Scientific Publications

With the accessibility of prevention of mother to child transmission (PMTCT) services in sub-Saharan Africa, more women are being tested for HIV in antenatal care settings. Involving partners in the counselling and testing process could help prevent horizontal and vertical transmission of HIV. This study was conducted to assess the feasibility of couples' voluntary counseling and testing (CVCT) in antenatal care and to measure compliance with PMTCT.

The importance of a broad CD8 T lymphocyte (CD8-TL) immune response to HIV is unknown. Ex vivo measurements of immunological activity directed at a limited number of defined epitopes provide an incomplete portrait of the actual immune response. We examined viral loads in simian immunodeficiency virus (SIV)-infected major histocompatibility complex (MHC)-homozygous and MHC-heterozygous Mauritian cynomolgus macaques. Chronic viremia in MHC-homozygous macaques was 80 times that in MHC-heterozygous macaques. Virus from MHC-homozygous macaques accumulated 11 to 14 variants, consistent with escape from CD8-TL responses after 1 year of SIV infection. The pattern of mutations detected in MHC-heterozygous macaques suggests that their epitope-specific CD8-TL responses are a composite of those present in their MHC-homozygous counterparts. These results provide the clearest example of MHC heterozygote advantage among individuals infected with the same immunodeficiency virus strain, suggesting that broad recognition of multiple CD8-TL epitopes should be a key feature of HIV vaccines.

During untreated, chronic HIV-1 infection, plasma viral load (VL) is a relatively stable quantitative trait that has clinical and epidemiological implications. Immunogenetic research has established various human genetic factors, especially human leukocyte antigen (HLA) variants, as independent determinants of VL set-point.

Most people infected with HIV-1 are dually infected with herpes simplex virus type 2. Daily suppression of this herpes virus reduces plasma HIV-1 concentrations, but whether it delays HIV-1 disease progression is unknown. We investigated the effect of acyclovir on HIV-1 progression.

We have characterized an assay measuring CD8 T cell-mediated inhibition of human immunodeficiency virus (HIV) type 1 replication, demonstrating specificity and reproducibility and employing a panel of primary HIV-1 isolates. The assay uses relatively simple autologous cell culture and enzyme-linked immunosorbent assay, avoids generation of T cell clones, and can be performed with 2 million peripheral blood mononuclear cells. Efficient CD8 T cell-mediated cross-clade inhibition of HIV-1 replication in vitro was demonstrated in antiretroviral therapy-naive HIV-1-infected subjects with controlled viral replication in vivo but not in viremic subjects. An HIV-1 vaccine candidate, consisting of DNA and recombinant adenovirus 5 vectors tested in a phase I clinical trial, induced CD8 T cells that efficiently inhibited HIV-1 in a HLA-I-dependent manner. Assessment of direct antiviral T cell function by this assay provides additional information to guide vaccine design and the prioritizing of candidates for further clinical trials.

The membrane-proximal external region (MPER) of the HIV-1 gp41 transmembrane glycoprotein is the target of the broadly neutralizing antibody 2F5. Prior studies have suggested a two-component mechanism for 2F5-mediated neutralization involving both structure-specific recognition of a gp41 protein epitope and nonspecific interaction with the viral lipid membrane. Here, we mutationally alter a hydrophobic patch on the third complementarity-determining region of the heavy chain (CDR H3) of the 2F5 antibody and assess the abilities of altered 2F5 variants to bind gp41 and to neutralize diverse strains of HIV-1. CDR H3 alterations had little effect on the affinity of 2F5 variants for a peptide corresponding to its gp41 epitope. In contrast, strong effects and a high degree of correlation (P < 0.0001) were found between virus neutralization and CDR H3 hydrophobicity, as defined by predicted free energies of transfer from water to a lipid bilayer interface or to octanol. The effect of CDR H3 hydrophobicity on neutralization was independent of isolate sensitivity to 2F5, and CDR H3 variants with tryptophan substitutions were able to neutralize HIV-1 approximately 10-fold more potently than unmodified 2F5. A threshold was observed for increased hydrophobicity of the 2F5 CDR H3 loop beyond which effects on 2F5-mediated neutralization leveled off. Together, the results provide a more complete understanding of the 2F5 mechanism of HIV-1 neutralization and indicate ways to enhance the potency of MPER-directed antibodies.

Design of an envelope glycoprotein (Env)-based vaccine against human immunodeficiency virus type-1 (HIV-1) is complicated by the large number of N-linked glycans that coat the protein and serve as a barrier to antibody-mediated neutralization. Compared to normal mammalian glycoproteins, high-mannose-type glycans are disproportionately represented on the gp120 subunit of Env. These N-glycans serve as a target for a number of anti-HIV molecules that bind terminal alpha1,2-linked mannose residues, including lectins and the monoclonal antibody 2G12. We created a Saccharomyces cerevisiae glycosylation mutant, Deltamnn1Deltamnn4, to expose numerous terminal Manalpha1,2-Man residues on endogenous hypermannosylated glycoproteins in the yeast cell wall. Immunization of rabbits with whole cells from this mutant induced antibodies that bound to a broad range of Env proteins, including clade A, B, and C of HIV and simian immunodeficiency virus (SIV). The gp120 binding activity of these immune sera was due to mannose-specific immunoglobulin, as removal of high-mannose glycans and alpha1,2-linked mannoses from gp120 abrogated serum binding. Glycan array analysis with purified IgG demonstrated binding mainly to glycans with Manalpha1,2-Manalpha1,2-Man trisaccharides. Altogether, these data demonstrate the immunogenicity of exposed polyvalent Manalpha1,2-Manalpha1,2-Man structures on the yeast cell wall mannan and their ability to induce antibodies that bind to the HIV Env protein. The yeast strain and sera from this study will be useful tools for determining the type of mannose-specific response that is needed to develop neutralizing antibodies to the glycan shield of HIV.

Most persons who are infected with human immunodeficiency virus type 1 (HIV-1) are also infected with herpes simplex virus type 2 (HSV-2), which is frequently reactivated and is associated with increased plasma and genital levels of HIV-1. Therapy to suppress HSV-2 reduces the frequency of reactivation of HSV-2 as well as HIV-1 levels, suggesting that suppression of HSV-2 may reduce the risk of transmission of HIV-1.

The introduction of antiretroviral (ARV) therapy in resource-poor settings is effective in suppressing HIV-1 replication and prolonging life of infected individuals. This has led to a demand for affordable HIV-1 drug resistance assays, since treatment failure due to development of drug resistance is common. This study developed and evaluated an affordable 'in-house' genotyping assay to monitor HIV-1 drug resistance in Africa, particularly South Africa. An 'in-house' assay using automated RNA extraction, and subtype C specific PCR and sequencing primers was developed and successfully evaluated 396 patient samples (viral load ranges 1000-1.6 million RNA copies/ml). The 'in-house' assay was validated by comparing sequence data and drug resistance profiles from 90 patient and 10 external quality control samples to data from the ViroSeq HIV-1 Genotyping kit. The 'in-house' assay was more efficient, amplifying all 100 samples, compared to 91 samples using Viroseq. The 'in house' sequences were 99.2% homologous to the ViroSeq sequences, and identical drug resistance mutation profiles were observed in 96 samples. Furthermore, the 'in-house' assay genotyped 260 of 295 samples from seven African sites, where 47% were non-subtype C. Overall, the newly validated 'in-house' drug resistance assay is suited for use in Africa as it overcomes the obstacle of subtype diversity.

We compared HIV-1 strains in incident and prevalent infections in a cohort of men having sex with men (MSM) and female sex workers (FSW) near Mombasa, Kenya and conducted a cross-sectional study of viral isolates from a sample of HIV-1-infected MSM and FSW in Kilifi, Coast Province, Kenya. RNA extracted from plasma of 13 MSM, 9 FSW, and one heterosexual male was amplified by nested RT-PCR and the products were directly sequenced. HIV-1 strains from 21 individuals were characterized with one or more complete genome sequences, and two were sequenced in the Nef gene. The envelope quasispecies was also studied in one individual. Among MSM, eight strains were subtype A and five were recombinant. There were two epidemiologically linked pairs of sequences; one pair was subtype A and the other pair was a complex AA2CD recombinant of identical structure. Another MSM was dually infected with DG recombinant strains of related, but nonidentical, structure. MSM also harbored AC and AD recombinant strains. The FSW harbored seven subtype A strains, an AD recombinant, and an AA2D strain related to CRF16_A2D. The one heterosexual male studied had a subtype A infection. This MSM epidemic in Kenya appears to be of local origin, harboring many strains typical of the broader Kenyan epidemic. Characteristics of a close social network were identified, with extended chains of transmission, novel recombinant strains possibly generated within the network, and a relatively high proportion of recombinant and dual infections.

The membrane-proximal external region (MPER) of HIV-1, located at the C terminus of the gp41 ectodomain, is conserved and crucial for viral fusion. Three broadly neutralizing monoclonal antibodies (bnMAbs), 2F5, 4E10, and Z13e1, are directed against linear epitopes mapped to the MPER, making this conserved region an important potential vaccine target. However, no MPER antibodies have been definitively shown to provide protection against HIV challenge. Here, we show that both MAbs 2F5 and 4E10 can provide complete protection against mucosal simian-human immunodeficiency virus (SHIV) challenge in macaques. MAb 2F5 or 4E10 was administered intravenously at 50 mg/kg to groups of six male Indian rhesus macaques 1 day prior to and again 1 day following intrarectal challenge with SHIV(Ba-L). In both groups, five out of six animals showed complete protection and sterilizing immunity, while for one animal in each group a low level of viral replication following challenge could not be ruled out. The study confirms the protective potential of 2F5 and 4E10 and supports emphasis on HIV immunogen design based on the MPER region of gp41.

The high-affinity in vivo interaction between soluble HIV-1 envelope glycoprotein (Env) immunogens and primate CD4 results in conformational changes that alter the immunogenicity of the gp120 subunit. Because the conserved binding site on gp120 that directly interacts with CD4 is a major vaccine target, we sought to better understand the impact of in vivo Env-CD4 interactions during vaccination. Rhesus macaques were immunized with soluble wild-type (WT) Env trimers, and two trimer immunogens rendered CD4 binding defective through distinct mechanisms. In one variant, we introduced a mutation that directly disrupts CD4 binding (368D/R). In the second variant, we introduced three mutations (423I/M, 425N/K, and 431G/E) that disrupt CD4 binding indirectly by altering a gp120 subdomain known as the bridging sheet, which is required for locking Env into a stable interaction with CD4. Following immunization, Env-specific binding antibody titers and frequencies of Env-specific memory B cells were comparable between the groups. However, the quality of neutralizing antibody responses induced by the variants was distinctly different. Antibodies against the coreceptor binding site were elicited by WT trimers but not the CD4 binding-defective trimers, while antibodies against the CD4 binding site were elicited by the WT and the 423I/M, 425N/K, and 431G/E trimers but not the 368D/R trimers. Furthermore, the CD4 binding-defective trimer variants stimulated less potent neutralizing antibody activity against neutralization-sensitive viruses than WT trimers. Overall, our studies do not reveal any potential negative effects imparted by the in vivo interaction between WT Env and primate CD4 on the generation of functional T cells and antibodies in response to soluble Env vaccination.