R. Keith Reeves

ADCC develops over time during persistent infection with live-attenuated SIV and is associated with complete protection against SIV(mac)251 challenge.

Live-attenuated strains of simian immunodeficiency virus (SIV) routinely confer apparent sterilizing immunity against pathogenic SIV challenge in rhesus macaques. Understanding the mechanisms of protection by live-attenuated SIV may provide important insights into the immune responses needed for protection against HIV-1. Here we investigated the development of antibodies that are functional against neutralization-resistant SIV challenge strains, and tested the hypothesis that these antibodies are associated with protection. In the absence of detectable neutralizing antibodies, Env-specific antibody-dependent cell-mediated cytotoxicity (ADCC) emerged by three weeks after inoculation with SIVΔnef, increased progressively over time, and was proportional to SIVΔnef replication. Persistent infection with SIVΔnef elicited significantly higher ADCC titers than immunization with a non-persistent SIV strain that is limited to a single cycle of infection. ADCC titers were higher against viruses matched to the vaccine strain in Env, but were measurable against viruses expressing heterologous Env proteins. In two separate experiments, which took advantage of either the strain-specificity or the time-dependent maturation of immunity to overcome complete protection against SIVmac251 challenge, measures of ADCC activity were higher among the SIVΔnef-inoculated macaques that remained uninfected than among those that became infected. These observations show that features of the antibody response elicited by SIVΔnef are consistent with hallmarks of protection by live-attenuated SIV, and reveal an association between Env-specific antibodies that direct ADCC and apparent sterilizing protection by SIVΔnef.

Antigen-specific NK cell memory in rhesus macaques.

Natural killer (NK) cells have traditionally been considered nonspecific components of innate immunity, but recent studies have shown features of antigen-specific memory in mouse NK cells. However, it has remained unclear whether this phenomenon also exists in primates. We found that splenic and hepatic NK cells from SHIVSF162P3-infected and SIVmac251-infected macaques specifically lysed Gag- and Env-pulsed dendritic cells in an NKG2-dependent fashion, in contrast to NK cells from uninfected macaques. Moreover, splenic and hepatic NK cells from Ad26-vaccinated macaques efficiently lysed antigen-matched but not antigen-mismatched targets 5 years after vaccination. These data demonstrate that robust, durable, antigen-specific NK cell memory can be induced in primates after both infection and vaccination, and this finding could be important for the development of vaccines against HIV-1 and other pathogens.

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.

Live Simian Immunodeficiency Virus Vaccine Correlate of Protection: Local Antibody Production and Concentration on the Path of Virus Entry

We sought design principles for a vaccine to prevent HIV transmission to women by identifying correlates of protection conferred by a highly effective live attenuated SIV vaccine in the rhesus macaque animal model. We show that SIVmac239Δnef vaccination recruits plasma cells and induces ectopic lymphoid follicle formation beneath the mucosal epithelium in the rhesus macaque female reproductive tract. The plasma cells and ectopic follicles produce IgG Abs reactive with viral envelope glycoprotein gp41 trimers, and these Abs are concentrated on the path of virus entry by the neonatal FcR in cervical reserve epithelium and in vaginal epithelium. This local Ab production and delivery system correlated spatially and temporally with the maturation of local protection against high-dose pathogenic SIV vaginal challenge. Thus, designing vaccines to elicit production and concentration of Abs at mucosal frontlines could aid in the development of an effective vaccine to protect women against HIV-1.

KIR polymorphisms modulate peptide-dependent binding to an MHC class I ligand with a Bw6 motif.

Molecular interactions between killer immunoglobulin-like receptors (KIRs) and their MHC class I ligands play a central role in the regulation of natural killer (NK) cell responses to viral pathogens and tumors. Here we identify Mamu-A1*00201 (Mamu-A*02), a common MHC class I molecule in the rhesus macaque with a canonical Bw6 motif, as a ligand for Mamu-KIR3DL05. Mamu-A1*00201 tetramers folded with certain SIV peptides, but not others, directly stained primary NK cells and Jurkat cells expressing multiple allotypes of Mamu-KIR3DL05. Differences in binding avidity were associated with polymorphisms in the D0 and D1 domains of Mamu-KIR3DL05, whereas differences in peptide-selectivity mapped to the D1 domain. The reciprocal exchange of the third predicted MHC class I-contact loop of the D1 domain switched the specificity of two Mamu-KIR3DL05 allotypes for different Mamu-A1*00201-peptide complexes. Consistent with the function of an inhibitory KIR, incubation of lymphocytes from Mamu-KIR3DL05+ macaques with target cells expressing Mamu-A1*00201 suppressed the degranulation of tetramer-positive NK cells. These observations reveal a previously unappreciated role for D1 polymorphisms in determining the selectivity of KIRs for MHC class I-bound peptides, and identify the first functional KIR-MHC class I interaction in the rhesus macaque. The modulation of KIR-MHC class I interactions by viral peptides has important implications to pathogenesis, since it suggests that the immunodeficiency viruses, and potentially other types of viruses and tumors, may acquire changes in epitopes that increase the affinity of certain MHC class I ligands for inhibitory KIRs to prevent the activation of specific NK cell subsets.

Inhibitory TCR Coreceptor PD-1 Is a Sensitive Indicator of Low-Level Replication of SIV and HIV-1

Ongoing antigenic stimulation appears to be an important prerequisite for the persistent expression of programmed death 1 (PD-1), an inhibitory TCR coreceptor of the CD28 family. Although recent publications have emphasized the utility of PD-1 as a marker for dysfunctional T cells in chronic viral infections, its dependence on antigenic stimulation potentially renders it a sensitive indicator of low-level viral replication. To explore the antigenic threshold for the maintenance of PD-1 expression on virus-specific T cells, we compared PD-1 expression on virus-specific and memory T cell populations in controlled and uncontrolled SIV and HIV-1 infection. In both controlled live attenuated SIV infection in rhesus macaques and HIV-1 infection in elite controllers, elevated levels of PD-1 expression were observed on SIV- and HIV-1–specific CD8+ T cells. However, in contrast to chronic wild-type SIV infection and uncontrolled HIV-1 infection, controlled SIV/HIV-1 infection did not result in increased expression of PD-1 on total memory T cells. PD-1 expression on SIV-specific CD8+ T cells rapidly decreased after the emergence of CTL escape in cognate epitopes, but was maintained in the setting of low or undetectable levels of plasma viremia in live attenuated SIV-infected macaques. After inoculation of naive macaques with a single-cycle SIV, PD-1 expression on SIV-specific CD8+ T cells initially increased, but was rapidly downregulated. These results demonstrate that PD-1 can serve as a sensitive indicator of persistent, low-level virus replication and that generalized PD-1 expression on T lymphocytes is a distinguishing characteristic of uncontrolled lentiviral infections.

Characterization of killer immunoglobulin-like receptor genetics and comprehensive genotyping by pyrosequencing in rhesus macaques

BackgroundHuman killer immunoglobulin-like receptors (KIRs) play a critical role in governing the immune response to neoplastic and infectious disease. Rhesus macaques serve as important animal models for many human diseases in which KIRs are implicated; however, the study of KIR activity in this model is hindered by incomplete characterization of KIR genetics.ResultsHere we present a characterization of KIR genetics in rhesus macaques (Macaca mulatta). We conducted a survey of KIRs in this species, identifying 47 novel full-length KIR sequences. Using this expanded sequence library to build upon previous work, we present evidence supporting the existence of 22 Mamu-KIR genes, providing a framework within which to describe macaque KIRs. We also developed a novel pyrosequencing-based technique for KIR genotyping. This method provides both comprehensive KIR genotype and frequency estimates of transcript level, with implications for the study of KIRs in all species.ConclusionsThe results of this study significantly improve our understanding of macaque KIR genetic organization and diversity, with implications for the study of many human diseases that use macaques as a model. The ability to obtain comprehensive KIR genotypes is of basic importance for the study of KIRs, and can easily be adapted to other species. Together these findings both advance the field of macaque KIRs and facilitate future research into the role of KIRs in human disease.

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.

Enhancement of Microbiota in Healthy Macaques Results in Beneficial Modulation of Mucosal and Systemic Immune Function.

Given the critical role of mucosal surfaces in susceptibility to infection, it is imperative that effective mucosal responses are induced when developing efficacious vaccines and prevention strategies for infection. Modulating the microbiota in the gastrointestinal (GI) tract through the use of probiotics (PBio) is a safe and well-tolerated approach to enhance mucosal and overall health. We assessed the longitudinal impact of daily treatment with the VSL#3 probiotic on cellular and humoral immunity and inflammation in healthy macaques. PBio therapy resulted in significantly increased frequencies of B cells expressing IgA in the colon and lymph node (LN), likely because of significantly increased LN T follicular helper cell frequencies and LN follicles. Increased frequencies of IL-23+ APCs in the colon were found post-PBio treatment, which correlated with LN T follicular helper cells. Finally, VSL#3 significantly downmodulated the response of TLR2-, TLR3-, TLR4-, and TLR9-expressing HEK293 cells to stimulation with Pam3CSK4, polyinosinic-polycytidylic acid, LPS, and ODN2006, respectively. These data provide a mechanism for the beneficial impact of PBio on mucosal health and implicates the use of PBio therapy in the context of vaccination or preventative approaches to enhance protection from mucosal infection by improving immune defenses at the mucosal portal of entry.