment of cell-associated virus load in T cell subsets in multiple anatomic compartments of chronically SIV-infected sabeus African green monkeys (AGMs) revealed that gastrointestinal memory CD4 + T lymphocytes are a major source of cell-associated virus and a significant contributor to SIV viremia in AGMs.

The design of immunologic interventions to prevent postnatal transmission of human immunodeficiency virus (HIV) will require identification of protective immune responses in this setting. Simian immunodeficiency virus (SIV)-infected rhesus monkeys (RMs), a species that develops an AIDS-like illness following experimental infection, transmit the virus at a high rate during breastfeeding. In contrast, postnatal transmission of SIV occurs rarely or not at all in natural, asymptomatic primate hosts of SIV. These contrasting transmission patterns provide a unique opportunity to study mechanisms that evolved to protect suckling infants from SIV infection. We compared the virologic and immunologic properties of milk of SIV-infected and uninfected natural hosts of SIV, African green monkeys (AGMs), to that of RMs. Interestingly, despite a low number of milk CD4 + T lymphocytes in uninfected AGMs, milk virus RNA load in SIV-infected AGMs was comparable to that of SIV-infected RMs and that in AGM plasma. This observation is in contrast to the relatively low virus load in milk compared to that in plasma of SIV-infected RMs and HIV-infected women. Milk of SIV-infected AGMs also displayed robust virus-specific cellular immune responses. Importantly, an autologous challenge virus-specific neutralization response was detected in milk of five of six SIV-infected AGMs that was comparable in magnitude to that in plasma. In contrast, autologous challenge virus neutralization was not detectable in milk of SIV-infected RMs. The autologous virus-specific adaptive immune responses in breast milk of AGMs may contribute to impedance of virus transmission in the infant oral/gastrointestinal tract and the rarity of postnatal virus transmission in natural hosts of SIV.

The design of an effective AIDS vaccine has eluded the efforts of the scientific community to the point that alternative approaches to classic vaccine formulations have to be considered. We propose here that HIV vaccine research could greatly benefit from the study of natural simian immunodeficiency virus (SIV) infections of African nonhuman primates. Natural SIV hosts (for example, sooty mangabeys, African green monkeys and mandrills) share many features of HIV infection of humans; however, they usually do not develop immunodeficiency. These natural, nonprogressive SIV infections represent an evolutionary adaptation that allows a peaceful coexistence of primate lentiviruses and the host immune system. This adaptation does not result in reduced viral replication but, rather, involves phenotypic changes to CD4(+) T cell subsets, limited immune activation and preserved mucosal immunity, all of which contribute to the avoidance of disease progression and, possibly, to the reduction of vertical SIV transmission. Here we summarize the current understanding of SIV infection of African nonhuman primates and discuss how unraveling these evolutionary adaptations may provide clues for new vaccine designs that might induce effective immune responses without the harmful consequences of excessive immune activation.

Depletion of CD8(+) lymphocytes during acute simian immunodeficiency virus (SIV) infection of rhesus macaques (RMs) results in irreversible prolongation of peak-level viral replication and rapid disease progression, consistent with a major role for CD8(+) lymphocytes in determining postacute-phase viral replication set points. However, we report that CD8(+) lymphocyte depletion is also associated with a dramatic induction of proliferation among CD4(+) effector memory T (T(EM)) cells and, to a lesser extent, transitional memory T (T(TrM)) cells, raising the question of whether an increased availability of optimal (activated/proliferating), CD4(+)/CCR5(+) SIV "target" cells contributes to this accelerated pathogenesis. In keeping with this, depletion of CD8(+) lymphocytes in SIV(-) RMs led to a sustained increase in the number of potential CD4(+) SIV targets, whereas such depletion in acute SIV infection led to increased target cell consumption. However, we found that the excess CD4(+) T(EM) cell proliferation of CD8(+) lymphocyte-depleted, acutely SIV-infected RMs was completely inhibited by interleukin (IL)-15 neutralization, and that this inhibition did not abrogate the rapidly progressive infection in these RMs. Moreover, although administration of IL-15 during acute infection induced robust CD4(+) T(EM) and T(TrM) cell proliferation, it did not recapitulate the viral dynamics of CD8(+) lymphocyte depletion. These data suggest that CD8(+) lymphocyte function has a larger impact on the outcome of acute SIV infection than the number and/or activation status of target cells available for infection and viral production.

Acute human immunodeficiency virus (HIV) infection represents a period of intense immune perturbation and activation of the host immune system. Study of the eclipse and viral expansion phases of infection is difficult in humans, but studies in nonprogressive and progressive nonhuman primate (NHP) infection models can provide significant insight into critical events occurring during this time. Cytokines, chemokines, and other soluble immune factors were measured in longitudinal samples from rhesus macaques infected with either SIVmac251 (progressive infection) or SIVmac239Δ nef (attenuated/nonprogressive infection) and from African green monkeys infected with SIVsab9315BR (nonpathogenic infection). Levels of acute-phase peak viral replication were highest in SIVmac251 infection but correlated positively with viremia at 3 months postinfection in all three infection models. SIVmac251 infection was associated with stronger corresponding acute-phase cytokine/chemokine responses than the nonprogressive infections. The production of interleukin 15 (IL-15), IL-18, gamma interferon (IFN-γ), granulocyte colony-stimulating factor (G-CSF), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1β (MIP-1β), and serum amyloid A protein (SAA) during acute SIVmac251 infection, but not during SIVmac239Δ nef or SIVsab9315BR infection, correlated positively with chronic viremia at 3 months postinfection. Acute-phase production of MCP-1 correlated with viremia at 3 months postinfection in both nonprogressive infections. Finally, a positive correlation between the acute-phase area under the curve (AUC) for IL-6 and soluble CD40 ligand (sCD40L) and chronic viremia was observed only for the nonprogressive infection models. While we observed dynamic acute inflammatory immune responses in both progressive and nonprogressive SIV infections, the responses in the nonprogressive infections were not only lower in magnitude but also qualitatively different biomarkers of disease progression. IMPORTANCE NHP models of HIV infection constitute a powerful tool with which to study viral pathogenesis in order to gain critical information for a better understanding of HIV infection in humans. Here we studied progressive and nonprogressive simian immunodeficiency virus (SIV) infection models in both natural and nonnatural host NHP species. Regardless of the pathogenicity of the virus infection and regardless of the NHP species studied, the magnitude of viremia, as measured by area under the curve, during the first 4 weeks of infection correlated positively with viremia in chronic infection. The magnitude of cytokine and chemokine responses during primary infection also correlated positively with both acute-phase and chronic viremia. However, the pattern and levels of specific cytokines and chemokines produced differed between nonprogressive and progressive SIV infection models. The qualitative differences in the early immune response in pathogenic and nonpathogenic infections identified here may be important determinants of the subsequent disease course.

To date, most therapeutic and vaccine candidates for human immunodeficiency virus type 1 (HIV-1) are evaluated preclinically for efficacy against cell-free viral challenges. However, cell-associated HIV-1 is suggested to be a major contributor to sexual transmission by mucosal routes. To determine if neutralizing antibodies or inhibitors block cell-free and cell-associated virus transmission of diverse HIV-1 strains with different efficiencies, we tested 12 different antibodies and five inhibitors against four green fluorescent protein (GFP)-labeled HIV-1 envelope (Env) variants from transmitted/founder (T/F) or chronic infection isolates. We evaluated antibody/inhibitor-mediated virus neutralization using either TZM-bl target cells, in which infectivity was determined by virus-driven luciferase expression, or A3R5 lymphoblastoid target cells, in which infectivity was evaluated by GFP expression. In both the TZM-bl and A3R5 assays, cell-free virus or infected CD4 + lymphocytes were used as targets for neutralization. We further hypothesized that the combined use of specific neutralizing antibodies targeting HIV-1 Env would more effectively prevent cell-associated virus transmission than the use of individual antibodies. The tested antibody combinations included two gp120-directed antibodies, VRC01 and PG9, or VRC01 with the gp41-directed antibody 10E8. Our results demonstrated that cell-associated virus was less sensitive to neutralizing antibodies and inhibitors, particularly using the A3R5 neutralization assay, and the potencies of these neutralizing agents differed among Env variants. A combination of different neutralizing antibodies that target specific sites on gp120 led to a significant reduction in cell-associated virus transmission. These assays will help identify ideal combinations of broadly neutralizing antibodies to use for passive preventive antibody administration and further characterize targets for the most effective neutralizing antibodies/inhibitors. IMPORTANCE Prevention of the transmission of human immunodeficiency virus type 1 (HIV-1) remains a prominent goal of HIV research. The relative contribution of HIV-1 within an infected cell versus cell-free HIV-1 to virus transmission remains debated. It has been suggested that cell-associated virus is more efficient at transmitting HIV-1 and more difficult to neutralize than cell-free virus. Several broadly neutralizing antibodies and retroviral inhibitors are currently being studied as potential therapies against HIV-1 transmission. The present study demonstrates a decrease in neutralizing antibody and inhibitor efficiencies against cell-associated compared to cell-free HIV-1 transmission among different strains of HIV-1. We also observed a significant reduction in virus transmission using a combination of two different neutralizing antibodies that target specific sites on the outermost region of HIV-1, the virus envelope. Therefore, our findings support the use of antibody combinations against both cell-free and cell-associated virus in future candidate therapy regimens.

HIV-1 mucosal transmission begins with virus or virus-infected cells moving through mucus across mucosal epithelium to infect CD4+ T cells. Although broadly neutralizing antibodies (bnAbs) are the type of HIV-1 antibodies that are most likely protective, they are not induced with current vaccine candidates. In contrast, antibodies that do not neutralize primary HIV-1 strains in the TZM-bl infection assay are readily induced by current vaccine candidates and have also been implicated as secondary correlates of decreased HIV-1 risk in the RV144 vaccine efficacy trial. Here, we have studied the capacity of anti-Env monoclonal antibodies (mAbs) against either the immunodominant region of gp41 (7B2 IgG1), the first constant region of gp120 (A32 IgG1), or the third variable loop (V3) of gp120 (CH22 IgG1) to modulate in vivo rectal mucosal transmission of a high-dose simian-human immunodeficiency virus (SHIV-BaL) in rhesus macaques. 7B2 IgG1 or A32 IgG1, each containing mutations to enhance Fc function, was administered passively to rhesus macaques but afforded no protection against productive clinical infection while the positive control antibody CH22 IgG1 prevented infection in 4 of 6 animals. Enumeration of transmitted/founder (T/F) viruses revealed that passive infusion of each of the three antibodies significantly reduced the number of T/F genomes. Thus, some antibodies that bind HIV-1 Env but fail to neutralize virus in traditional neutralization assays may limit the number of T/F viruses involved in transmission without leading to enhancement of viral infection. For one of these mAbs, gp41 mAb 7B2, we provide the first co-crystal structure in complex with a common cyclical loop motif demonstrated to be critical for infection by other retroviruses., Author Summary Antibodies specifically recognize antigenic sites on pathogens and can mediate multiple antiviral functions through engagement of effector cells via their Fc region. Current HIV-1 vaccine candidates induce polyclonal antibody responses with multiple antiviral functions, but do not induce broadly neutralizing antibodies. An improved understanding of whether certain types of non-neutralizing HIV-1 specific antibodies can individually protect against HIV-1 infection may facilitate vaccine development. Here, we test whether non-neutralizing antibodies with multiple antiviral functions mediated through FcR engagement and recognition of virus particles or virus-infected cells can limit infection, despite lacking classical virus neutralization activity. In a passive antibody infusion-rhesus macaque challenge model, we tested the ability of non-neutralizing monoclonal antibodies to limit virus acquisition. We demonstrate that two different types of non-neutralizing antibodies, one that recognizes both virus particles and infected cells (7B2) and another that recognizes only infected cells (A32) were capable of decreasing the number of transmitted founder viruses. Further, we provide the structure of 7B2 in complex with the gp41 cyclical loop motif, a motif critical for entry. These findings provide insights into the role that antibodies with antiviral properties, including virion capture and FcR mediated effector function, may play in protecting against HIV-1 acquisition.

Vaccination with live vaccinia virus affords long-lasting protection against variola virus, the agent of smallpox. Its mode of protection in humans, however, has not been clearly defined. Here we report that vaccinia-specific B-cell responses are essential for protection of macaques from monkeypox virus, a variola virus ortholog. Antibody-mediated depletion of B cells, but not CD4+ or CD8+ T cells, abrogated vaccine-induced protection from a lethal intravenous challenge with monkeypox virus. In addition, passive transfer of human vaccinia-neutralizing antibodies protected nonimmunized macaques from severe disease. Thus, vaccines able to induce long-lasting protective antibody responses may constitute realistic alternatives to the currently available smallpox vaccine (Dryvax).

Since most human immunodeficiency virus (HIV) infections are initiated following mucosal exposure to the virus, the anatomic containment or abortion of an HIV infection is likely to require vaccine-elicited cellular immune responses in those mucosal sites. Studying vaccine-elicited mucosal immune responses has been problematic because of the difficulties associated with sampling T lymphocytes from those anatomic compartments. In the present study, we demonstrate that mucosal cytotoxic T lymphocytes (CTL) specific for simian immunodeficiency virus (SIV) and simian HIV can be reproducibly sampled from intestinal mucosal tissue of rhesus monkeys obtained under endoscopic guidance. These lymphocytes recognize peptide-major histocompatibility complex class I complexes and express gamma interferon on exposure to peptide antigen. Interestingly, systemic immunization of monkeys with plasmid DNA immunogens followed by live recombinant attenuated poxviruses or adenoviruses with genes deleted elicits high-frequency SIV-specific CTL responses in these mucosal tissues. These studies therefore suggest that systemic delivery of potent HIV immunogens may suffice to elicit substantial mucosal CTL responses.

Objective To directly examine the role of CD8+ T cells in controlling viremia and disease during chronic, low-level primate immunodeficiency virus infection in DNA prime/protein boost-vaccinated macaques. Background A cohort of macaques, vaccinated with either a DNA prime/HIV-1 gp160 boost regimen or with gp160 alone was previously protected partially from sequential challenges with non-pathogenic and pathogenic strains of chimeric simian/human immunodeficiency virus (SHIV). In this study, the effect of temporary ablation of CD8+ T cells in these animals was examined. Methods Animals were treated with an anti-CD8 antibody and CD8+ T-cell levels in peripheral blood, plasma viral loads, peripheral blood mononuclear cell-associated virus levels, neutralizing antibody (nAb) titers and simian immunodeficiency virus Gag-specific CD8+ T-cell numbers were followed. Results Plasma viremia rose sharply in direct synchrony with a rapid but transient drop in CD8+ T cells. However, although levels of cell-associated virus also rose concomitantly, peak levels were much lower than those in virus-challenged, naive animals. In addition, despite a rise of pathogenic SHIV89.6P RNA levels in three animals, CD4+ T-cell counts remained unchanged. In each of these animals, neutralizing antibody titers against the pathogenic SHIV89.6P strain were high. Conclusions The results indicate that CD8+ T cells play a key role in suppressing viremia in a chronically infected host. In addition, the results suggest that in the absence of CD8+ T cells, nAb may act as an effective second line of defense by limiting both the spread of infectious virus to new target cells and CD4+ T-cell loss.

Mycobacteria, the etiological agents of tuberculosis and leprosy, have coevolved with mammals for millions of years and have numerous ways of suppressing their host's immune response. It has been suggested that mycobacteria may contain genes that reduce the host's ability to elicit CD8 + T cell responses. We screened 3,290 mutant Mycobacterium bovis bacillus Calmette Guerin (BCG) strains to identify genes that decrease major histocompatibility complex (MHC) class I presentation of mycobacterium-encoded epitope peptides. Through our analysis, we identified 16 mutant BCG strains that generated increased transgene product-specific CD8 + T cell responses. The genes disrupted in these mutant strains had disparate predicted functions. Reconstruction of strains via targeted deletion of genes identified in the screen recapitulated the enhanced immunogenicity phenotype of the original mutant strains. When we introduced the simian immunodeficiency virus (SIV) gag gene into several of these novel BCG strains, we observed enhanced SIV Gag-specific CD8 + T cell responses in vivo . This study demonstrates that mycobacteria carry numerous genes that act to dampen CD8 + T cell responses and suggests that genetic modification of these genes may generate a novel group of recombinant BCG strains capable of serving as more effective and immunogenic vaccine vectors.

Live attenuated nonpathogenic Mycobacterium bovis bacillus Calmette-Guérin (BCG) mediates long-lasting immune responses, has been safely administered as a tuberculosis vaccine to billions of humans, and is affordable to produce as a vaccine vector. These characteristics make it very attractive as a human immunodeficiency virus (HIV) vaccine vector candidate. Here, we assessed the immunogenicity of recombinant BCG (rBCG) constructs with different simian immunodeficiency virus (SIV) gag expression cassettes as priming agents followed by a recombinant replication-incompetent New York vaccinia virus (NYVAC) boost in rhesus macaques. Unmutated rBCG constructs were used in comparison to mutants with gene deletions identified in an in vitro screen for augmented immunogenicity. We demonstrated that BCG-SIV gag is able to elicit robust transgene-specific priming responses, resulting in strong SIV epitope-specific cellular immune responses. While enhanced immunogenicity was sustained at moderate levels for >1 year following the heterologous boost vaccination, we were unable to demonstrate a protective effect after repeated rectal mucosal challenges with pathogenic SIVmac251. Our findings highlight the potential for rBCG vaccines to stimulate effective cross-priming and enhanced major histocompatibility complex class I presentation, suggesting that combining this approach with other immunogens may contribute to the development of effective vaccine regimens against HIV.

In this review, the roles of Fc-gamma receptor polymorphisms are discussed in regards to HIV-1 vaccine efficacy, HIV acquisition, and disease progression. In addition, the significance of the neonatal immunoglobulin G (IgG) Fc receptor and potential effects of the aggregated immunoglobulin A Fc receptor (FcalphaR) are addressed.Fc receptors undoubtedly play an important role in antibody-mediated action in HIV infection and vaccines. Several studies have determined an association between polymorphic variants of Fc-gamma-RIIA and Fc-gamma-RIIIA in the acquisition and progression of HIV-1 infection, and in responses to vaccination regimens. A rather complex relationship exists between the relative affinity of these molecules and their impact on HIV disease acquisition and progression and HIV vaccine efficacy.The discrepancies between different investigations of the role of Fc receptor polymorphisms appear to derive from the complex nature of the Fc receptor functions, including factors such as epistatic interactions and the race, sex, age, and relative risk behavior of the investigated individuals. Furthermore, Fc receptors in nonhuman primates (NHPs), the key model to study an AIDS-like disease in an animal model, appear to be even more diverse than in humans, and the function of these proteins has not been extensively explored. Given the critical role of Fc receptors in antibody-mediated function in humans and NHP, more investigations are needed to fully understand and exploit these functions for vaccine design.

The T cell-stimulatory cytokine interleukin 2 (IL-2) is being evaluated as a therapeutic in the clinical settings of HIV infection and cancer. However, the clinical utility of IL-2 may be mitigated by its short in vivo half-life, toxic effects, and high production costs. We show here that an IL-2/Ig fusion protein possesses IL-2 immunostimulatory activity in vitro and a long in vivo half-life. IL-2/Ig treatment of healthy rhesus monkeys induced significant increases in CD4(+) T lymphocyte counts and expression of CD25 by these cells. Short courses of IL-2/Ig treatment of simian immunodeficiency virus (SIV)-infected rhesus monkeys in conjunction with antiretroviral drugs resulted in increased CD25 expression on T lymphocytes, and transient increases in CD4(+) T lymphocyte counts. Plasma viremia did not increase in these treated animals. Treatment of healthy or SIV-infected rhesus monkeys with a plasmid encoding the IL-2/Ig protein did not affect CD4(+) T lymphocytes. These results demonstrate that IL-2/Ig has potential utility as an immunostimulatory therapeutic.

A costimulatory signal in addition to an Ag-specific stimulus is required for optimal activation of T lymphocytes. CD28, the primary positive costimulatory receptor on T cells, has two identified ligands, B7-1 and B7-2. Whether B7-1 and B7-2 have identical, overlapping, or distinct functions remains unresolved. In this study, we show that mice lacking B7-2 were unable to generate CTL responses following immunization with a plasmid DNA vaccine. The ability of these B7-2-deficient mice to generate CTL responses following plasmid gp120 DNA vaccination was fully reconstituted by coadministering either a plasmid expressing B7-2 or B7-1. Moreover, the ability to generate CTL responses following plasmid DNA vaccination in mice lacking both B7-1 and B7-2 could be reconstituted by administering either plasmid B7-1 or plasmid B7-2 with the vaccine construct. These data demonstrate that either B7-1 or B7-2 administered concurrently with a plasmid DNA vaccine can fully costimulate vaccine-elicited CTL responses. Functional differences between B7-1 and B7-2 observed in vivo therefore may not reflect inherent differences in the interactions of CD28 with these ligands.

Recombinant Mycobacterium bovis bacillus Calmette-Guerin (rBCG) has been explored as a vector for vaccines against HIV because of its ability to induce long lasting humoral and cell mediated immune responses. To maximize the potential for rBCG vaccines to induce effective immunity against HIV, various strategies are being employed to improve its ability to prime CD8+ T cells, which play an important role in the control of HIV infections. In this study we adopted a previously described approach of incorporating glycolipids that activate CD1d-restricted natural killer T (NKT) cells to enhance priming of CD8+ T cells by rBCG strains expressing an SIV Gag antigen (rBCG-SIV gag). We found that the incorporation of the synthetic NKT activating glycolipid α-galactosylceramide (α-GC) into rBCG-SIV gag significantly enhanced CD8+ T cell responses against an immunodominant Gag epitope, compared to responses primed by unmodified rBCG-SIV gag. The abilities of structural analogues of α-GC to enhance CD8+ T cell responses to rBCG were compared in both wild type and partially humanized mice that express human CD1d molecules in place of mouse CD1d. These studies identified an α-GC analogue known as 7DW8-5, which has previously been used successfully as an adjuvant in non-human primates, as a promising compound for enhancing immunogenicity of antigens delivered by rBCG.vectors. Our findings support the incorporation of synthetic glycolipid activators of NKT cells as a novel approach to enhance the immunogenicity of rBCG-vectored antigens for induction of CD8+ T cell responses. The glycolipid adjuvant 7DW8-5 may be a promising candidate for advancing to non-human primate and human clinical studies for the development of HIV vaccines based on rBCG vectors.

Granzyme B (GrzB) is expressed by activated T cells and mediates cellular apoptosis. GrzB also acts as an extracellular protease involved in tissue degradation. We hypothesized that GrzB production from activated memory CD4 T cells may be associated with HIV pathogenesis. We found that stimulated memory CD4 T cells (via costimulation, cytokines, and TLR ligands) concomitantly produced GrzB and HIV. Both GrzB and HIV expression were mainly restricted to CCR5-expressing memory CD4+CD45RO+ T cells, including Th1 and Th17 subsets. Activated memory CD4 T cells also mediated tissue damage, such as disruption of intestinal epithelial monolayers. In non-human primates, CD4 T cells of rhesus macaques (pathogenic SIV hosts) expressed higher GrzB compared to African green monkeys (non-pathogenic SIV hosts). These results suggest that GrzB from CCR5+ memory CD4 T cells may have a role in cellular and tissue pathologies during HIV infection.

Soon after the discovery of HIV-infected humans, rhesus macaques in a colony at the New England Primate Research Center showed similar signs of a progressive immune suppression. The discovery of the simian immunodeficiency virus (SIV)-associated disease opened the door to study an AIDS-like illness in nonhuman primates (NHP). Even after 3 decades, this animal model remains an invaluable tool to provide a greater insight into HIV immunopathogenesis. In this review, recent progress in deciphering pathways of immunopathogenesis in SIV-infected NHP is discussed.The immense diversity of mutations in SIV stocks prepared at different laboratories has recently been realized. The massive expansion of the enteric virome is a key finding in SIV-induced immunopathogenesis. Defining the function of host restriction factors, like the recently discovered SAMHD1, helps to evaluate the impact of the innate immune responses on virus replication. Utilization of pyrosequencing and defining molecular mechanisms of major histocompatibility complex (MHC) class I restriction helps to understand how the virus evades CD8 T-cell responses. The definition of MHC class I molecules in different NHP species provides new animal models to study SIV immunopathogenesis. T follicular helper cells have gained major interest in characterizing humoral immune responses in SIV infection and AIDS vaccine strategies. The ability of natural hosts to remain disease-free despite ongoing replication of SIV is continuing to puzzle the field.The HIV research field continues to realize the immense complexity of the host virus interaction. NHP present an invaluable tool to make progress towards an effective AIDS vaccine.

Vaccination and SIV challenge of macaque species is the best animal model for evaluating candidate HIV vaccines in pre-clinical studies. As such, robust assays optimized for use in nonhuman primates are necessary for reliable ex vivo measurement of immune responses and identification of potential immune correlates of protection. We optimized and qualified an 8-color intracellular cytokine staining assay for the measurement of IFNγ, IL-2, and TNF from viable CD4 and CD8 T cells from cryopreserved rhesus macaque PBMC stimulated with peptides. After optimization, five laboratories tested assay performance using the same reagents and PBMC samples; similar results were obtained despite the use of flow cytometers with different configurations. The 8-color assay was then subjected to a pre-qualification study to quantify specificity and precision. These data were used to set positivity thresholds and to design the qualification protocol. Upon completion of the qualification study, the assay was shown to be highly reproducible with low inter-aliquot, inter-day, and inter-operator variability according to the qualification criteria with an overall variability of 20–40% for each outcome measurement. Thus, the 8-color ICS assay was formally qualified according to the ICH guidelines Q2 (R1) for specificity and precision indicating that it is considered a standardized/robust assay acceptable for use in pre-clinical trial immunogenicity testing.

While CD8+ T cells are clearly important in controlling virus replication during HIV and SIV infections, the mechanisms underlying this antiviral effect remain poorly understood. In this study, we assessed the in vivo effect of CD8+ lymphocyte depletion on the lifespan of productively infected cells during chronic SIVmac239 infection of rhesus macaques. We treated two groups of animals that were either CD8+ lymphocyte-depleted or controls with antiretroviral therapy, and used mathematical modeling to assess the lifespan of infected cells either in the presence or absence of CD8+ lymphocytes. We found that, in both early (day 57 post-SIV) and late (day 177 post-SIV) chronic SIV infection, depletion of CD8+ lymphocytes did not result in a measurable increase in the lifespan of either short- or long-lived productively infected cells in vivo. This result indicates that the presence of CD8+ lymphocytes does not result in a noticeably shorter lifespan of productively SIV-infected cells, and thus that direct cell killing is unlikely to be the main mechanism underlying the antiviral effect of CD8+ T cells in SIV-infected macaques with high virus replication., Author Summary Despite overwhelming evidence that CD8+ T cells are important in controlling virus replication during HIV and simian immunodeficiency virus (SIV) infections, the mechanisms responsible for this antiviral effect in vivo remain poorly understood. This lack of knowledge represents a key obstacle to our efforts to develop a CD8+ T cell-based AIDS vaccine. In this study, we implemented a new experimental system in which we determined the lifespan of productively SIV-infected cells in vivo, either in the presence or absence of CD8+ lymphocytes. The lifespan of productively infected cells was calculated based on the slope of the decline of SIV plasma viremia after initiation of ART using a widely accepted mathematical model. Using this novel approach, we determined that CD8+ lymphocytes control virus replication without noticeably decreasing the lifespan of productively infected cells, thus suggesting that the major mechanism of antiviral activity by CD8+ lymphocytes during pathogenic SIV infection may not be direct cell killing of productively SIV-infected cells.

The immunologic basis for the potential enhanced HIV-1 acquisition in adenovirus serotype 5 (Ad5)-seropositive individuals who received the Merck recombinant Ad5 HIV-1 vaccine in the STEP study remains unclear. Here we show that baseline Ad5-specific neutralizing antibodies are not correlated with Ad5-specific T lymphocyte responses and that Ad5-seropositive subjects do not develop higher vector-specific cellular immune responses as compared with Ad5-seronegative subjects after vaccination. These findings challenge the hypothesis that activated Ad5-specific T lymphocytes were the cause of the potential enhanced HIV-1 susceptibility in the STEP study.

Virus-specific CD4+ T cell responses have been shown to play a critical role in controlling HIV-1 replication. Candidate HIV-1 vaccines should therefore elicit potent CD4+ as well as CD8+ T cell responses. In this report we investigate the ability of plasmid GM-CSF to augment CD4+ T cell responses elicited by an HIV-1 gp120 DNA vaccine in mice. Coadministration of a plasmid expressing GM-CSF with the gp120 DNA vaccine led to only a marginal increase in gp120-specific splenocyte CD4+ T cell responses. However, immunization with a bicistronic plasmid that coexpressed gp120 and GM-CSF under control of a single promoter led to a dramatic augmentation of vaccine-elicited CD4+ T cell responses, as measured by both cellular proliferation and ELISPOT assays. This augmentation of CD4+ T cell responses was selective, since vaccine-elicited Ab and CD8+ T cell responses were not significantly changed by the addition of GM-CSF. A 100-fold lower dose of the gp120/GM-CSF bicistronic DNA vaccine was required to elicit detectable gp120-specific splenocyte proliferative responses compared with the monocistronic gp120 DNA vaccine. Consistent with these findings, i.m. injection of the gp120/GM-CSF bicistronic DNA vaccine evoked a more extensive cellular infiltrate at the site of inoculation than the monocistronic gp120 DNA vaccine. These results demonstrate that bicistronic DNA vaccines containing GM-CSF elicit remarkably potent CD4+ T cell responses and suggest that optimal Th cell priming requires the precise temporal and spatial codelivery of Ag and GM-CSF.

The TCR repertoire of an epitope-specific CD8+ T cell population remains poorly characterized. To determine the breadth of the TCR repertoire of a CD8+ T cell population that recognizes a dominant epitope of the AIDS virus, the CD8+ T cells recognizing the tetrameric Mamu-A*01/p11C,CM complex were isolated from simian immunodeficiency virus (SIV)-infected Mamu-A*01+ rhesus monkeys. This CD8+ T cell population exhibited selected usage of TCR Vβ families and complementarity-determining region 3 (CDR3) segments. Although the epitope-specific CD8+ T cell response was clearly polyclonal, a dominance of selected Vβ+ cell subpopulations and clones was seen in the TCR repertoire. Interestingly, some of the selected Vβ+ cell subpopulations and clones maintained their dominance in the TCR repertoire over time after infection with SIV of macaques. Other Vβ+ cell subpopulations declined over time in their relative representation and were replaced by newly evolving clones that became dominant. The present study provides molecular evidence indicating that the TCR repertoire shaped by a single viral epitope is dominated at any point in time by selected Vβ+ cell subpopulations and clones and suggests that dominant Vβ+ cell subpopulations and clones can either be stable or evolve during a chronic infection.

Major histocompatibility class I-peptide tetramer technology and simian immunodeficiency virus of macaques (SIVmac)-infected rhesus monkeys were used to clarify the distribution of acquired immunodeficiency syndrome virus-specific cytotoxic T lymphocytes (CTL) in secondary lymphoid organs and to assess the relationship between these CTL and the extent of viral replication in the various anatomic compartments. SIVmac Gag epitope-specific CD8(+) T cells were evaluated in the spleen, bone marrow, tonsils, thymus, and 5 different lymph node compartments of 4 SIVmac-infected rhesus monkeys. The average percentage of CD8(+) T lymphocytes that bound this tetramer in all the different lymph node compartments was similar to that in peripheral blood lymphocytes in individual monkeys. The percentage of CD8(+) T cells that bound the tetramer in the thymus was uniformly low in the monkeys. However, the percentage of CD8(+) T cells that bound the tetramer in bone marrow and spleen was consistently higher than that seen in lymph nodes and peripheral blood. The phenotypic profile of the tetramer-binding CD8(+) T lymphocytes in the different lymphoid compartments was similar, showing a high expression of activation-associated adhesion molecules and a low level expression of naive T-cell-associated molecules. Surprisingly, no correlation was evident between the percentage of tetramer-binding CD8(+) T lymphocytes and the magnitude of the cell-associated SIV RNA level in each lymphoid compartment of individual monkeys. These studies suggest that a dynamic process of trafficking may obscure the tendency of CTL to localize in particular regional lymph nodes or that some lymphoid organs may provide milieus that are particularly conducive to CTL expansion. (Blood. 2000;96:1474-1479)

We report on the preclinical results of an immunotherapeutic approach of AIDS mediated by ex vivo propagated CD4+ and CD8+ T-cells. A mean yield of 6.23 x 10(9) lymphocytes, containing 1.82 x 10(9) CD4+, 3.23 x 10(9) CD8+ T-lymphocytes and 8.39 x 10(6) CD34+ peripheral blood progenitor cells (PBPC) were be obtained by continuous flow cytapheresis (CFC) in 15 asymptomatic HIV infected patients (CD4-count350/mm3). The CD4/CD8 ratio (mean: 0.53, SD: +/- 0.15) in the cell concentrates reflected the distribution of the circulating lymphocyte subsets in vivo. Absolute lymphocyte counts decreased at a mean of 404/microliter (25%) immediately after CFC but were replaced from the extravascular pool within one hour. Neither the CD4/CD8 ratio nor p24-antigen and neopterin levels did change significantly after cell separation. No alteration of the number of proviral DNA copies (1/10(3)-1/10(6)) could be detected in peripheral T-helper cells by semiquantitative PCR after lymphapheresis. Cells were cryopreserved in liquid nitrogen without substantial loss of viability or function. Ex vivo propagation of T-cells in a strictly autologous manner in the presence of PHA + IL-2 for 14d resulted in a 50-fold expansion rate (140-fold in healthy controls, p0.001). Viral replication could be controlled but not completely eliminated by cocultivation with autologous CD8+ T-lymphocytes as measured by limiting dilution nested PCR (NPCR). The expanded cells showed the typical phenotype of highly activated memory type T-lymphocytes (CD3+ CD45RO+ CD25+ HLA-DR+). The distribution of CD4+ and CD8+ T-cells did not reveal significant changes before and after culture indicating that both subsets were equally expanded. Functionally important membrane or intracellular epitopes which were found to be decreased in HIV infected subjects (CD7, CD55, CD59) before culture were reconstituted after ex vivo propagation of T-cells. The functional importance of the up-regulation of complement regulating epitopes (CD55, CD59) after culture could be proven by a significant inhibition of cytolysis of T-cells in the presence of autologous complement. The majority (75%) of expanded CD8+ T-cells stained positive with mAb TIA-1 which is directed to intracellular granules within cytotoxic T-cells. Furthermore, programmed cell death of expanded T-cells could be prevented by cocultivation with fibroblasts which are believed to secrete a cytokine pattern preventing activated T-cells from apoptosis after withdrawal of IL-2 and other stimuli.(ABSTRACT TRUNCATED AT 400 WORDS)

While CD8+ T cells are clearly important in controlling virus replication during HIV and SIV infections, the mechanisms underlying this antiviral effect remain poorly understood. In this study, we assessed the in vivo effect of CD8+ lymphocyte depletion on the lifespan of productively infected cells during chronic SIVmac239 infection of rhesus macaques. We treated two groups of animals that were either CD8+ lymphocyte-depleted or controls with antiretroviral therapy, and used mathematical modeling to assess the lifespan of infected cells either in the presence or absence of CD8+ lymphocytes. We found that, in both early (day 57 post-SIV) and late (day 177 post-SIV) chronic SIV infection, depletion of CD8+ lymphocytes did not result in a measurable increase in the lifespan of either short- or long-lived productively infected cells in vivo. This result indicates that the presence of CD8+ lymphocytes does not result in a noticeably shorter lifespan of productively SIV-infected cells, and thus that direct cell killing is unlikely to be the main mechanism underlying the antiviral effect of CD8+ T cells in SIV-infected macaques with high virus replication.

Peptides derived from the heptad repeat 2 region (HR2) of HIV gp41 (C-peptides) effectively inhibit entry of HIV at the level of virus membrane fusion. A retroviral vector (M87o) expressing a membrane-anchored C-peptide was developed that also has strong antiviral activity in T cell lines and primary T lymphocytes (Egelhofer et al., J.Virol., 2004). M87o was shown to inhibit viral entry more than 10,000-fold in single round infection assays.

CD8(+) T lymphocytes play a pivotal role in controlling human immunodeficiency virus (HIV)-1 replication in vivo. We have performed four-color flow cytometric analysis of CD8(+) peripheral blood lymphocytes (PBL) from 21 HIV-1 seronegative and 103 seropositive individuals to explore the phenotypic heterogeneity of CD8beta-chain expression on CD8(+) T lymphocytes and to clarify how its expression on CD8(+) T lymphocytes may relate to acquired immunodeficiency syndrome (AIDS) clinical progression. We showed that the single monoclonal antibody (MoAb) 2ST8-5H7, directed against the CD8alpha beta-heterodimer, identifies CD8(+) T lymphocytes as effectively as the conventional combination of anti-CD3 and anti-CD8alpha antibodies. However, we detected a significantly lower mean fluorescence (MF) of anti-CD8alpha beta staining on PBL from HIV-1 seropositive donors as compared with seronegative donors. In fact, CD8(+) T lymphocytes from HIV-1-infected individuals with the lowest CD4 counts showed the lowest levels of CD8alpha beta MF. To explore further this change in CD8alpha beta expression, we assessed the expression of 14 different cell surface molecules on CD8alpha beta+ T lymphocytes of PBL from 11 HIV-1 seronegative and 22 HIV-1 seropositive individuals. The MF of anti-CD8alpha beta staining was significantly reduced on CD8(+) T lymphocyte subsets that showed immunophenotypic evidence of activation. The subset of lymphocytes expressing low levels of CD8alpha beta expressed higher levels of activation, adhesion, and cytotoxic-associated molecules and was predominantly CD45RO+ and CD28(-). Finally, we monitored the expression of the CD8alpha beta-heterodimer on PBL of eight HIV-1-infected individuals over a 16-week period after the initiation of highly active antiretroviral therapy (HAART), including zidovudine (ZDV), lamivudine (3TC), and indinavir (IDV), and found a significant increase in the expression of the CD8alpha beta-heterodimer. These results suggest that antibodies recognizing the CD8alpha beta-heterodimer are useful tools to specifically identify CD8(+) T lymphocytes. Moreover, the quantitative monitoring of CD8alpha beta expression allows the detection of discrete CD8(+) T lymphocyte subsets and may be useful for assessing the immune status of individuals infected with HIV-1.

Blood monocytes as well as cells of the macrophage-phagocytic system in several tissues are targets for HIV-1 in vivo and in vitro. However, the data on HIV-1 infection of liver macrophages/Kupffer cells (KCs), which make up the main pool of fixed-tissue macrophages, is controversial. We therefore studied HIV-1 infection of KCs in vivo. Blood and liver tissue was obtained from seven AIDS patients shortly after death. Liver tissue was minced before processing. Cell suspensions were further purified by density gradient centrifugation, stained with anti-CD14 (blood monocytes) or anti-macrophage 25F9 (KCs), and separated by fluorescence-activated cell sorting (FACS). These highly purified cell populations were then analyzed for HIV-1 proviral DNA by the polymerase chain reaction (PCR). By performing PCR on FACS-purified cell populations, we could show that both KCs and peripheral blood monocytes were HIV-1-infected in 3 of 7 patients. KCs harbored HIV-1 proviral DNA only if peripheral blood monocytes were infected. These data show that KCs of the liver are infected with HIV-1 in vivo.

Antiretroviral therapy for HIV infection requires life-long access and strict adherence to regimens that are both expensive and associated with toxicities. There is growing recognition that a curative intervention will be needed to fully stop the epidemic. The failure to eradicate HIV infection during long-term antiretroviral therapy reflects the intrinsic stability of the viral genome in latently infected CD4+ T cells and other cells and perhaps ongoing low-level viral replication. Heterogeneity in latently infected cell populations and homeostatic proliferation of infected cells may influence the dynamics of virus production and persistence. Chronic immune activation, inflammation and immune dysfunction persist despite potent antiretroviral therapy and likely have important effects on the size and distribution of the viral reservoir. The inability of the immune system to recognize cells harboring latent virus and to eliminate cells actively producing virus represents the biggest challenge to finding a cure. In this perspective, we highlight new approaches toward unraveling the complex virus-host interactions that lead to persistent infection and latency and discuss the rationale for combining novel therapeutic strategies with current antiretroviral treatment options with the goal of curing HIV disease.