Scientific Publications

Currently there is limited information about the quality of immune responses elicited by candidate human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env)-based immunogens in primates. Here we describe a comprehensive analysis of neutralizing antibody and T-cell responses obtained in cynomolgus macaques by three selected immunization regimens. We used the previously described YU2-based gp140 protein trimers administered in an adjuvant, preceded by two distinct priming strategies: either alphavirus replicon particles expressing matched gp140 trimers or gp120 core proteins stabilized in the CD4-bound conformation. The rationale for priming with replicon particles was to evaluate the impact of the expression platform on trimer immunogenicity. The stable core proteins were chosen in an attempt to expand selectively lymphocytes recognizing common determinants between the core and trimers to broaden the immune response. The results presented here demonstrate that the platform by which Env trimers were delivered in the priming (either protein or replicon vector) had little impact on the overall immune response. In contrast, priming with stable core proteins followed by a trimer boost strikingly focused the T-cell response on the core sequences of HIV-1 Env. The specificity of the T-cell response was distinctly different from that of the responses obtained in animals immunized with trimers alone and was shown to be mediated by CD4(+) T cells. However, this regimen showed limited or no improvement in the neutralizing antibody responses, suggesting that further immunogen design efforts are required to successfully focus the B-cell response on conserved neutralizing determinants of HIV-1 Env.

The Partners HSV-2/HIV-1 Transmission Study (Partners Study) is a phase III, placebo-controlled trial of daily acyclovir for genital herpes (HSV-2) suppression among HIV-1/HSV-2 co-infected persons to reduce HIV-1 transmission to their HIV-1 susceptible partners, which requires recruitment of HIV-1 serodiscordant heterosexual couples. We describe the baseline characteristics of this cohort.

During human immunodeficiency virus type 1 (HIV-1) infection, patients develop various levels of neutralizing antibody (NAb) responses. In some cases, patient sera can potently neutralize diverse strains of HIV-1, but the antibody specificities that mediate this broad neutralization are not known, and their elucidation remains a formidable challenge. Due to variable and nonneutralizing determinants on the exterior envelope glycoprotein (Env), nonnative Env protein released from cells, and the glycan shielding that assembles in the context of the quaternary structure of the functional spike, HIV-1 Env elicits a myriad of binding antibodies. However, few of these antibodies can neutralize circulating viruses. We present a systematic analysis of the NAb specificities of a panel of HIV-1-positive sera, using methodologies that identify both conformational and continuous neutralization determinants on the HIV-1 Env protein. Characterization of sera included selective adsorption with native gp120 and specific point mutant variants, chimeric virus analysis, and peptide inhibition of viral neutralization. The gp120 protein was the major neutralizing determinant for most sera, although not all neutralization activity against all viruses could be identified. In some broadly neutralizing sera, the gp120-directed neutralization mapped to the CD4 binding region of gp120. In addition, we found evidence that regions of the gp120 coreceptor binding site may also be a target of neutralizing activity. Sera displaying limited neutralization breadth were mapped to the immunogenic V3 region of gp120. In a subset of sera, we also identified NAbs directed against the conserved, membrane-proximal external region of gp41. These data allow a more detailed understanding of the humoral responses to the HIV-1 Env protein and provide insights regarding the most relevant targets for HIV-1 vaccine design.

Clinical laboratory reference intervals have not been established in many African countries, and non-local intervals are commonly used in clinical trials to screen and monitor adverse events (AEs) among African participants. Using laboratory reference intervals derived from other populations excludes potential trial volunteers in Africa and makes AE assessment challenging. The objective of this study was to establish clinical laboratory reference intervals for 25 hematology, immunology and biochemistry values among healthy African adults typical of those who might join a clinical trial.

The HIV-1 epidemic in sub-Saharan Africa is driven largely by heterosexual transmission of non-subtype B viruses, of which subtypes C and A are predominant. Previous studies of subtype B and subtype C transmission pairs have suggested that a single variant from the chronically infected partner can establish infection in their newly infected partner. However, in subtype A infected individuals from a sex worker cohort and subtype B individuals from STD clinics, infection was frequently established by multiple variants. This study examined over 1750 single-genome amplified viral sequences derived from epidemiologically linked subtype C and subtype A transmission pairs very early after infection. In 90% (18/20) of the pairs, HIV-1 infection is initiated by a single viral variant that is derived from the quasispecies of the transmitting partner. In addition, the virus initiating infection in individuals who were infected by someone other than their spouse was characterized to determine if genital infections mitigated the severe genetic bottleneck observed in a majority of epidemiologically linked heterosexual HIV-1 transmission events. In nearly 50% (3/7) of individuals infected by someone other than their spouse, multiple genetic variants from a single individual established infection. A statistically significant association was observed between infection by multiple genetic variants and an inflammatory genital infection in the newly infected individual. Thus, in the vast majority of HIV-1 transmission events in cohabiting heterosexual couples, a single genetic variant establishes infection. Nevertheless, this severe genetic bottleneck can be mitigated by the presence of inflammatory genital infections in the at risk partner, suggesting that this restriction on genetic diversity is imposed in large part by the mucosal barrier.

An HIV vaccine remains elusive despite the concerted efforts of investigators and clinicians over the past two decades. Animal models are regularly used to obtain new insights on disease pathogenesis and have become invaluable tools in the translation of treatments from basic research laboratories to the clinic. Vaccination of macaques with live, attenuated simian immunodeficiency virus is currently the most effective method of garnering protection against subsequent pathogenic SIV challenge. However, immunization of humans with live, attenuated HIV is not feasible due to safety concerns. Therefore, clues to an effective and safe vaccine against HIV may be found by studying immune correlates of protection in the live, attenuated, vaccinated macaque model. Previous studies have identified the immune correlates of protection against Friend retrovirus in live, attenuated vaccinated mice using allogeneic adoptive transfers. Similar experiments in macaques have thus far been hindered due to the vast genetic diversity found within outbred populations. Here we review the current state of SIV adoptive transfer research and present a novel macaque model that allows for allogeneic adoptive transfers.

IAVI-006 was the first large randomised, double-blinded, placebo-controlled Phase I clinical trial to systematically investigate the prime-boost strategy for induction of HIV-1 specific CD8+ cytotoxic T-lymphocytes (CTL) in a factorial trial design using (i) priming with 0.5 mg or 2 mg of pTHr.HIVA DNA vaccine, followed by (ii) two booster vaccinations with 5 x 10(7) MVA.HIVA at weeks 8 and 12 (early boost) or weeks 20 and 24 (late boost). This study set the basis for later clinical trials and demonstrated the safety of these candidate HIV vaccines. The safety and immunogenicity results are presented and the lessons derived from this clinical trial are discussed.

Human immunodeficiency virus type 1 and simian immunodeficiency virus possess three closely spaced, highly conserved sites for N-linked carbohydrate attachment in the extracellular domain of the transmembrane protein gp41. We infected rhesus monkeys with a variant of cloned SIVmac239 lacking the second and third sites or with a variant strain lacking all three of SIVmac239's glycosylation sites in gp41. For each mutation, asparagine (N) in the canonical N-X-S/T recognition sequence for carbohydrate attachment was changed to the structurally similar glutamine such that two nucleotide changes would be required for a reversion of the mutated codon. By 16 weeks, experimentally infected monkeys made antibodies that neutralized the mutant viruses to high titers. Such antibodies were not observed in monkeys infected with the parental virus. Thus, new specificities were revealed as a result of the carbohydrate attachment mutations, and antibodies of these specificities had neutralizing activity. Unlike monkeys infected with the parental virus, monkeys infected with the mutant viruses made antibodies that reacted with peptides corresponding to the sequences in this region. Furthermore, there was strong selective pressure for the emergence of variant sequences in this region during the course of infection. By analyzing the neutralization profiles of sequence variants, we were able to define three mutations (Q625R, K631N, and Q634H) in the region of the glycosylation site mutations that conferred resistance to neutralization by plasma from the monkeys infected with mutant virus. Based on the reactivity of antibodies to peptides in this region and the colocalization of neutralization escape mutations, we conclude that N-linked carbohydrates in the ectodomain of the transmembrane protein shield underlying epitopes that would otherwise be the direct targets of neutralizing antibodies.

The V3 loop of HIV-1 gp120 is considered occluded on many primary viruses. However, virus sensitivity to neutralization by different V3 mAbs often varies, indicating that access to V3 is not restricted equally for all antibodies. Here, we have sought to gain a better understanding of these restrictions by determining the neutralizing activities of 7 V3 mAbs (19b, 39F, CO11, F2A3, F530, LA21, and LE311) against 15 subtype B primary isolates and relating these activities to the fine specificity of the mAbs. Not surprisingly, we found that most mAbs neutralized the same 2-3 viruses, with only mAb F530 able to neutralize 2 additional viruses not neutralized by the other mAbs. Epitope mapping revealed that positively-charged residues in or near the V3 stem are important for the binding of all the mAbs and that most mAbs seem to require the Pro residue that forms the GPGR beta hairpin turn in the V3 tip for binding. Based on the mapping, we determined that V3 sequence variation accounted for neutralization resistance of approximately half the viruses tested. Comparison of these results to those of select V3 mAbs with overall better neutralizing activities in the light of structural information illustrates how an antibody's mode of interaction with V3, driven by contact residue requirements, may restrict the antibody from accessing its epitope on different viruses. Based on the data we propose an angle of interaction with V3 that is less stringent on access for antibodies with cross-neutralizing activity compared to antibodies that neutralize relatively fewer viruses.

Plasma samples from individuals infected with human immunodeficiency virus type 1 (HIV-1) are known to be highly strain specific in their ability to neutralize HIV-1 infectivity. Such plasma samples exhibit significant neutralizing activity against autologous HIV-1 isolates but typically exhibit little or no activity against heterologous strains, although some cross-neutralizing activity can develop late in infection. Monkeys infected with the simian-human immunodeficiency virus (SHIV) clone DH12 generated antibodies that neutralized SHIV DH12, but not SHIV KB9. Conversely, antibodies from monkeys infected with the SHIV clone KB9 neutralized SHIV KB9, but not SHIV DH12. To investigate the role of the variable loops of the HIV-1 envelope glycoprotein gp120 in determining this strain specificity, variable loops 1 and 2 (V1/V2), V3, or V4 were exchanged individually or in combination between SHIV DH12 and SHIV KB9. Despite the fact that both parental viruses exhibited significant infectivity and good replication in the cell lines examined, 3 of the 10 variable-loop chimeras exhibited such poor infectivity that they could not be used further for neutralization assays. These results indicate that a variable loop that is functional in the context of one particular envelope background will not necessarily function within another. The remaining seven replication-competent chimeras allowed unambiguous assignment of the sequences principally responsible for the strain specificity of the neutralizing activity present in SHIV-positive plasma. Exchange of the V1/V2 loop sequences conferred a dominant loss of sensitivity to neutralization by autologous plasma and a gain of sensitivity to neutralization by heterologous plasma. Substitution of V3 or V4 had little or no effect on the sensitivity to neutralization. These data demonstrate that the V1/V2 region of HIV-1 gp120 is principally responsible for the strain specificity of the neutralizing antibody response in monkeys infected with these prototypic SHIVs.

Killer Ig-like receptors (KIRs) are implicated in protection from multiple pathogens including HIV, human papillomavirus, and malaria. Nonhuman primates such as rhesus and cynomolgus macaques are important models for the study of human pathogens; however, KIR genetics in nonhuman primates are poorly defined. Understanding KIR allelic diversity and genomic organization are essential prerequisites to evaluate NK cell responses in macaques. In this study, we present a complete characterization of KIRs in Mauritian cynomolgus macaques, a geographically isolated population. In this study we demonstrate that only eight KIR haplotypes are present in the entire population and characterize the gene content of each. Using the simplified genetics of this population, we construct a model for macaque KIR genomic organization, defining four putative KIR3DL loci, one KIR3DH, two KIR2DL, and one KIR1D. We further demonstrate that loci defined in Mauritian cynomolgus macaques can be applied to rhesus macaques. The findings from this study fundamentally advance our understanding of KIR genetics in nonhuman primates and establish a foundation from which to study KIR signaling in disease pathogenesis.

An effective AIDS vaccine will need to protect against globally diverse isolates of HIV. To address this issue in macaques, we administered a live-attenuated simian immunodeficiency virus (SIV) vaccine and challenged with a highly pathogenic heterologous isolate. Vaccinees reduced viral replication by approximately 2 logs between weeks 2-32 (P < or = 0.049) postchallenge. Remarkably, vaccinees expressing MHC-I (MHC class I) alleles previously associated with viral control completely suppressed acute phase replication of the challenge virus, implicating CD8(+) T cells in this control. Furthermore, transient depletion of peripheral CD8(+) lymphocytes in four vaccinees during the chronic phase resulted in an increase in virus replication. In two of these animals, the recrudescent virus population contained only the vaccine strain and not the challenge virus. Alarmingly, however, we found evidence of recombinant viruses emerging in some of the vaccinated animals. This finding argues strongly against an attenuated virus vaccine as a solution to the AIDS epidemic. On a more positive note, our results suggest that MHC-I-restricted CD8(+) T cells contribute to the protection induced by the live-attenuated SIV vaccine and demonstrate that vaccine-induced CD8(+) T cell responses can control replication of heterologous challenge viruses.