Jacqueline Gillis

Effect of CD8+ Lymphocyte Depletion on Virus Containment after Simian Immunodeficiency Virus SIVmac251 Challenge of Live Attenuated SIVmac239Δ3-Vaccinated Rhesus Macaques

ABSTRACT Although live attenuated vaccines can provide potent protection against simian immunodeficiency virus (SIV) and simian-human immunodeficiency virus challenges, the specific immune responses that confer this protection have not been determined. To test whether cellular immune responses mediated by CD8+ lymphocytes contribute to this vaccine-induced protection, we depleted rhesus macaques vaccinated with the live attenuated virus SIVmac239Δ3 of CD8+ lymphocytes and then challenged them with SIVmac251 by the intravenous route. While vaccination did not prevent infection with the pathogenic challenge virus, the postchallenge levels of virus in the plasmas of vaccinated control animals were significantly lower than those for unvaccinated animals. The depletion of CD8+ lymphocytes at the time of challenge resulted in virus levels in the plasma that were intermediate between those of the vaccinated and unvaccinated controls, suggesting that CD8+ cell-mediated immune responses contributed to protection. Interestingly, at the time of challenge, animals expressing the Mamu-A*01 major histocompatibility complex class I allele showed significantly higher frequencies of SIV-specific CD8+ T-cell responses and lower neutralizing antibody titers than those in Mamu-A*01− animals. Consistent with these findings, the depletion of CD8+ lymphocytes abrogated vaccine-induced protection, as judged by the peak postchallenge viremia, to a greater extent in Mamu-A*01+ than in Mamu-A*01− animals. The partial control of postchallenge viremia after CD8+ lymphocyte depletion suggests that both humoral and cellular immune responses induced by live attenuated SIV vaccines can contribute to protection against a pathogenic challenge and that the relative contribution of each of these responses to protection may be genetically determined.

Mucosal Priming of Simian Immunodeficiency Virus-Specific Cytotoxic T-Lymphocyte Responses in Rhesus Macaques by the Salmonella Type III Secretion Antigen Delivery System

ABSTRACT Nearly all human immunodeficiency virus (HIV) infections are acquired mucosally, and the gut-associated lymphoid tissues are important sites for early virus replication. Thus, vaccine strategies designed to prime virus-specific cytotoxic T lymphocyte (CTL) responses that home to mucosal compartments may be particularly effective at preventing or containing HIV infection. The Salmonella type III secretion system has been shown to be an effective approach for stimulating mucosal CTL responses in mice. We therefore tested ΔphoP-phoQ attenuated strains of Salmonella enterica serovar Typhimurium and S. enterica serovar Typhi expressing fragments of the simian immunodeficiency virus (SIV) Gag protein fused to the type III-secreted SopE protein for the ability to prime virus-specific CTL responses in rhesus macaques. Mamu-A*01+ macaques were inoculated with three oral doses of recombinant Salmonella, followed by a peripheral boost with modified vaccinia virus Ankara expressing SIV Gag (MVA Gag). Transient low-level CTL responses to the Mamu-A*01 Gag181-189 epitope were detected following each dose of Salmonella. After boosting with MVA Gag, strong Gag-specific CTL responses were consistently detected, and tetramer staining revealed the expansion of Gag181-189-specific CD8+ T-cell responses in peripheral blood. A significant percentage of the Gag181-189-specific T-cell population in each animal also expressed the intestinal homing receptor α4β7. Additionally, Gag181-189-specific CD8+ T cells were detected in lymphocytes isolated from the colon. Yet, despite these responses, Salmonella-primed/MVA-boosted animals did not exhibit improved control of virus replication following a rectal challenge with SIVmac239. Nevertheless, this study demonstrates the potential of mucosal priming by the Salmonella type III secretion system to direct SIV-specific cellular immune responses to the gastrointestinal mucosa in a primate model.

SIV Infection Induces Accumulation of Plasmacytoid Dendritic Cells in the Gut Mucosa

Multiple studies suggest that plasmacytoid dendritic cells (pDCs) are depleted and dysfunctional during human immunodeficiency virus/simian immunodeficiency virus (HIV/SIV) infection, but little is known about pDCs in the gut-the primary site of virus replication. Here, we show that during SIV infection, pDCs were reduced 3--fold in the circulation and significantly upregulated the gut-homing marker α4β7, but were increased 4-fold in rectal biopsies of infected compared to naive macaques. These data revise the understanding of pDC immunobiology during SIV infection, indicating that pDCs are not necessarily depleted, but instead may traffic to and accumulate in the gut mucosa.

Simian Immunodeficiency Virus Infection Induces Expansion of α4β7+ and Cytotoxic CD56+ NK Cells

ABSTRACT Herein we demonstrate that chronic simian immunodeficiency virus (SIV) infection induces significant upregulation of the gut-homing marker α4β7 on macaque NK cells, coupled with downregulation of the lymph node-trafficking marker, CCR7. Interestingly, in naïve animals, α4β7 expression was associated with increased NK cell activation and, on CD16+ NK cells, delineated a unique dual-function cytotoxic-CD107a+/gamma interferon (IFN-γ)-secreting population. However, while SIV infection increased CD107a expression on stimulated CD56+ NK cells, α4β7+ and α4β7− NK cells were affected similarly. These findings suggest that SIV infection redirects NK cells away from the lymph nodes to the gut mucosae but alters NK cell function independent of trafficking repertoires.

Hypercytotoxicity and rapid loss of NKp44+ innate lymphoid cells during acute SIV infection.

HIV/SIV infections break down the integrity of the gastrointestinal mucosa and lead to chronic immune activation and associated disease progression. Innate lymphoid cells (ILCs), distinguishable by high expression of NKp44 and RORγt, play key roles in mucosal defense and homeostasis, but are depleted from gastrointestinal (GI) tract large bowel during chronic SIV infection. However, less is known about the kinetics of ILC loss, or if it occurs systemically. In acute SIV infection, we found a massive, up to 8-fold, loss of NKp44+ILCs in all mucosae as early as day 6 post-infection, which was sustained through chronic disease. Interestingly, no loss of ILCs was observed in mucosa-draining lymph nodes. In contrast, classical NK cells were not depleted either from gut or draining lymph nodes. Both ILCs and NK cells exhibited significantly increased levels of apoptosis as measured by increased Annexin-V expression, but while classical NK cells also showed increased proliferation, ILCs did not. Interestingly, ILCs, which are normally noncytolytic, dramatically upregulated cytotoxic functions in acute and chronic infection and acquired a polyfunctional phenotype secreting IFN-γ, MIP1-β, and TNF-α, but decreased production of the prototypical cytokine, IL-17. Classical NK cells had less dramatic functional change, but upregulated perforin expression and increased cytotoxic potential. Finally, we show that numerical and functional loss of ILCs was due to increased apoptosis and ROR γt suppression induced by inflammatory cytokines in the gut milieu. Herein we demonstrate the first evidence for acute, systemic, and permanent loss of mucosal ILCs during SIV infection associated with reduction of IL-17. The massive reduction of ILCs involves apoptosis without compensatory de novo development/proliferation, but the full mechanism of depletion and the impact of functional change so early in infection remain unclear.

The values of quantitative serum HIV-1 RNA levels and CD4 cell counts for predicting survival time among HIV-positive individuals with CD4 counts of ≤ 50 × 106 cells/l.

ObjectiveTo evaluate the HIV-1 RNA level as a predictor of survival time among individuals with advanced AIDS. MethodsThe serum HIV-1 RNA level, the CD4 cell count, and other clinical variables were evaluated at baseline, as predictors of survival time, among 56 retrospectively identified HIV-1 positive individuals with ⩽ 50 × 106 CD4 cells/l who attended the Beth Israel Deaconess Medical Center, Division of Infectious Diseases, between 1 July 1989 and 30 September 1993. ResultsDuring follow-up, 55 of these 56 patients died. The median survival time was 20.5 months. In univariate Cox proportional hazard modeling neither the baseline HIV-1 RNA level nor the CD4 cell count were predictive of survival time. However, in multivariate models longer survival time was associated with the use of trimethoprim-sulphamethoxazole at entry [hazard ratio (HR), 0.42;P  = 0.007], whereas shorter survival time was associated with a history of an AIDS-defining illness other than Pneumocystis carinii pneumonia (HR, 2.87;P  = 0.007). Correlative analysis revealed a modest correlation of the baseline CD4 cell count with survival time (Spearman ρ = 0.41;P  = 0.002). However, no correlation was found between HIV RNA levels and survival time (P  = 0.5). ConclusionsIn this population with very advanced disease, the HIV-1 RNA level was a poor discriminator of survival time, being inferior to the CD4 cell count and to specific clinical variables such as the nature of the prior AIDS-defining illness and the type of Pneumocystis carinii pneumonia prophylaxis employed. Among individuals with advanced AIDS, these data emphasize the relative importance of the CD4 cell count and of specific clinical factors, over the HIV-1 RNA level in predicting survival time.

Multi-functional plasmacytoid dendritic cells redistribute to gut tissues during simian immunodeficiency virus infection.

The objective of this study was to determine the systemic effects of chronic simian immunodeficiency virus (SIV) infection on plasmacytoid dendritic cells (pDCs). pDCs play a critical role in antiviral immunity, but current data are conflicting on whether pDCs inhibit HIV/SIV replication, or, alternatively, contribute to chronic immune activation and disease. Furthermore, previous pDC studies have been complicated by incomplete descriptions of generalized depletion during HIV/SIV infection, and the effects of infection on pDCs outside peripheral blood remain unclear. In scheduled‐sacrifice studies of naive and chronically SIV‐infected rhesus macaques we evaluated the distribution and functionality of pDCs in multiple tissues using surface and intracellular polychromatic flow cytometry. As previously observed, pDCs were reduced in peripheral blood and spleens, but were also depleted in non‐lymphoid organs such as the liver. Interestingly, pDCs accumulated up to fourfold in jejunum, colon and gut‐draining lymph nodes, but not in peripheral lymph nodes. Most unexpectedly, SIV infection induced a multi‐functional interferon‐α, tumour necrosis factor‐α, and macrophage inflammatory protein‐1β cytokine secretion phenotype, whereas in normal animals these were generally distinct and separate functions. Herein we show a systemic redistribution of pDCs to gut tissues and gut‐draining lymph nodes during chronic SIV infection, coupled to a novel multi‐functional cytokine‐producing phenotype. While pDC accumulation in the mucosa could aid in virus control, over‐production of cytokines from these cells could also contribute to the increased immune activation in the gut mucosa commonly associated with progressive lentivirus infections.

Diverse recognition of conserved orthopoxvirus CD8+ T cell epitopes in vaccinated rhesus macaques

Vaccinia virus (VACV) induces a vigorous virus-specific CD8+ T cell response that plays an important role in control of poxvirus infection. To identify immunodominant poxvirus proteins and to facilitate future testing of smallpox vaccines in non-human primates, we used an algorithm for the prediction of VACV peptides able to bind to the common macaque MHC class I molecule Mamu-A*01. We synthesized 294 peptides derived from 97 VACV ORFs; 100 of these peptides did not contain the canonical proline at position three of the Mamu-A*01 binding motif. Cellular immune responses in PBMC from two vaccinia-vaccinated Mamu-A*01+ macaques were assessed by IFNgamma ELISPOT assays. Vaccinated macaques recognized 17 peptides from 16 different ORFs with 6 peptides recognized by both macaques. Comparison with other orthopoxvirus sequences revealed that 12 of these epitopes are strictly conserved between VACV, variola, and monkeypoxvirus. ELISPOT responses were also observed to eight epitopes that did not contain the canonical P3 proline. These results suggest that the virus-specific CD8+ T cell response is broadly directed against multiple VACV proteins and that a subset of these T cell epitopes is highly conserved among orthopoxviruses.

Persistent Low-Level Replication of SIVΔnef Drives Maturation of Antibody and CD8 T Cell Responses to Induce Protective Immunity against Vaginal SIV Infection

Defining the correlates of immune protection conferred by SIVΔnef, the most effective vaccine against SIV challenge, could enable the design of a protective vaccine against HIV infection. Here we provide a comprehensive assessment of immune responses that protect against SIV infection through detailed analyses of cellular and humoral immune responses in the blood and tissues of rhesus macaques vaccinated with SIVΔnef and then vaginally challenged with wild-type SIV. Despite the presence of robust cellular immune responses, animals at 5 weeks after vaccination displayed only transient viral suppression of challenge virus, whereas all macaques challenged at weeks 20 and 40 post-SIVΔnef vaccination were protected, as defined by either apparent sterile protection or significant suppression of viremia in infected animals. Multiple parameters of CD8 T cell function temporally correlated with maturation of protection, including polyfunctionality, phenotypic differentiation, and redistribution to gut and lymphoid tissues. Importantly, we also demonstrate the induction of a tissue-resident memory population of SIV-specific CD8 T cells in the vaginal mucosa, which was dependent on ongoing low-level antigenic stimulation. Moreover, we show that vaginal and serum antibody titers inversely correlated with post-challenge peak viral load, and we correlate the accumulation and affinity maturation of the antibody response to the duration of the vaccination period as well as to the SIVΔnef antigenic load. In conclusion, maturation of SIVΔnef-induced CD8 T cell and antibody responses, both propelled by viral persistence in the gut mucosa and secondary lymphoid tissues, results in protective immune responses that are able to interrupt viral transmission at mucosal portals of entry as well as potential sites of viral dissemination.

Bone Marrow–Imprinted Gut-Homing of Plasmacytoid Dendritic Cells (pDCs) in Acute Simian Immunodeficiency Virus Infection Results in Massive Accumulation of Hyperfunctional CD4+ pDCs in the Mucosae

Plasmacytoid dendritic cells (pDCs), a primary source of interferon α (IFN-α), provide a first line of innate immune defense against human immunodeficiency virus infection. However, their kinetics and functions during acute infection are poorly understood. In mucosal tissues of normal rhesus macaques, we found CD4(+) pDCs to be the subset responsible for most IFN-α and tumor necrosis factor α (TNF-α) production in response to Toll-like receptor (TLR) 7/8 stimulation, compared with relatively anergic CD4(-) pDCs. During acute simian immunodeficiency virus (SIV) infection, gut homing was imprinted on pDCs in the bone marrow, resulting in a decline in pDCs from circulation and secondary lymphoid tissues. Although the accumulated pDCs in the gut mucosae had robust cytokine responses to TLR7/8 stimulation in vitro, pDC gut migration occurred after infection and detection of SIV in plasma. Our data suggest that innate pDC responses do not control initial SIV seeding and dissemination but instead may contribute to ongoing immune activation in the gut.