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HIV-1 infection greatly alters the NK cell phenotypic and functional repertoire. This is highlighted by the expansion of a rare population of FcRγ- NK cells exhibiting characteristics of traditional immunologic memory in people with HIV (PWH). Although current antiretroviral therapy (ART) effectively controls HIV-1 viremia and disease progression, its impact on HIV-1-associated NK cell abnormalities remains unclear. To address this, we performed a longitudinal analysis detailing conventional and memory-like NK cell characteristics in n = 60 PWH during the first 4 y of ART. Throughout this regimen, a skewed repertoire of cytokine unresponsive FcRγ- memory-like NK cells persisted and accompanied an overall increase in NK surface expression of CD57 and KLRG1, suggestive of progression toward immune senescence. These traits were linked to elevated serum inflammatory biomarkers and increasing Ab titers to human CMV, with human CMV viremia detected in approximately one-third of PWH at years 1-4 of ART. Interestingly, 40% of PWH displayed atypical NK cell subsets, representing intermediate stages of NK-poiesis based on single-cell multiomic trajectory analysis. Our findings indicate that NK cell irregularities persist in PWH despite long-term ART, underscoring the need to better understand the causative mechanisms that prevent full restoration of immune health in PWH., (Copyright © 2024 by The American Association of Immunologists, Inc.)

Ovarian tumor domain-containing 6B ( OTUD6B ) belongs to the OTU deubiquitylating enzyme family. In this study, we report that zebrafish otud6b is induced upon viral infection, and overexpression of otud6b suppresses cellular antiviral response. Disruption of otud6b in zebrafish increases the survival rate upon spring viremia of carp virus and grass carp reovirus exposure. Further assays indicate that otud6b interacts with irf3 and irf7 and diminishes traf6-mediated K63-linked polyubiquitination of irf3 and irf7. In addition, the OTU domain is required for otud6b to repress IFN-1 activation and K63-linked polyubiquitination of irf3 and irf7. Moreover, otud6b also attenuates tbk1 to bind to irf3 and irf7, resulting in the impairment of irf3 and irf7 phosphorylation. This study provides, to our knowledge, novel insights into otud6b function and sheds new lights on the regulation of irf3 and irf7 by deubiquitination in IFN-1 signaling., (Copyright © 2021 by The American Association of Immunologists, Inc.)

The 2019 coronavirus disease (COVID-19) pandemic caused by the virus severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has created an unprecedented global crisis for the infrastructure sectors, including economic, political, healthcare, education, and research systems. Although over 90% of infected individuals are asymptomatic or manifest noncritical symptoms and will recover from the infection, those individuals presenting with critical symptoms are in urgent need of effective treatment options. Emerging data related to mechanism of severity and potential therapies for patients presenting with severe symptoms are scattered and therefore require a comprehensive analysis to focus research on developing effective therapeutics. A critical literature review suggests that the severity of SARS-CoV-2 infection is associated with dysregulation of inflammatory immune responses, which in turn inhibits the development of protective immunity to the infection. Therefore, the use of therapeutics that modulate inflammation without compromising the adaptive immune response could be the most effective therapeutic strategy., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Follicular CD8 + T (fCD8) cells reside within B cell follicles and are thought to be immune-privileged sites of HIV/SIV infection. We have observed comparable levels of fCD8 cells between chronically SIV-infected rhesus macaques with low viral loads (LVL) and high viral loads (HVL), raising the question concerning their contribution to viremia control. In this study, we sought to clarify the role of SIV-specific fCD8 cells in lymph nodes during the course of SIV infection in rhesus macaques. We observed that fCD8 cells, T follicular helper (Tfh) cells, and T follicular regulatory cells (Tfreg) were all elevated in chronic SIV infection. fCD8 cells of LVL animals tended to express more Gag-specific granzyme B and exhibited significantly greater killing than did HVL animals, and their cell frequencies were negatively correlated with viremia, suggesting a role in viremia control. Env- and Gag-specific IL-21 + Tfh of LVL but not HVL macaques negatively correlated with viral load, suggesting better provision of T cell help to fCD8 cells. Tfreg positively correlated with fCD8 cells in LVL animals and negatively correlated with viremia, suggesting a potential benefit of Tfreg via suppression of chronic inflammation. In contrast, in HVL macaques, Tfreg and fCD8 cell frequencies tended to be negatively correlated, and a positive correlation was seen between Tfreg number and viremia, suggesting possible dysfunction and suppression of an effective fCD8 cell immune response. Our data suggest that control of virus-infected cells in B cell follicles not only depends on fCD8 cell cytotoxicity but also on complex fCD8 cell associations with Tfh cells and Tfreg., (Copyright © 2018 by The American Association of Immunologists, Inc.)

NK cells are key components of the immune system because of their rapid response potential and their ability to mediate cytotoxic and immunomodulatory functions. Additionally, NK cells have recently been shown to persist for long periods in vivo and to have the capacity to establish immunologic memory. In the current study, we assessed the phenotype and function of circulatory and tissue-resident NK cells in a unique cohort of SIV-controlling rhesus macaques that maintained low to undetectable levels of viremia in the chronic phase of infection. By contrasting NK responses of these macaques with those observed in SIV-noncontrolling and uninfected macaques, we aimed to identify markers and activities of NK subpopulations associated with disease control. We show in this article that most differences among NK cells of the three groups of macaques were observed in tissue-resident cells. Although SIV infection resulted in NK cell dysfunction, double-negative NK cells and those expressing CXCR3, NKG2D, and IL-18Rα were associated with viremia control, as was Ab-dependent cytotoxic function. Our results suggest several novel targets for therapeutic intervention., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Zika virus (ZIKV) is a mosquito-borne and sexually transmitted flavivirus that is associated with fetal CNS-damaging malformations during pregnancy in humans. This study documents the viral kinetics and immune responses in rhesus macaques infected with a clinical ZIKV Brazilian isolate. We evaluated the viral kinetics and immune responses induced after an i.v. infection with a Brazilian ZIKV clinical isolate (HS-2015-BA-01) in rhesus macaques for up to 142 d. ZIKV-specific Ab-secreting cells, germinal center reactions, and monocyte, dendritic cell, NK, and T cell frequencies were monitored. ZIKV loads were readily detected in plasma (until day 5 or 7), semen and urine (until days 7 and 14), and saliva (until day 42), but the viremia was rapidly controlled. No detectable clinical manifestations were observed. However, lymph node hyperplasia was clearly visible postviremia but was associated with low frequencies of ZIKV-specific Ab-secreting cells in lymph nodes and bone marrow, correlating with low Ab titers. CD14 + /CD16 - monocytes and myeloid CD11c hi dendritic cells decreased in blood, whereas NK and T cell numbers were only marginally altered during the course of the study. ZIKV infection caused a significant lymphoid tissue activation but limited induction of ZIKV-specific B cells, suggesting that these parameters need to be considered for ZIKV vaccine design., (Copyright © 2017 by The American Association of Immunologists, Inc.)

CMV infection in kidney transplant recipients (KTRs) has been associated with an increased risk for graft loss and reduced host survival. CMV promotes persistent expansions of NK cells expressing the CD94/NKG2C receptor. The NKG2C (KLRC2) gene is frequently deleted, and copy number influences the adaptive response of NKG2C + NK cells. The distribution of NKG2C + NK cells and NKG2C genotypes (NKG2C +/+ , NKG2C +/del , NKG2C del/del ) were studied in cross-sectional (n = 253) and prospective (n = 122) KTR cohorts. Assessment of CMV viremia was restricted to symptomatic cases in the retrospective study, but was regularly monitored in the prospective cohort. Overall, the proportions of NKG2C + NK cells were significantly higher in KTRs who had suffered posttransplant symptomatic CMV infection in the cross-sectional study. Yet, along the prospective follow-up (3, 6, 12, and 24 mo), posttransplant NKG2C + NK cell expansions were not observed in every patient with detectable viremia who received preemptive antiviral therapy, suggesting that the adaptive NK cell response may be inversely related with the degree of CMV control. Remarkably, the incidence of posttransplant viremia was reduced among cases with high pretransplant levels of NKG2C + NK cells. The NKG2C genotype distribution was comparable in KTR and healthy controls, and greater proportions of NKG2C + cells were detected in NKG2C +/+ than in NKG2C +/del patients. Yet, a trend toward increased NKG2C +/del and reduced NKG2C +/+ frequencies associated with symptomatic infection was appreciated in both cohorts. Altogether, our results indirectly support that adaptive NKG2C + NK cells are involved in the control of CMV in KTRs., (Copyright © 2016 by The American Association of Immunologists, Inc.)

An established sex bias in HIV pathogenesis is linked to immune responses. Recently we reported a vaccine-induced sex bias: vaccinated female but not male rhesus macaques exhibited delayed SIV acquisition. This outcome was correlated with SIV Env-specific rectal IgA, rectal memory B cells, and total rectal plasma cells. To uncover additional contributing factors, using samples from the same study, we investigated memory B cell population dynamics in blood, bone marrow, and rectal tissue during immunization and postchallenge; IgG subtypes and Ab avidity; and regulatory B (Breg) cell frequency and function. Few sex differences were seen in Env-specific memory B cell, plasmablast, or plasma cell frequencies in the three compartments. Males had higher IgG Ab titers and avidity indices than females. However, females had elevated levels of Env-specific IgG1, IgG2, and IgG3 Abs compared with males. gp140-specific IgG3 Abs of females but not males were correlated with Ab-dependent cell-mediated cytotoxicity activity against gp120 targets (p = 0.026) and with Ab-dependent phagocytic activity (p = 0.010). IgG3 Ab of females but not males also correlated with decreased peak viremia (p = 0.028). Peripheral blood CD19(+)CD25(+) Breg cells suppressed T cell proliferation compared with CD19(+)CD25(-) cells (p = 0.031) and exhibited increased IL-10 mRNA expression (p = 0.031). Male macaques postvaccination (p = 0.018) and postinfection (p = 0.0048) exhibited higher Breg frequencies than females. Moreover, male Breg frequencies correlated with peak viremia (p = 0.0071). Our data suggest that vaccinated females developed better Ab quality, contributing to better functionality. The elevated Breg frequencies in males may have facilitated SIV acquisition.

Immunological competence declines progressively with age, resulting in increased susceptibility of the elderly to infection and impaired responses to vaccines. Underlying mechanisms remain largely obscure as they have been related to complex, individual systemic immune properties that are challenging to investigate. In this study, we explored age-related changes in human immunity during a primary virus infection experimentally induced by immunization with live-attenuated yellow fever (YF) vaccine. Applying detailed serology, advanced FACS analysis, and systems biology, we discovered that aged subjects developed fewer neutralizing Abs, mounted diminished YF-specific CD8(+) T cell responses, and showed quantitatively and qualitatively altered YF-specific CD4(+) T cell immunity. Among numerous immune signatures, low in vivo numbers of naive CD4(+) recent thymic emigrants and peripheral dendritic cells correlated well with reduced acute responsiveness and altered long-term persistence of human cellular immunity to YF vaccination. Hence, we reveal in this article that essential elements of immune responses such as recent thymic emigrants and dendritic cells strongly relate to productive immunity in the elderly, providing a conceivable explanation for diminished responsiveness to vaccination with neoantigens and infection with de novo pathogens in the aged population., (Copyright © 2015 by The American Association of Immunologists, Inc.)

HIV affects the function of all lymphocyte populations, including B cells. Phenotypic and functional defects of B cells in HIV-infected adults have been well characterized, but defects in children have not been studied to the same extent. We determined the proportion of B cell subsets and frequencies of Ag-specific memory B cells in peripheral blood from HIV-infected children and healthy controls, using flow cytometry and B cell ELISPOT, respectively. In addition, we measured the quantities and avidities of plasma Abs against various Ags by ELISA. We also determined plasma levels of BAFF and expression of BAFF receptors on B cells. Children with high HIV viremia had increased proportions of activated mature B cells, tissue-like memory B cells and plasmablasts, and low proportions of naive B cells when compared with community controls and children with low HIV viremia, similar to adults infected with HIV. HIV-infected groups had lower proportions of resting memory B cells than did community controls. Notably, high HIV viremia prevented the age-dependent accumulation of class-switched resting memory B cells. HIV-infected children, regardless of the level of viremia, showed lower quantities and avidities of IgG and lower frequencies of memory B cells against Expanded Program on Immunization vaccines. The HIV-infected children had an altered BAFF profile that could have affected their B cell compartment. Therefore, B cell defects in HIV-infected children are similar to those seen in HIV-infected adults. However, control of HIV viremia is associated with normalization of activated B cell subsets and allows age-dependent accumulation of resting memory B cells., (Copyright © 2015 The Authors.)

The goal of an HIV vaccine is to generate robust and durable protective Ab. Vital to this goal is the induction of CD4(+) T follicular helper (TFH) cells. However, very little is known about the TFH response to HIV vaccination and its relative contribution to magnitude and quality of vaccine-elicited Ab titers. In this study, we investigated these questions in the context of a DNA/modified vaccinia virus Ankara SIV vaccine with and without gp140 boost in aluminum hydroxide in rhesus macaques. In addition, we determined the frequency of vaccine-induced CD4(+) T cells coexpressing chemokine receptor, CXCR5 (facilitates migration to B cell follicles) in blood and whether these responses were representative of lymph node TFH responses. We show that booster modified vaccinia virus Ankara immunization induced a distinct and transient accumulation of proliferating CXCR5(+) and CXCR5(-) CD4 T cells in blood at day 7 postimmunization, and the frequency of the former but not the latter correlated with TFH and B cell responses in germinal centers of the lymph node. Interestingly, gp140 boost induced a skewing toward CXCR3 expression on germinal center TFH cells, which was strongly associated with longevity, avidity, and neutralization potential of vaccine-elicited Ab response. However, CXCR3(+) cells preferentially expressed the HIV coreceptor CCR5, and vaccine-induced CXCR3(+)CXCR5(+) cells showed a moderate positive association with peak viremia following SIV251 infection. Taken together, our findings demonstrate that vaccine regimens that elicit CXCR3-biased TFH cell responses favor Ab persistence and avidity but may predispose to higher acute viremia in the event of breakthrough infections., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Identifying the mechanisms of natural control of HIV-1 infection could lead to novel approaches to prevent or cure HIV infection. Several studies have associated natural control of HIV-1 infection with IgG Abs against HIV-1 Gag proteins (e.g., p24) and/or production of IgG2 Abs against HIV-1 proteins. These Abs likely exert their effect by activating antiviral effector cell responses rather than virus neutralization. We hypothesized that an opsonophagocytic IgG Ab response against HIV-1 p24 that activates plasmacytoid dendritic cells (pDCs) through FcγRIIa would be associated with control of HIV and that this would be enhanced by Ab isotype diversification. Using the Gen2.2 pDC cell line, we demonstrated that pDC-reactive opsonophagocytic IgG Ab responses against HIV-1 p24 were higher in HIV controllers (HIV RNA < 2000 copies/ml) than noncontrollers (HIV RNA > 10,000 copies/ml), particularly in controllers with low but detectable viremia (HIV RNA 75-2000 copies/ml). Opsonophagocytic Ab responses correlated with plasma levels of IgG1 and IgG2 anti-HIV-1 p24 and, notably, correlated inversely with plasma HIV RNA levels in viremic HIV patients. Phagocytosis of these Abs was mediated via FcγRIIa. Isotype diversification (toward IgG2) was greatest in HIV controllers, and depletion of IgG2 from Ig preparations indicated that IgG2 Abs to HIV-1 p24 do not enhance phagocytosis, suggesting that they enhance other aspects of Ab function, such as Ag opsonization. Our findings emulate those for pDC-reactive opsonophagocytic Ab responses against coxsackie, picorna, and influenza viruses and demonstrate a previously undefined immune correlate of HIV-1 control that may be relevant to HIV vaccine development., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Elevated levels of the chemokine CXCL13 have been observed in the plasma of chronically HIV-1-infected subjects and have been correlated with plasma viremia, which in turn has been linked to progressive dysregulation of humoral responses. In this study we sought to identify mechanisms of CXCL13 induction in response to HIV-1 infection. Plasma levels of CXCL13 in HIV-1-infected antiretroviral therapy-naive subjects correlated with viral load and were higher compared with antiretroviral therapy-treated HIV-1-infected and HIV-1-uninfected subjects. To elucidate the relationship between HIV-1 viremia and CXCL13 plasma levels, PBMCs from uninfected donors were stimulated with HIV-1 infectious virions, HIV-1 ssRNA, TLR 7 and 8 agonists, or IFN-α. The cellular sources of CXCL13 were determined by intracellular cytokine staining of cell populations. CXCL13 was produced by monocytes after stimulation with TLR 7 and 8 ligands or HIV-1-derived ssRNA. CXCL13 production by monocytes required TLR7 activation of plasmacytoid dendritic cells and secretion of type I IFN. IFN-α alone was sufficient to induce CXCL13 expression in human monocytes. In sum, we identified a novel mechanism of HIV-1-induced CXCL13 secretion-one caused by TLR7 induction of type I IFN by plasmacytoid dendritic cells and subsequent IFN stimulation of monocytes. Our findings are relevant in understanding how HIV-1 infection leads to immune dysregulation and provide the opportunity to develop and test potential therapeutic interventions., (Copyright © 2015 by The American Association of Immunologists, Inc.)

The human papillomavirus pseudovirions (HPV-PsVs) approach is an effective gene-delivery system that can prime or boost an immune response in the vaginal tract of nonhuman primates and mice. Intravaginal vaccination with HPV-PsVs expressing SIV genes, combined with an i.m. gp120 protein injection, induced humoral and cellular SIV-specific responses in macaques. Priming systemic immune responses with i.m. immunization with ALVAC-SIV vaccines, followed by intravaginal HPV-PsV-SIV/gp120 boosting, expanded and/or recruited T cells in the female genital tract. Using a stringent repeated low-dose intravaginal challenge with the highly pathogenic SIVmac251, we show that although these regimens did not demonstrate significant protection from virus acquisition, they provided control of viremia in a number of animals. High-avidity Ab responses to the envelope gp120 V1/V2 region correlated with delayed SIVmac251 acquisition, whereas virus levels in mucosal tissues were inversely correlated with antienvelope CD4(+) T cell responses. CD8(+) T cell depletion in animals with controlled viremia caused an increase in tissue virus load in some animals, suggesting a role for CD8(+) T cells in virus control. This study highlights the importance of CD8(+) cells and antienvelope CD4(+) T cells in curtailing virus replication and antienvelope V1/V2 Abs in preventing SIVmac251 acquisition., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Tetherin/BST-2 is a host restriction factor that could directly inhibit retroviral particle release by tethering nascent virions to the plasma membrane. However, the immunological impact of Tetherin during retrovirus infection remains unknown. We now show that Tetherin influences antiretroviral cell-mediated immune responses. In contrast to the direct antiviral effects of Tetherin, which are dependent on cell surface expression, the immunomodulatory effects are linked to the endocytosis of the molecule. Mice encoding endocytosis-competent C57BL/6 Tetherin exhibited lower viremia and pathology at 7 d postinfection with Friend retrovirus (FV) compared with mice encoding endocytosis-defective NZW/LacJ Tetherin. Notably, antiretroviral protection correlated with stronger NK cell responses. In addition, Friend retrovirus infection levels were significantly lower in wild-type C57BL/6 mice than in Tetherin knockout mice at 2 wk postinfection, and antiretroviral protection correlated with stronger NK cell and virus-specific CD8+ T cell responses. The results demonstrate that Tetherin acts as a modulator of the cell-mediated immune response against retrovirus infection in vivo.

The programmed cell death-1 (PD-1)/programmed cell death ligand-1 pathway has been shown to limit cell-mediated effector functions during chronic viral infections impeding clearance of pathogens. As a strategy to reverse this exhaustion and increase T cell polyfunctionality, PD-1 ligands were blocked in vivo using a recombinant macaque PD-1 fused to a macaque Ig-Fc (rPD-1-Fc) in SIVmac239-infected rhesus macaques during the early chronic phase of infection, either alone or in combination with antiretroviral therapy. In vitro blockade showed improvement of Ag-specific CD4(+) and CD8(+) T cells from monkeys chronically infected with SIV. Of note, a prolonged 5-d blockade in culture was beneficial for both gag-specific CD4(+) and CD8(+) T cells based on proliferation and dual cytokine production. Although the in vivo administration of rPD-1-Fc induced enhanced SIV-specific CD4 and CD8 T cell proliferation both in the blood and gut, it failed to alter plasma viremia. However, rPD-1-Fc administration in the context of antiretroviral therapy interruption induced a significant delay of viral load rebound. In addition, rPD-1-Fc administration in MamuA*001(+) monkeys led to both an increase in the frequencies and Ki67 expression of GagCM9(+) CD8(+) T cells in the blood and rectal mucosa and polyfunctionality of GagCM9(+) CD8(+) T cells in blood. In conclusion, however, our data suggest that PD-1/programmed cell death ligand-1 blockade using soluble rPD-1-Fc instead of anti-PD-1 mAb, although effective in rescuing the effector function of SIV-specific CD4(+) and CD8(+) T cells during the early chronic phase of infection, has limited clinical benefit.

Therapeutic administration of IFN-α in clinical trials significantly reduced HIV-1 plasma viral load and human T-lymphotropic virus type I proviral load in infected patients. The mechanism may involve the concerted action of multiple antiretroviral effectors collectively known as "restriction factors," which could vary in relative importance according to the magnitude of transcriptional induction. However, direct genetic approaches to identify the relevant IFN-α restriction factors will not be feasible in humans in vivo. Meanwhile, mice encode an analogous set of restriction factor genes and could be used to obtain insights on how IFN-α could inhibit retroviruses in vivo. As expected, IFN-α treatment of mice significantly upregulated the transcription of multiple restriction factors including Tetherin/BST2, SAMHD1, Viperin, ISG15, OAS1, and IFITM3. However, a dominant antiretroviral factor, Apobec3, was only minimally induced. To determine whether Apobec3 was necessary for direct IFN-α antiretroviral action in vivo, wild-type and Apobec3-deficient mice were infected with Friend retrovirus, then treated with IFN-α. Treatment of infected wild-type mice with IFN-α significantly reduced acute plasma viral load 28-fold, splenic proviral load 5-fold, bone marrow proviral load 14-fold, and infected bone marrow cells 7-fold, but no inhibition was observed in Apobec3-deficient mice. These findings reveal that IFN-α inhibits acute Friend retrovirus infection primarily through the antiviral effector Apobec3 in vivo, demonstrate that transcriptional induction levels did not predict the mechanism of IFN-α-mediated control, and highlight the potential of the human APOBEC3 proteins as therapeutic targets against pathogenic retrovirus infections.

Hepatitis C virus (HCV) is remarkable at disrupting human immunity to establish chronic infection. Upregulation of inhibitory signaling pathways (such as T cell Ig and mucin domain protein-3 [Tim-3]) and accumulation of regulatory T cells (Tregs) play pivotal roles in suppressing antiviral effector T cell (Teff) responses that are essential for viral clearance. Although the Tim-3 pathway has been shown to negatively regulate Teffs, its role in regulating Foxp3(+) Tregs is poorly explored. In this study, we investigated whether and how the Tim-3 pathway alters Foxp3(+) Treg development and function in patients with chronic HCV infection. We found that Tim-3 was upregulated, not only on IL-2-producing CD4(+)CD25(+)Foxp3(-) Teffs, but also on CD4(+)CD25(+)Foxp3(+) Tregs, which accumulate in the peripheral blood of chronically HCV-infected individuals when compared with healthy subjects. Tim-3 expression on Foxp3(+) Tregs positively correlated with expression of the proliferation marker Ki67 on Tregs, but it was inversely associated with proliferation of IL-2-producing Teffs. Moreover, Foxp3(+) Tregs were found to be more resistant to, and Foxp3(-) Teffs more sensitive to, TCR activation-induced cell apoptosis, which was reversible by blocking Tim-3 signaling. Consistent with its role in T cell proliferation and apoptosis, blockade of Tim-3 on CD4(+)CD25(+) T cells promoted expansion of Teffs more substantially than Tregs through improving STAT-5 signaling, thus correcting the imbalance of Foxp3(+) Tregs/Foxp3(-) Teffs that was induced by HCV infection. Taken together, the Tim-3 pathway appears to control Treg and Teff balance through altering cell proliferation and apoptosis during HCV infection.

A small number of HIV-infected individuals known as elite controllers experience low levels of chronic phase viral replication and delayed progression to AIDS. Specific HLA class I alleles are associated with elite control, implicating CD8(+) T lymphocytes in the establishment of these low levels of viral replication. Most HIV-infected individuals that express protective HLA class I alleles, however, do not control viral replication. Approximately 50% of Mamu-B*00801(+) Indian rhesus macaques control SIVmac239 replication in the chronic phase in a manner that resembles elite control in humans. We followed both the immune response and viral evolution in SIV-infected Mamu-B*00801(+) animals to better understand the role of CD8(+) T lymphocytes during the acute phase of viral infection, when viral control status is determined. The virus escaped from immunodominant Vif and Nef Mamu-B*00801-restricted CD8(+) T lymphocyte responses during the critical early weeks of acute infection only in progressor animals that did not control viral replication. Thus, early CD8(+) T lymphocyte escape is a hallmark of Mamu-B*00801(+) macaques who do not control viral replication. By contrast, virus in elite controller macaques showed little evidence of variation in epitopes recognized by immunodominant CD8(+) T lymphocytes, implying that these cells play a role in viral control.

The potential contribution of HLA-A alleles to viremic control in chronic HIV type 1 (HIV-1) infection has been relatively understudied compared with HLA-B. In these studies, we show that HLA-A*7401 is associated with favorable viremic control in extended southern African cohorts of >2100 C-clade-infected subjects. We present evidence that HLA-A*7401 operates an effect that is independent of HLA-B*5703, with which it is in linkage disequilibrium in some populations, to mediate lowered viremia. We describe a novel statistical approach to detecting additive effects between class I alleles in control of HIV-1 disease, highlighting improved viremic control in subjects with HLA-A*7401 combined with HLA-B*57. In common with HLA-B alleles that are associated with effective control of viremia, HLA-A*7401 presents highly targeted epitopes in several proteins, including Gag, Pol, Rev, and Nef, of which the Gag epitopes appear immunodominant. We identify eight novel putative HLA-A*7401-restricted epitopes, of which three have been defined to the optimal epitope. In common with HLA-B alleles linked with slow progression, viremic control through an HLA-A*7401-restricted response appears to be associated with the selection of escape mutants within Gag epitopes that reduce viral replicative capacity. These studies highlight the potentially important contribution of an HLA-A allele to immune control of HIV infection, which may have been concealed by a stronger effect mediated by an HLA-B allele with which it is in linkage disequilibrium. In addition, these studies identify a factor contributing to different HIV disease outcomes in individuals expressing HLA-B*5703.

The influence of preexisting immunity to viral vectors is a major issue for the development of viral-vectored vaccines. In this study, we investigate the effect of preexisting vaccinia virus immunity on the immunogenicity and efficacy of a DNA/modified vaccinia Ankara (MVA) SIV vaccine in rhesus macaques using a pathogenic intrarectal SIV251 challenge. Preexisting immunity decreased SIV-specific CD8 and CD4 T cell responses but preserved the SIV-specific humoral immunity. In addition, preexisting immunity did not diminish the control of an SIV challenge mediated by the DNA/MVA vaccine. The peak and set point viremia was 150- and 17-fold lower, respectively, in preimmune animals compared with those of control animals. The peak and set point viremia correlated directly with colorectal virus at 2 wk postchallenge suggesting that early control of virus replication at the site of viral challenge was critical for viral control. Factors that correlated with early colorectal viral control included 1) the presence of anti-SIV IgA in rectal secretions, 2) high-avidity binding Ab for the native form of Env, and 3) low magnitude of vaccine-elicited SIV-specific CD4 T cells displaying the CCR5 viral coreceptor. The frequency of SIV-specific CD8 T cells in blood and colorectal tissue at 2 wk postchallenge did not correlate with early colorectal viral control. These results suggest that preexisting vaccinia virus immunity may not limit the potential of recombinant MVA vaccines to elicit humoral immunity and highlight the importance of immunodeficiency virus vaccines achieving early control at the mucosal sites of challenge.

Indian rhesus macaques infected with the Rev-independent live-attenuated SIVmac239 strains control viremia to undetectable levels, have persistent but low cellular and humoral anti-SIV responses, and show no signs of immune deficiency. To analyze the immune mechanisms responsible for viral control, five macaques infected at day 1 after birth were subjected to CD8(+) cell depletion at 6.7 y postinfection. This resulted in viremia increases to 3.7-5.5 log(10) RNA copies, supporting a role of CD8-mediated responses in the control of viral replication. The rebounding viremia was rapidly controlled to levels below the threshold of detection, and occurred in the absence of SIV-specific CD8(+) T cells and significant CD8(+) T cell recovery in four of the five animals, suggesting that other mechanisms are involved in the immunological control of viremia. Monitoring immune responses at the time of viral control demonstrated a burst of circulating SIV-specific CD4(+) T cells characterized as CD45RA(-)CD28(+)CD95(+)CCR7(-) and also granzyme B(+), suggesting cytotoxic ability. Control of viremia was also concomitant with increases in humoral responses to Gag and Env, including a transient increase in neutralizing Abs against the neutralization-resistant SIVmac239 in four of five animals. These data demonstrate that a combination of cellular responses mediated by CD4(+) T cells and humoral responses was associated with the rapid control of the rebounding viremia in macaques infected by the Rev-independent live-attenuated SIV, even in the absence of measurable SIV-specific CD8(+) T cells in the blood, emphasizing the importance of different components of the immune response for full control of SIV infection.

Chikungunya virus (CHIKV) is an alphavirus that causes chronic and incapacitating arthralgia in humans. To date, interactions between the immune system and the different stages of the virus life cycle remain poorly defined. We demonstrated for the first time that CHIKV Ags could be detected in vivo in the monocytes of acutely infected patients. Using in vitro experimental systems, whole blood and purified monocytes, we confirmed that monocytes could be infected and virus growth could be sustained. CHIKV interactions with monocytes, and with other blood leukocytes, induced a robust and rapid innate immune response with the production of specific chemokines and cytokines. In particular, high levels of IFN-alpha were produced rapidly after CHIKV incubation with monocytes. The identification of monocytes during the early phase of CHIKV infection in vivo is significant as infected monocyte/macrophage cells have been detected in the synovial tissues of chronically CHIKV-infected patients, and these cells may behave as the vehicles for virus dissemination. This may explain the persistence of joint symptoms despite the short duration of viremia. Our results provide a better understanding on the basic mechanisms of infection and early antiviral immune responses and will help in the development of future effective control strategies.

Alphaviruses, including Chikungunya virus (CHIKV), produce a transient illness in humans, but severe forms leading to chronic incapacitating arthralgia/arthritis have been reported by mechanisms largely ill-characterized. The pathogenesis of CHIKV was addressed in a prospective cohort study of 49 hospitalized patients from Reunion Island subsequently categorized into two distinct groups at 12 mo postinfection. Comprehensive analyses of the clinical and immunological parameters throughout the disease course were analyzed in either the "recovered" or the "chronic" groups to identify prognostic markers of arthritis-like pathology after CHIKV disease. We found that the chronic group consisted mainly of more elderly patients (>60 y) and with much higher viral loads (up to 10(10) viruses per milliliter of blood) during the acute phase. Remarkably, a rapid innate immune antiviral response was demonstrated by robust dendritic/NK/CD4/CD8 cell activation and accompanied by a rather weak Th1/Th2 cytokine response in both groups. Interestingly, the antiviral immune response witnessed by high levels of IFN-alpha mRNA in PBMCs and circulating IL-12 persisted for months only in the chronic group. CHIKV (RNA and proteins) was found in perivascular synovial macrophages in one chronic patient 18 mo postinfection surrounded by infiltrating NK and T cells (CD4(++) but rare cytotoxic CD8). Fibroblast hyperplasia, strong angiogenesis, tissue lesions given the high levels of matrix metalloproteinase 2, and acute cell death [high cleaved poly(ADP-ribose) polymerase staining] were observed in the injured synovial tissue. These observed cellular and molecular events may contribute to chronic arthralgia/arthritis targeted by methotrexate used empirically for effective treatment but with immunosuppressive function in a context of viral persistence.

The functional integrity of CD4(+) T cells is crucial for well-orchestrated immunity and control of HIV-1 infection, but their selective depletion during infection creates a paradox for understanding a protective response. We used multiparameter flow cytometry to measure activation, memory maturation, and multiple functions of total and Ag-specific CD4(+) T cells in 14 HIV-1- and CMV- coinfected individuals at 3 and 12 mo post HIV-1 infection. Primary HIV-1 infection was characterized by elevated levels of CD38, HLA-DR, and Ki67 in total memory and Gag-specific CD4(+) and CD8(+) T cells. In both HIV-infected and 15 uninfected controls, the frequency of activated cells was uniformly distributed among early differentiated (ED; CD45RO(+)CD27(+)), late differentiated (CD45RO(+)CD27(-)), and fully differentiated effector (CD45RO(-)CD27(-)) memory CD4(+) T cells. In HIV-1-infected individuals, activated CD4(+) T cells significantly correlated with viremia at 3 mo postinfection (r = 0.79, p = 0.0007) and also harbored more gag provirus DNA copies than nonactivated cells (p = 0.04). Moreover, Gag-specific ED CD4(+) T cells inversely associated with plasma viral load (r = -0.87, p < 0.0001). Overall, we show that low copy numbers of gag provirus and plasma RNA copies associated with low CD4 activation as well as accumulation of ED HIV-specific CD4(+) memory. Significant positive correlations between 3 and 12 mo activation and memory events highlighted that a steady state of CD4(+) T cell activation and memory maturation was established during primary infection and that these cells were unlikely to be involved in influencing the course of viremia in the first 12 mo of HIV-1 infection.

FOXP3(+)CD8(+) T cells are present at low levels in humans; however, the function of these cells is not known. In this study, we demonstrate a rapid expansion of CD25(+)FOXP3(+)CD8(+) regulatory T cells (Tregs) in the blood and multiple tissues following a pathogenic SIV infection in rhesus macaques. The expansion was pronounced in lymphoid and colorectal mucosal tissues, preferential sites of virus replication. These CD8 Tregs expressed molecules associated with immune suppressor function such as CTLA-4 and CD39 and suppressed proliferation of SIV-specific T cells in vitro. They also expressed low levels of granzyme B and perforin, suggesting that these cells do not possess killing potential. Expansion of CD8 Tregs correlated directly with acute phase viremia and inversely with the magnitude of antiviral T cell response. Expansion was also observed in HIV-infected humans but not in SIV-infected sooty mangabeys with high viremia, suggesting a direct role for hyperimmune activation and an indirect role for viremia in the induction of these cells. These results suggest an important but previously unappreciated role for CD8 Tregs in suppressing antiviral immunity during immunodeficiency virus infections. These results also suggest that CD8 Tregs expand in pathogenic immunodeficiency virus infections in the nonnatural hosts and that therapeutic strategies that prevent expansion of these cells may enhance control of HIV infection.

Plasma viremia decreases coincident with the appearance of virus-specific CD8(+) T cells during acute HIV or SIV infection. This finding, along with demonstrations of viral mutational escape from CD8(+) T cell responses and transient increase in plasma viremia after depletion of CD8(+) T cells in SIV-infected monkeys strongly suggest a role for CD8(+) T cells in controlling HIV/SIV. However, direct quantitative or qualitative correlates between CD8(+) T cell activity and virus control have not been established. To directly assess the impact of large numbers of virus-specific CD8(+) T cells present at time of SIV infection, we transferred in vitro expanded autologous central and effector memory-derived Gag CM9-, Nef YY9-, and Vif WY8-specific CD8(+) T cell clones to acutely infected rhesus macaques. The cells persisted in PBMCs between 4 and 9 d, but were not detected in gut-associated lymphoid tissue or lymph nodes. Interestingly, a high frequency of the infused cells localized to the lungs, where they persisted at high frequency for >6 wk. Although persisting cells in the lungs were Ag reactive, there was no measurable effect on virus load. Sequencing of virus from the animal receiving Nef YY9-specific CD8(+) T cells demonstrated an escape mutation in this epitope <3 wk postinfection, consistent with immune selection pressure by the infused cells. These studies establish methods for adoptive transfer of autologous SIV-specific CD8(+) T cells for evaluating immune control during acute infection and demonstrate that infused cells retain function and persist for at least 2 mo in specific tissues.

Mechanisms that may allow circulating monocytes to persist as CD4 T cells diminish in HIV-1 infection have not been investigated. We have characterized steady-state gene expression signatures in circulating monocytes from HIV-infected subjects and have identified a stable antiapoptosis gene signature comprised of 38 genes associated with p53, CD40L, TNF, and MAPK signaling networks. The significance of this gene signature is indicated by our demonstration of cadmium chloride- or Fas ligand-induced apoptosis resistance in circulating monocytes in contrast to increasing apoptosis in CD4 T cells from the same infected subjects. As potential mechanisms in vivo, we show that monocyte CCR5 binding by HIV-1 virus or agonist chemokines serves as independent viral and host modulators resulting in increased monocyte apoptosis resistance in vitro. We also show evidence for concordance between circulating monocyte apoptosis-related gene expression in HIV-1 infection in vivo and available datasets following viral infection or envelope exposure in monocyte-derived macrophages in vitro. The identification of in vivo gene expression associated with monocyte resistance to apoptosis is of relevance to AIDS pathogenesis since it would contribute to: 1) maintaining viability of infection targets and long-term reservoirs of HIV-1 infection in the monocyte/macrophage populations, and 2) protecting a cell subset critical to host survival despite sustained high viral replication.

The IFNs and their receptors have existed in early chordates for approximately 500 million years and represent the early elements in innate and adaptive immunity. Both types I and II IFNs have been discovered in fish, and type I has recently been classified into two groups based on their primary protein sequences. However, the biological activities of fish IFNs and their roles in infection are largely unknown. Using the zebrafish and manageable bacterial (Streptococcus iniae) and viral (spring viremia of carp virus) infection models, we are reporting in this study that zebrafish IFN (zfIFN) gamma failed to induce antiviral and proinflammatory genes when administered in vivo, which correlates with its inability to protect the fish against bacterial and viral infections. We also found that, although both group I (i.e., zfIFN1) and group II zfIFNs (i.e., zfIFN2 and zfIFN3) displayed strong in vivo antiviral activities, only group I zfIFN was able to protect the fish against bacterial infection, which may reflect the different patterns and kinetics of immune-related genes elicited by these two groups of IFNs. Thus, group II zfIFNs induced a rapid and transient expression of antiviral genes, whereas group I zfIFN exerted a slow but more powerful induction of several antiviral and proinflammatory genes. Collectively, our results suggest nonredundant, complementary roles of type I zfIFNs in viral infections and provide evidence for a pivotal role of the recently identified group II IFN of fish in the early stages of viral infections.

Acquisition of T cell responses during primary CMV infection in lung transplant recipients (LTRs) appear critical for host defense and allograft durability, with increased mortality in donor+/recipient- (D+R-) individuals. In 15 D+R- LTRs studied, acute primary CMV infection was characterized by viremia in the presence or absence of pneumonitis, with viral loads higher in the lung airways/allograft compared with the blood. A striking influx of CD8+ T cells into the lung airways/allograft was observed, with inversion of the CD4+:CD8+ T cell ratio. De novo CMV-specific CD8+ effector frequencies in response to pooled peptides of pp65 were strikingly higher in lung mononuclear cells compared with the PBMC and predominated over IE1-specific responses and CD4+ effector responses in both compartments. The frequencies of pp65-specific cytokine responses were significantly higher in lung mononuclear cells compared with PBMC and demonstrated marked contraction with long-term persistence of effector memory CD8+ T cells in the lung airways following primary infection. CMV-tetramer+CD8+ T cells from PBMC were CD45RA- during viremia and transitioned to CD45RA+ following resolution. In contrast, CMV-specific CD8+ effectors in the lung airways/allograft maintained a CD45RA- phenotype during transition from acute into chronic infection. Together, these data reveal differential CMV-specific CD8+ effector frequencies, immunodominance, and polyfunctional cytokine responses predominating in the lung airways/allograft compared with the blood during acute primary infection. Moreover, we show intercompartmental phenotypic differences in CMV-specific memory responses during the transition to chronic infection.

Evidences have recently suggested that the preservation of vaccine-induced memory rather than effector T cells is essential for better outcome and survival following pathogenic SIV challenge in macaques. However, an equivalent demonstration in humans is missing, and the immune correlates of HIV-1 control have been only partially characterized. We focused on the quantification of Ag-specific T cell precursors with high proliferative capacity (PHPC) using a peptide-based cultured IFN-gamma ELISPOT assay (PHPC assay), which has been shown to identify expandable memory T cells. To determine which responses correlate with viral suppression and positive immunologic outcome, PBMC from 32 chronically untreated HIV-1-infected individuals were evaluated in response to peptide pools, representing the complete HIV-1 Gag, Nef, and Rev proteins, by PHPC and IFN-gamma ELISPOT assay, which instead identifies effector T cells with low proliferative capacity. High magnitude of Gag-specific PHPC, but not ELISPOT, responses significantly correlated with low plasma viremia, due to responses directed toward p17 and p15 subunits. Only Gag p17-specific PHPC response significantly correlated with high CD4 counts. Analysis of 20 additional PBMC samples from an independent cohort of chronically untreated HIV-1-infected individuals confirmed the correlation between Gag p17-specific PHPC response and either plasma viremia (inverse correlation) or CD4 counts (direct correlation). Our results indicate that the PHPC assay is quantitatively and qualitatively different from the ELISPOT assay, supporting that different T cell populations are being evaluated. The PHPC assay might be an attractive option for individual patient management and for the design and testing of therapeutic and prophylactic vaccines.

In this study, we examined the effect of in vivo treatment of acutely SIV-infected Mamu-A*01+ rhesus macaques with IL-15. IL-15 treatment during acute infection increased viral set point by 3 logs and accelerated the development of simian AIDS in two of six animals with one developing early minimal lesion SIV meningoencephalitis. Although IL-15 induced a 2- to 3-fold increase in SIV-specific CD8+ T cell and NK cell numbers at peak viremia and reduced lymph node (LN) SIV-infected cells, this had no impact on peak viremia and did not lower viral set point. At viral set point, however, activated SIV-specific CD8+ T cells and NK cells were reduced in the blood of IL-15-treated animals and LN SIV-infected cells were increased. Week 30 LN from IL-15-treated animals had significantly increased Gag-specific CD8+ T cell numbers, whereas total cell, lymphocyte, and CD4+ T cell numbers were reduced. IL-15 treatment significantly reduced anti-SIV Ab concentrations at week 3 and viral set point. IL-15 increased Ki-67+CD4+ T cells at week 1 of treatment and reduced blood CCR5+ and CD45RA-CD62L- CD4+ T cells. The frequency of day 7 Ki-67+CD4+ T cells strongly correlated with viral set point. These findings suggest that CD4+ T cell activation during acute infection determines subsequent viral set point and IL-15 treatment by increasing such activation elevates viral set point. Finally, IL-15-treated acutely SIV-infected primates may serve as a useful model to investigate the poorly understood mechanisms that control viral set point and disease progression in HIV infection.

We analyzed dendritic cell (DC) and NK cell compartments in relation to CD4 recovery in 21 HIV-infected subjects followed to <50 copies/ml once starting antiretroviral therapy (ART) and observed for 52 wk of sustained suppression. Although CD4 counts increased in all subjects in response to ART, we observed a restoration of functional plasmacytoid DC (PDC) after 52 wk of sustained suppression under ART (from 1850 cells/ml to 4550 cells/ml) to levels comparable to controls (5120 cells/ml) only in subjects with a low baseline viral load, which also rapidly suppressed to <50 copies/ml upon

KIR3DL1 is a highly polymorphic killer cell Ig-like receptor gene with at least 23 alleles described, including its activating counterpart, KIR3DS1. Recently, the KIR3DS1 allele has been shown to slow progression to AIDS in individuals expressing HLA-Bw4 with isoleucine at position 80. However, due to the lack of a specific Ab, KIR3DS1 expression and function is not well characterized. In this study, we demonstrate KIR3DS1 expression on a substantial subset of peripheral natural killer cells through its recognition by the mAb Z27. The fidelity of this detection method was confirmed by analysis of KIR3DS1 transfectants and the identification of a novel KIR3DS1 null allele. Interestingly, KIR3DS1 is also expressed by a small proportion of CD56(+) T cells. We show that ligation of KIR3DS1 by Z27 leads to NK cell IFN-gamma production and degranulation as assessed by expression of CD107a. Furthermore, we document the persistence of KIR3DS1(+) NK cells in HIV-1 viremic patients. The high frequency of KIR3DS1 expression, along with its ability to activate NK cells, and its maintenance during HIV-1 viremia are consistent with the epidemiological data suggesting a critical role for this receptor in controlling HIV-1 pathogenesis.

Persistent immune activation is a hallmark of chronic viremic HIV-1 infection. Activation of cells of the innate immune system, such as NK cells, occurs rapidly upon infection, and is sustained throughout the course of the disease. However, the precise underlying mechanism accounting for the persistent HIV-1-induced activation of NK cells is poorly understood. In this study, we assessed the role of uridine-rich ssRNA derived from the HIV-1 long terminal repeat (ssRNA40) on activation of NK cells via TLR7/8. Although dramatic activation of NK cells was observed following stimulation of PBMC with ssRNA40, negligible activation was observed following stimulation of purified NK cells despite their expression of TLR8 mRNA and protein. The functional activation of NK cells by this HIV-1-encoded TLR7/8 ligand could not be reconstituted with exogenous IL-12, IFN-alpha, or TNF-alpha, but was critically dependent on the direct contact of NK cells with plasmacytoid dendritic cells or CD14(+) monocytes, indicating an important level of NK cell cross-talk and regulation by accessory cells during TLR-mediated activation. Coincubation of monocyte/plasmacytoid dendritic cells, NK cells, and ssRNA40 potentiated NK cell IFN-gamma secretion in response to MHC-devoid target cells. Studies using NK cells derived from individuals with chronic HIV-1 infection demonstrated a reduction of NK cell responsiveness following stimulation with TLR ligands in viremic HIV-1 infection. These data demonstrate that HIV-1-derived TLR ligands can contribute to the immune activation of NK cells and may play an important role in HIV-1-associated immunopathogenesis and NK cell dysfunction observed during acute and chronic viremic HIV-1 infection.

The loss of CD4(+) T cells and the impairment of CD8(+) T cell function in HIV infection suggest that pharmacological treatment with IL-7 and IL-15, cytokines that increase the homeostatic proliferation of T cells and improve effector function, may be beneficial. However, these cytokines could also have a detrimental effect in HIV-1-infected individuals, because both cytokines increase HIV replication in vitro. We assessed the impact of IL-7 and IL-15 treatment on viral replication and the immunogenicity of live poxvirus vaccines in SIV(mac251)-infected macaques (Macaca mulatta). Neither cytokine augmented the frequency of vaccine-expanded CD4(+) or CD8(+) memory T cells, clonal recruitment to the SIV-specific CD8(+) T cell pool, or CD8(+) T cell function. Vaccination alone transiently decreased the viral set point following antiretroviral therapy suspension. IL-15 induced massive proliferation of CD4(+) effector T cells and abrogated the ability of vaccination to decrease set point viremia. In contrast, IL-7 neither augmented nor decreased the vaccine effect and was associated with a decrease in TGF-beta expression. These results underscore the importance of testing immunomodulatory approaches in vivo to assess potential risks and benefits for HIV-1-infected individuals.

Despite efficient antiretroviral therapy (ART), CD4+ T cell counts often remain low in HIV-1-infected patients. This has led to IL-7, a crucial cytokine involved in both thymopoiesis and peripheral T cell homeostasis, being suggested as an additional therapeutic strategy. We investigated whether recombinant simian IL-7-treatment enhanced the T cell renewal initiated by ART in rhesus macaques chronically infected with SIVmac251. Six macaques in the early chronic phase of SIV infection received antiretroviral treatment. Four macaques also received a 3-wk course of IL-7 injections. Viral load was unaffected by IL-7 treatment. IL-7 treatment increased the number of circulating CD4+ and CD8+ memory T cells expressing activation (HLA-DR+, CD25+) and proliferation (Ki-67+) markers. It also increased naive (CD45RAbrightCD62L+) T cell counts by peripheral proliferation and enhanced de novo thymic production. The studied parameters returned to pretreatment values by day 29 after the initiation of treatment, concomitantly to the appearance of anti-IL-7 neutralizing Abs, supporting the need for a nonimmunogenic molecule for human treatment. Thus, IL-7, which increases T cell memory and de novo renewal of naive T cells may have additional benefits in HIV-infected patients receiving ART.

Chronic infection with the HIV results in poor HIV-specific CD4 T cell proliferation, but more recent analyses using intracellular cytokine staining demonstrated that IFN-gamma-producing, HIV-specific CD4 T cells can be detected for years in HIV-infected subjects. Because it is not known whether the majority of HIV-specific T cells are lost or become dysfunctional, we examined the kinetics of the T cell response over an extended period of time using a panel of 10 HLA-DR tetramers loaded with HIV p24 peptides. Tetramer+ CD4 T cells were present at a relatively high frequency during acute infection, but the size of these populations substantially contracted following suppression of viral replication. Short-term cessation of antiretroviral therapy resulted in a burst of viral replication and concomitant expansion of tetramer+ CD4 T cells, and these populations again contracted following reinitiation of therapy. The kinetics with which these cell populations contracted were characteristic of effector T cells, a conclusion that was supported by their phenotypic (CCR7-CD45RA-) and functional properties (IFN-gamma+). Continued high-level viremia resulted in the physical loss of the majority of tetramer+ CD4 T cells, and the decline of HIV p24-specific CD4 T cells occurred more rapidly and was more substantial than the reduction of total CD4 T cell numbers. We conclude that the population of HIV p24-specific CD4 T cells is initially responsive to changes in the levels of viral Ags, but that the majority of these cells are lost in a setting of chronic viremia.

Bone marrow transplantation (BMT) is commonly used in the treatment of leukemia, however its therapeutic application is partly limited by the high incidence of associated opportunistic infections. We modeled this clinical situation by infecting mice that underwent BMT with lymphocytic choriomeningitis virus (LCMV) and investigated the potential of immunotherapeutic strategies to counter such infections. All mice that received BMT survived LCMV infection and developed a virus carrier status. Immunotherapy by adoptive transfer of naive splenocytes protected against low (200 PFU), but not high (2 x 10(6) PFU), doses of LCMV. Attempts to control infection of high viral titers using strongly elevated frequencies of activated LCMV-specific T cells failed to control virus and resulted in immunopathology and death. In contrast, virus neutralizing Abs combined with naive splenocytes were able to efficiently control high-dose LCMV infection without associated side effects. Thus, cell transfer combined with neutralizing Abs represented the most effective means of controlling BMT-associated opportunistic viral infection in our in vivo model. These data underscore the in vivo efficacy and immunopathological "safety" of neutralizing antibodies.

A vigorous hepatitis C virus (HCV)-specific Th cell response is regarded as essential to the immunological control of HCV viremia. The aim of this study was to comprehensively define the breadth and specificity of dominant HCV-specific CD4(+) T cell epitopes in large cohorts of subjects with chronic and spontaneously resolved HCV viremia. Following in vitro stimulation of PBMC, HCV-specific cell cultures from each subject were screened with an overlapping panel of synthetic 20-mer peptides spanning the entire HCV polyprotein. Of 22 subjects who spontaneously controlled HCV viremia, all recognized at least one of a group of six epitopes situated within the nonstructural (NS) proteins NS3, NS4, and NS5, each of which was detected by >30% of subjects, but most subjects recognized additional, more heterogeneous specificities. In contrast, none of the most frequently targeted epitopes was detected by >5% of persons with chronic infection. The most frequently recognized peptides showed promiscuous binding to multiple HLA-DR molecules in in vitro binding assays and were restricted by different HLA-DR molecules in functional assays in different persons. These data demonstrate that predominant CD4(+) T cell epitopes in persons with resolved HCV infection are preferentially located in the nonstructural proteins and are immunogenic in the context of multiple class II molecules. This comprehensive characterization of CD4(+) T cell epitopes in resolved HCV infection provides important information to facilitate studies of immunopathogenesis and HCV vaccine design and evaluation.

Effector cells armed with Abs can eliminate virus-infected target cells by Ab-dependent cellular cytotoxicity (ADCC), an immune mechanism that has been largely overlooked in HIV vaccine development. Here, we show that a prime/boost AIDS vaccine approach elicits potent ADCC activity correlating with protection against SIV in rhesus macaques (Macacca mulatta). Priming with replicating adenovirus type 5 host range mutant-SIV recombinants, followed by boosting with SIV gp120, elicited Abs with ADCC activity against SIV(mac251)-infected cells. In vitro ADCC activity correlated with in vivo reduced acute viremia after a mucosal challenge with pathogenic SIV. Our findings expose ADCC activity as an immune correlate that may be relevant in the rational design of an efficacious vaccine against HIV.

NK cells are a subset of granular lymphocytes that are critical in the innate immune response to infection. These cells are capable of killing infected cells and secreting integral cytokines and chemokines. The role that this subset of cytolytic cells plays in HIV infection is not well understood. In this study, we dissected the function of NK cells in viremic and aviremic HIV-1-infected subjects, as well as HIV-1-negative control individuals. Despite reduced NK cell numbers in subjects with ongoing viral replication, these cells were significantly more active in secreting both IFN-gamma and TNF-alpha than NK cells from aviremic subjects or HIV-1-negative controls. In addition, NK cells in subjects with detectable viral loads expressed significantly higher levels of CD107a, a marker of lysosomal granule exocytosis. The expression of CD107a correlated with NK cell-mediated cytokine secretion and cytolytic activity as well as with the level of viral replication, suggesting that CD107a represents a good marker for the functional activity of NK cells. Finally, killer Ig-related receptor+ NK cells were stable or elevated in viremic subjects, while the numbers of CD3-/CD56+/CD94+ and CD3-/CD56+/CD161+ NK cells were reduced. Taken together, these data demonstrate that viremic HIV-1 infection is associated with a reduction in NK cell numbers and a perturbation of NK cell subsets, but increased overall NK cell activity.

A successful HIV vaccine may need to stimulate antiviral immunity in mucosal and systemic immune compartments, because HIV transmission occurs predominantly at mucosal sites. We report here the results of a combined DNA-modified vaccinia virus Ankara (MVA) vaccine approach that stimulated simian/human immunodeficiency virus (SHIV)-specific immune responses by vaccination at the nasal mucosa. Fifteen male rhesus macaques, divided into three groups, received three nasal vaccinations on day 1, wk 9, and wk 25 with a SHIV DNA plasmid producing noninfectious viral particles (group 1), or SHIV DNA plus IL-2/Ig DNA (group 2), or SHIV DNA plus IL-12 DNA (group 3). On wk 33, all macaques were boosted with rMVA expressing SIV Gag-Pol and HIV Env 89.6P, administered nasally. Humoral responses were evaluated by measuring SHIV-specific IgG and neutralizing Abs in plasma, and SHIV-specific IgA in rectal secretions. Cellular responses were monitored by evaluating blood-derived virus-specific IFN-gamma-secreting cells and TNF-alpha-expressing CD8+ T cells, and blood- and rectally derived p11C tetramer-positive T cells. Many of the vaccinated animals developed both mucosal and systemic humoral and cell-mediated anti-SHIV immune responses, although the responses were not homogenous among animals in the different groups. After rectal challenge of vaccinated and naive animals with SHIV89.6P, all animals became infected. However a subset, including all group 2 animals, were protected from CD4+ T cell loss and AIDS development. Taken together, these data indicate that nasal vaccination with SHIV-DNA plus IL-2/Ig DNA and rMVA can provide significant protection from disease progression.

An in vitro proliferative defect has been observed in HIV-1-specific CD4(+) T cells from infected subjects with high-level plasma HIV-1 viremia. To determine the mechanism of this defect, HIV-1 Gag-specific CD4(+) T cells from treated and untreated HIV-1-infected subjects were analyzed for cytokine profile, proliferative capacity, and maturation state. Unexpectedly high frequencies of HIV-1-specific, IL-2-producing CD4(+) T cells were measured in subjects with low or undetectable plasma HIV-1 loads, regardless of treatment status, and IL-2 frequencies correlated inversely with viral loads. IL-2-producing CD4(+) T cells also primarily displayed a central memory (T(Cm); CCR7(+)CD45RA(-)) maturation phenotype, whereas IFN-gamma-producing cells were mostly effector memory (T(Em), CCR7(-)CD45RA(-)). Among Gag-specific, IFN-gamma-producing CD4(+) T cells, higher T(Em) frequencies and lower T(Cm) frequencies were observed in untreated, high viral load subjects than in subjects with low viral loads. The percentage of HIV-1 Gag-specific CD4(+) T(Cm) correlated inversely with HIV-1 viral load and directly with Gag-specific CD4(+) T cell proliferation, whereas the opposite relationships were observed for HIV-1-specific CD4(+) T(Em). These results suggest that HIV-1 viremia skews Gag-specific CD4(+) T cells away from an IL-2-producing T(Cm) phenotype and toward a poorly proliferating T(Em) phenotype, which may limit the effectiveness of the HIV-1-specific immune response.

Gag-specific CD4 proliferative responses correlate inversely with HIV-1 RNA levels in infected adults, and robust responses are characteristic of long-term nonprogressive infection. However, strong responses are seldom detected in adult subjects with progressive infection and are not generally reconstituted on highly active antiretroviral therapy (HAART). To date, the role of HIV-1-specific Th responses in children has not been thoroughly examined. We characterized Gag-specific CD4 responses among 35 perinatally infected subjects, including 2 children who spontaneously control viremia without antiretroviral therapy, 21 children with viral loads (VL) of <400 on HAART, and 12 viremic children. Gag-specific Th activity was assessed by lymphoproliferative assay, and responses were mapped using overlapping Gag peptides in an IFN-gamma ELISPOT. Robust proliferative responses were detected in the children exhibiting spontaneous control of viremia, and mapping of targeted Gag regions in one such subject identified multiple epitopes. Among children >or=5 years old, 14 of 17 subjects with VL of <400 on HAART demonstrated a significant p24 proliferative response (median p24 stimulation index, 20), in contrast with only 1 of 9 viremic children (median p24 stimulation index, 2.0; p = 0.0008). However, no subject younger than 5 years of age possessed a significant response, even when viremia was fully suppressed. When compared with adults with VL of <400 on HAART, Th responses among children with VL of <400 were both more frequent (p = 0.009) and of greater magnitude (p = 0.002). These data suggest that children may have a greater intrinsic capacity to reconstitute HIV-1-specific immunity than adults, and may be excellent candidates for immune-based therapies.

At least two distinct human dendritic cell (DC) subsets are produced in the bone marrow and circulate in the peripheral blood-precursor myeloid DCs (pre-mDCs) and plasmacytoid DCs (PDCs). Both lineages of DCs are instrumental in antiviral innate immunity and shaping Th1 adaptive immune responses. PDCs are the most potent IFN-alpha-producing cells to viral pathogens. Dengue, an acute flavivirus disease, provides a model to study DC responses to a self-limited human viral infection. We analyzed circulating DC subsets in a prospective study of children with dengue across a broad range of illness severities: healthy controls; mild, nondengue, presumed viral infections; moderately ill dengue fever; and, the most severe form of illness, dengue hemorrhagic fever. We also examined PDC responses in monkeys with asymptomatic dengue viremia and to dengue virus exposure in vitro. The absolute number and frequency of circulating pre-mDCs early in acute viral illness decreased as illness severity increased. Depressed pre-mDC blood levels appeared to be part of the typical innate immune response to acute viral infection. The frequency of circulating PDCs trended upward and the absolute number of circulating PDCs remained stable early in moderately ill children with dengue fever, mild other, nondengue, febrile illness, and monkeys with asymptomatic dengue viremia. However, there was an early decrease in circulating PDC levels in children who subsequently developed dengue hemorrhagic fever. A blunted blood PDC response to dengue virus infection was associated with higher viremia levels, and was part of an altered innate immune response and pathogenetic cascade leading to severe disease.

HIV infection leads to numerous immunologic defects, including impaired B cell function. An effective humoral response requires bidirectional interactions between B cells and CD4(+) T cells, critical of which are interactions between CD80/CD86 expressed on activated B cells and CD28 expressed on responder CD4(+) T cells. In the present study, we examined the effect of active HIV replication on B cell costimulatory function. Induction of CD80/CD86 on B cells following B cell receptor and CD40 triggering and responsiveness of CD4(+) T cells to activated B cells were investigated in a system where B cells of HIV-infected patients were compared concurrently to B cells of HIV-negative donors. In contrast to HIV-aviremic patients, B cells of HIV-viremic patients were ineffective at stimulating CD4(+) T cells, as measured by the induction of activation markers and proliferation. The importance of interactions of CD80/CD86 and CD28 in activating CD4(+) T cells was clear; the ablation of a normal response following the addition of neutralizing anti-CD86/CD80 Abs mirrored the response of CD4(+) T cells to B cells of HIV-viremic patients, while the addition of exogenous CD28 ligands partially restored the poor CD4(+) T cell response to the B cells of HIV-viremic patients. Ineffective B cell costimulatory function in HIV-viremic patients was associated with low induction of CD80/CD86 expression on B cells. Our findings further delineate the scope of defects associated with cognate B cell-CD4(+) T cell interactions in HIV infection and suggest that therapeutic interventions designed to enhance CD28-dependent costimulatory pathways may help restore immune functions.

The neutralizing Ab response after primary HIV-1 infection is delayed relative to the virus-specific CD8(+) T cell response and the initial decline in plasma viremia. Because nearly all HIV-1 infections result in AIDS, it would be instructive to study cases where neutralizing Ab production commenced sooner. This was done in subject AC10, an individual treated during early infection and in whom a rapid autologous neutralizing Ab response was detected after therapy cessation as rebound viremia declined and remained below 1000 RNA copies/ml of blood for over 2.5 years. This subject's Abs were capable of reducing the infectivity of his rebound virus by >4 logs in vitro at a time when rebound viremia was down-regulated and virus-specific CD8(+) T cells were minimal, suggesting that neutralizing Abs played an important role in the early control of viremia. The rebound virus did not exhibit an unusual phenotype that might explain its high sensitivity to neutralization by autologous sera. Neutralization escape occurred within 75 days and was proceeded by neutralizing Ab production to the escape variant and subsequent escape. Notably, escape was not associated with a significant rise in plasma viremia, perhaps due to increasing CD8(+) T cell responses. Sequence analysis of gp160 revealed a growing number of mutations over time, suggesting ongoing viral evolution in the face of potent antiviral immune responses. We postulate that an early effective neutralizing Ab response can provide long-term clinical benefits despite neutralization escape.

Macaques infected with the SIV strain SIVmac251 develop a disease closely resembling human AIDS characterized by high viremia, progressive loss of CD4(+) T cells, occurrence of opportunistic infection, cachexia, and lymphomas. We report in this study that vaccination with the genetically attenuated poxvirus vector expressing the structural Ags of SIVmac (NYVAC-SIV-gag, pol, env) in combination with priming with DNA-SIV-gag, env resulted in significant suppression of viremia within 2 mo after mucosal exposure to the highly pathogenic SIVmac251 in the majority of vaccinated macaques. The control of viremia in these macaques was long lasting and inversely correlated to the level of both pre- and postchallenge Gag-specific lymphoproliferative responses, as well as to the level of total SIV-specific CD4(+) T lymphocyte responses at the peak of acute viremia as detected by intracellular cytokine-staining assay. Viremia containment also correlated with the frequency of the immunodominant Gag(181-189)CM9 epitope-specific CD8(+) T cells present before the challenge or expanded during acute infection. These data indicate, for the first time, the importance of vaccine-induced CD4(+) Th cell responses as an immune correlate of viremia containment. The results presented in this work also further demonstrate the potential of a DNA-prime/attenuated poxvirus-boost vaccine regimen in an animal model that well mirrors human AIDS.

A cohort of rhesus macaques with long-standing SIVmac251 infection (> or =5 mo) was treated with continuous antiretroviral therapy (ART). A group of eight macaques was vaccinated with or without simultaneous administration of low dose IL-2 with the highly attenuated poxvirus vector (NYVAC) vaccine candidate expressing the SIVmac structural gag-pol-env (gpe) genes and a novel chimeric fusion protein derived from the rev-tat-nef (rtn) regulatory genes. Control groups consisted of mock-vaccinated macaques or animals treated only with IL-2. Vaccination significantly expanded both virus-specific CD4(+) and CD8(+) T cell responses, and IL-2 further increased the vaccine-induced response to an immunodominant Gag epitope. Following antiretroviral treatment interruption, the viral set point was significantly lower in vaccinated than in control macaques for at least 4 consecutive mo, and viral containment was inversely correlated with vaccine-induced, virus-specific CD4(+) and CD8(+) T cell responses. These data provide the proof of concept that therapeutic vaccination before cessation of ART may be a feasible approach in the clinical management of HIV-1 infection.