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HIV-1-induced cytopathicity of thymocytes is a major cause of reduced peripheral T cells and rapid disease progression observed in HIV-1-infected infants. Understanding the virulence factors responsible for thymocyte depletion has paramount importance in addressing the pathogenesis of disease progression in children. In this study, thymocyte depletion was analyzed following infection with two primary CXCR4-tropic HIV-1 pediatric isolates (PI), PI-2 and PI-2.1, which were serially derived from an in utero-infected infant. Although highly similar to each other, PI-2 showed markedly decreased thymocyte depletion in vitro compared with PI-2.1. Further analysis showed a novel deletion in the Nef protein (NefΔK7S) of PI-2, which was absent in PI-2.1. This deletion inhibited Nef-mediated major histocompatibility complex class I (MHC-I) downregulation in infected thymocytes in vitro and in vivo; in contrast, the mutated Nef continued to downregulate CD4 surface expression in vitro. These results suggest that HIV-1 Nef contributes to thymic damage in infants through selective functions.

HIV-1–specific broadly neutralizing antibodies (bNAbs) can protect rhesus monkeys against simian-human immunodeficiency virus (SHIV) challenge. However, the site of antibody interception of virus and the mechanism of antibody-mediated protection remain unclear. We administered a fully protective dose of the bNAb PGT121 to rhesus monkeys and challenged them intravaginally with SHIV-SF162P3. In PGT121-treated animals, we detected low levels of viral RNA and viral DNA in distal tissues for seven days following challenge. Viral RNA–positive tissues showed transcriptomic changes indicative of innate immune activation, and cells from these tissues initiated infection after adoptive transfer into naïve hosts. These data demonstrate that bNAb-mediated protection against a mucosal virus challenge can involve clearance of infectious virus in distal tissues.

Natural killer (NK) cell responses in primates are regulated in part through interactions between two highly polymorphic molecules, the killer-cell immunoglobulin-like receptors (KIRs) on NK cells and their major histocompatibility complex (MHC) class I ligands on target cells. We previously reported that the binding of a common MHC class I molecule in the rhesus macaque, Mamu-A1*002, to the inhibitory receptor Mamu-KIR3DL05 is stabilized by certain simian immunodeficiency virus (SIV) peptides, but not by others. Here we investigated the functional implications of these interactions by testing SIV peptides bound by Mamu-A1*002 for the ability to modulate Mamu-KIR3DL05+ NK cell responses. Twenty-eight of 75 SIV peptides bound by Mamu-A1*002 suppressed the cytolytic activity of primary Mamu-KIR3DL05+ NK cells, including three immunodominant CD8+ T cell epitopes previously shown to stabilize Mamu-A1*002 tetramer binding to Mamu-KIR3DL05. Substitutions at C-terminal positions changed inhibitory peptides into disinhibitory peptides, and vice versa, without altering binding to Mamu-A1*002. The functional effects of these peptide variants on NK cell responses also corresponded to their effects on Mamu-A1*002 tetramer binding to Mamu-KIR3DL05. In assays with mixtures of inhibitory and disinhibitory peptides, low concentrations of inhibitory peptides dominated to suppress NK cell responses. Consistent with the inhibition of Mamu-KIR3DL05+ NK cells by viral epitopes presented by Mamu-A1*002, SIV replication was significantly higher in Mamu-A1*002+ CD4+ lymphocytes co-cultured with Mamu-KIR3DL05+ NK cells than with Mamu-KIR3DL05- NK cells. These results demonstrate that viral peptides can differentially affect NK cell responses by modulating MHC class I interactions with inhibitory KIRs, and provide a mechanism by which immunodeficiency viruses may evade NK cell responses., Author Summary Natural killer (NK) cells recognize and kill infected cells without prior antigenic stimulation, and thus provide an important early defense against virus infection. NK cell responses in primates are regulated in part through interactions between two highly polymorphic molecules, the killer-cell immunoglobulin-like receptors (KIRs) on NK cells and their major histocompatibility complex (MHC) class I ligands on target cells. Inhibitory KIRs normally suppress NK cell responses through interactions with their MHC class I ligands on the surface of healthy cells. However, when these interactions are perturbed, this inhibition is lost resulting in NK cell activation and killing of the target cell. We investigated the functional implications of simian immunodeficiency virus (SIV) peptides bound by a common MHC class I molecule in the rhesus macaque that stabilize or disrupt binding to an inhibitory KIR. Whereas SIV peptides that stabilized KIR-MHC class I binding suppressed NK cell activation, peptides that disrupted this interaction did not and resulted in NK cell lysis. These findings demonstrate that viral peptides can modulate NK cell responses through KIR-MHC class I interactions, and are consistent with the possibility that human and simian immunodeficiency viruses may acquire changes in epitopes that increase the binding of MHC class I ligands to inhibitory KIRs as a mechanism to suppress NK cell responses.

To defeat SIV, add a protein boost Despite 30 years of effort, no HIV-1 vaccine exists. Barouch et al. evaluated one promising strategy in rhesus macaques, a preclinical model commonly used to test potential HIV-1 vaccine candidates. They immunized monkeys with adenovirus-36 vectors engineered to express SIV (simian immunodeficiency virus) genes and then boosted them with a recombinant gp120 envelope glycoprotein (Env) from SIV. This regimen afforded greater protection than a strategy that instead used a viral vector–based boost. A parallel trial using a SHIV (simian/human immunodeficiency virus)–based vaccine and challenge model produced similar results. Whether this particular approach will be equally successful in humans remains to be tested. Science , this issue p. 320

The identification of MHC class I ligands for rhesus macaque killer cell Ig-like receptors (KIRs) is fundamental to our basic understanding of KIR and MHC class I coevolution and to the study of NK cell responses in this nonhuman primate model for AIDS and other viral diseases. In this study, we show that Mamu-KIR3DL01, which is expressed by ∼90% of rhesus macaques, recognizes MHC class I molecules with a Bw4 motif. Primary NK cells expressing Mamu-KIR3DL01 were identified by staining with a mAb which, in this study, was shown to bind Mamu-KIR3DL01 allotypes with an aspartic acid at position 233. The cytolytic activity of Mamu-KIR3DL01+ NK cells was suppressed by cell lines expressing the Bw4 molecules Mamu-B*007:01, -B*041:01, -B*058:02, and -B*065:01. The Bw4 motif was necessary for Mamu-KIR3DL01 recognition because substitutions in this region abrogated Mamu-KIR3DL01+ NK cell inhibition. However, the presence of a Bw4 motif was not sufficient for recognition because another Bw4 molecule, Mamu-B*017:01, failed to suppress the cytolytic activity of these NK cells. Replacement of three residues in Mamu-B*017:01, predicted to be KIR contacts based on the three-dimensional structure of the human KIR3DL1-HLA-Bw4 complex, with the corresponding residues at these positions for the other Mamu-Bw4 ligands restored Mamu-KIR3DL01+ NK cell inhibition. These results define the ligand specificity of one of the most polymorphic and commonly expressed KIRs in the rhesus macaque and reveal similarities in Bw4 recognition by Mamu-KIR3DL01 and human KIR3DL1, despite the absence of an orthologous relationship between these two KIRs or conservation of surface residues predicted to interact with MHC class I ligands.

SIV infection of macaques is the most widely employed model for preclinical AIDS vaccine and pathogenesis research. In macaques, high-titer virus-specific antibodies are induced by infection, and antibody responses can drive evolution of viral escape variants. However, neutralizing antibodies (Nabs) induced in response to SIVmac239 and SIVmac251 infection or immunization are generally undetectable or of low titer, and the identification and cloning of potent Nabs from SIVmac-infected macaques remains elusive. Based on recent advances in labeling HIV-specific B lymphocytes [1–3], we have generated recombinant, secreted, soluble SIVmac envelope (Env) proteins (gp120 and gp140) for detection and quantification of SIVmac Env-specific B lymphocytes. In contrast to HIV-1, we found that soluble SIVmac239 gp140 retains the ability to form stable oligomers without the necessity for introducing additional, stabilizing modifications. Soluble oligomeric gp140 reacted with rhesus anti-SIV Env-specific monoclonal antibodies (MAbs), and was used to deplete Env-specific antibodies with SIV neutralization capability from plasma taken from a rhesus macaque immunized with live attenuated SIVmac239∆nef. Soluble gp120 and gp140 bound to SIV-specific immortalized B cells, and to SIV Env-specific B lymphocytes in peripheral blood of immunized animals. These reagents will be useful for analyzing development of Env-specific B cell responses in preclinical studies using SIV-infected or vaccinated rhesus macaques.

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., Author Summary NK cells provide an important first line of defense against infectious diseases and tumors by virtue of their ability to kill infected or malignant cells without prior sensitization. NK cell activation is regulated in part through interactions between KIRs expressed on the surface of NK cells and their MHC class I ligands on target cells. Here we identify Mamu-A1*00201 (Mamu-A*02), a common MHC class I molecule in the rhesus macaque, as a ligand for Mamu-KIR3DL05. We show that this interaction is peptide-dependent, since soluble Mamu-A1*00201 tetramers folded with certain SIV peptides, but not others, stained cells expressing Mamu-KIR3DL05. Differences in binding avidity were associated with polymorphisms in the D0 and D1 domains of Mamu-KIR3DL05, whereas differences in peptide-specificity mapped to the D1 domain. 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. These observations suggest that SIV, and potentially also HIV-1, may acquire changes in epitopes that increase the avidity of MHC class I ligands for inhibitory KIRs as a mechanism of immune evasion to prevent the activation of certain NK cell subsets.