Arnaud D. Colantonio

Protective Efficacy of Adenovirus/Protein Vaccines Against SIV Challenges in Rhesus Monkeys

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 A viral vector–recombinant envelope glycoprotein–based HIV-1 vaccine strategy protected 50% of monkeys from infection. Preclinical studies of viral vector–based HIV-1 vaccine candidates have previously shown partial protection against neutralization-resistant virus challenges in rhesus monkeys. In this study, we evaluated the protective efficacy of adenovirus serotype 26 (Ad26) vector priming followed by purified envelope (Env) glycoprotein boosting. Rhesus monkeys primed with Ad26 vectors expressing SIVsmE543 Env, Gag, and Pol and boosted with AS01B-adjuvanted SIVmac32H Env gp140 demonstrated complete protection in 50% of vaccinated animals against a series of repeated, heterologous, intrarectal SIVmac251 challenges that infected all controls. Protective efficacy correlated with the functionality of Env-specific antibody responses. Comparable protection was also observed with a similar Ad/Env vaccine against repeated, heterologous, intrarectal SHIV-SF162P3 challenges. These data demonstrate robust protection by Ad/Env vaccines against acquisition of neutralization-resistant virus challenges in rhesus monkeys.

Endogenous IFN-α Production by Plasmacytoid Dendritic Cells Exerts an Antiviral Effect on Thymic HIV-1 Infection

Plasmacytoid dendritic cells (pDC) are the principal producers of IFN-α in response to viral infection. Because pDC are present in the thymus, we investigated the consequences of HIV-1-induced IFN-α production by thymic pDC. We observed that thymic pDC as well as thymocytes express intracellular IFN-α upon infection with HIV-1. However, only the pDC could suppress HIV-1 replication, because depletion of pDC resulted in enhancement of HIV-1 replication in thymocytes. Thymic pDC could also produce IFN-α in response to CpG oligonucleotides, consistent with the observations of others that peripheral pDC produce IFN-α upon engagement of TLR-9. Importantly, CpG considerably increased IFN-α production induced by HIV-1, and addition of CpG during HIV-1 infection enhanced expression of the IFN response protein MxA in thymocytes and strongly reduced HIV-replication. Our data indicate that thymic pDC modulate HIV-1 replication through secretion of IFN-α. The degree of inhibition depends on the level of IFN-α produced by the thymic pDC.

Antibody-mediated protection against SHIV challenge includes systemic clearance of distal virus

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.

Persistent gammaherpesvirus replication and dynamic interaction with the host in vivo.

ABSTRACT Gammaherpesviruses establish life-long persistency inside the host and cause various diseases during their persistent infection. However, the systemic interaction between the virus and host in vivo has not been studied in individual hosts continuously, although such information can be crucial to control the persistent infection of the gammaherpesviruses. For the noninvasive and continuous monitoring of the interaction between gammaherpesvirus and the host, a recombinant murine gammaherpesvirus 68 (MHV-68, a gammaherpesvirus 68) was constructed to express a firefly luciferase gene driven by the viral M3 promoter (M3FL). Real-time monitoring of M3FL infection revealed novel sites of viral replication, such as salivary glands, as well as acute replication in the nose and the lung and progression to the spleen. Continuous monitoring of M3FL infection in individual mice demonstrated the various kinetics of transition to different organs and local clearance, rather than systemically synchronized clearance. Moreover, in vivo spontaneous reactivation of M3FL from latency was detected after the initial clearance of acute infection and can be induced upon treatment with either a proteasome inhibitor Velcade or an immunosuppressant cyclosporine A. Taken together, our results demonstrate that the in vivo replication and reactivation of gammaherpesvirus are dynamically controlled by the locally defined interaction between the virus and the host immune system and that bioluminescence imaging can be successfully used for the real-time monitoring of this dynamic interaction of MHV-68 with its host in vivo.

The role of the thymus in HIV infection: a 10 year perspective.

Despite substantial progress over the last 10 years the exact role of the thymus in HIV-1 infection and HIV-1 pathogenesis is still under investigation. Much has been learned of the types of cells in the thymus that are targets for CXCR4 and CCR5 HIV-1 isolates. In addition, it has become clear that even the adult thymus continues to function, although at a much lower level in uninfected patients, and is able to export naive T cells to the periphery. Changes in thymus function can be evaluated by several methods, including determination of naive T-cell subsets using multicolor flow cytometry and T-cell receptor excision circles (TREC), as well as thymus size and metabolic labeling assays [1–5]. Although each of these measures has its advantages and drawbacks [6], combinations of these parameters provide a picture of the contribution of thymic output and homeostatic proliferative expansion (HPE) to peripheral blood T-cell homeostasis. The importance of the thymus in regenerating a functional immune system has been clearly shown after chemotherapy, bone marrow transplantation and highly active retroviral therapy (HAART) in HIV infection [5,7–10]. The data published during the last 10 years also show that a possible increase in thymic output has an instrumental role in the immunopathogenesis that takes place during the clinically asymptomatic phase of HIV-1 infection.

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.

KIR3DL01 Recognition of Bw4 Ligands in the Rhesus Macaque: Maintenance of Bw4 Specificity since the Divergence of Apes and Old World Monkeys

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.

Suppression of a Natural Killer Cell Response by Simian Immunodeficiency Virus Peptides.

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.

CD8 T Cell Response Maturation Defined by Anentropic Specificity and Repertoire Depth Correlates with SIVΔnef-induced Protection

The live attenuated simian immunodeficiency virus (LASIV) vaccine SIVΔnef is one of the most effective vaccines in inducing protection against wild-type lentiviral challenge, yet little is known about the mechanisms underlying its remarkable protective efficacy. Here, we exploit deep sequencing technology and comprehensive CD8 T cell epitope mapping to deconstruct the CD8 T cell response, to identify the regions of immune pressure and viral escape, and to delineate the effect of epitope escape on the evolution of the CD8 T cell response in SIVΔnef-vaccinated animals. We demonstrate that the initial CD8 T cell response in the acute phase of SIVΔnef infection is mounted predominantly against more variable epitopes, followed by widespread sequence evolution and viral escape. Furthermore, we show that epitope escape expands the CD8 T cell repertoire that targets highly conserved epitopes, defined as anentropic specificity, and generates de novo responses to the escaped epitope variants during the vaccination period. These results correlate SIVΔnef-induced protection with expanded anentropic specificity and increased response depth. Importantly, these findings render SIVΔnef, long the gold standard in HIV/SIV vaccine research, as a proof-of-concept vaccine that highlights the significance of the twin principles of anentropic specificity and repertoire depth in successful vaccine design.

Reproducing SIVΔnef vaccine correlates of protection: trimeric gp41 antibody concentrated at mucosal front lines

Vaccination with SIVmac239&Dgr;nef provides robust protection against subsequent challenge with wild-type simian immunodeficiency virus (SIV), but safety issues have precluded designing an HIV-1 vaccine based on a live-attenuated virus concept. Safe immunogens and adjuvants that could reproduce identified immune correlates of SIVmac239&Dgr;nef protection therefore offer an alternative path for development of an HIV vaccine. Here we describe SIV envelope trimeric gp41 (gp41t) immunogens based on a protective correlate of antibodies to gp41t concentrated on the path of virus entry by the neonatal Fc receptor (FcRn) in cervical vaginal epithelium. We developed a gp41t immunogen-monophosphoryl lipid A adjuvant liposomal nanoparticle for intramuscular (i.m.) immunization and a gp41t-Fc immunogen for intranasal immunization for pilot studies in mice, rabbits, and rhesus macaques. Repeated immunizations to mimic persistent antigen exposure in infection elicited gp41t antibodies in rhesus macaques that were detectable in FcRn+ cervical vaginal epithelium, thus recapitulating one key feature of SIVmac239&Dgr;nef vaccinated and protected animals. Although this strategy did not reproduce the system of local production of antibody in SIVmac239&Dgr;nef-vaccinated animals, passive immunization experiments supported the concept that sufficiently high levels of antibody can be concentrated by the FcRn at mucosal frontlines, thus setting the stage for assessing protection against vaginal challenge by gp41t immunization.