HIV type 1 subtype C gag and nef diversity in Southern Africa.

Several HIV-1 subtype C-specific gag- and/or nef-based vaccines are currently intended for clinical trial in southern Africa. Here we provide sequences of 64 gag and 45 nef genes sampled in Malawi, Zambia, Zimbabwe, and South Africa and assess the degree of southern African HIV-1 diversity that will confront these vaccines. Whereas reasonable phylogenetic evidence exists for geographical clustering of subtype C gag and nef sequences from various other parts of the world, there is little evidence of similar population founder effects in the southern African epidemic. The entire breadth of subtype C diversity is represented in the southern African genes suggesting there may be no advantage in producing region- or country-specific subtype C vaccines. We do not, however, find much evidence of intersubtype recombination in the Southern African genes, implying that the likelihood of vaccine failure due to the emergence of intersubtype recombinants is probably low.

Characterization of gp120 hydrolysis by IgA antibodies from humans without HIV infection.

Antibody hydrolysis of the superantigenic gp120 site and HIV-1 neutralization was studied as a potential anti-HIV mechanism in uninfected humans. gp120 hydrolysis by purified serum and salivary antibodies was determined by electrophoresis and peptide sequencing, the proteolytic mechanism was analyzed using electrophilic peptide analogs, and viral neutralization was studied using peripheral blood mononuclear cells as hosts. Polyclonal and monoclonal IgA but not IgG preparations selectively catalyzed the cleavage of HIV gp120 at rates sufficient to predict biologically relevant protection against the virus. The IgA hydrolytic reaction proceeded by noncovalent recognition of gp120 residues 421-433, a component of the superantigenic site of gp120, coordinated with peptide bond cleavage via a serine protease-like mechanism. The Lys-432-Ala-433 bond was one of the cleavage sites. Infection of peripheral blood mononuclear cells by a primary isolate of HIV was neutralized by the IgA but not IgG fractions. The neutralizing activity was specifically inhibited by an electrophilic inhibitor of the catalytic activity. The existence of catalytic IgAs to gp120 in uninfected humans suggests their role in resistance to HIV.

Viral dynamics and CD4+ T cell counts in subtype C human immunodeficiency virus type 1-infected individuals from southern Africa.

Defining viral dynamics in natural infection is prognostic of disease progression and could prove to be important for vaccine trial design as viremia may be a likely secondary end point in phase III HIV efficacy trials. There are limited data available on the early course of plasma viral load in subtype C HIV-1 infection in Africa. Plasma viral load and CD4+ T cell counts were monitored in 51 recently infected subjects for 9 months. Individuals were recruited from four southern African countries: Zambia, Malawi, Zimbabwe, and South Africa and the median estimated time from seroconversion was 8.9 months (interquartile range, 5.7-14 months). All were infected with subtype C HIV-1 and median viral loads, measured using branched DNA, ranged from 3.82-4.02 log10 RNA copies/ml from 2-24 months after seroconversion. Viral loads significantly correlated with CD4+ cell counts (r=-0.5, p<0.0001; range, 376-364 cells/mm3) and mathematical modeling defined a median set point of 4.08 log10 (12 143 RNA copies/ml), which was attained approximately 17 months after seroconversion. Comparative measurements using three different viral load platforms (bDNA, Amplicor, and NucliSens) confirmed that viremia in subtype C HIV-1-infected individuals within the first 2 years of infection did not significantly differ from that found in early subtype B infection. In conclusion, the course of plasma viremia, as described in this study, will allow a useful baseline comparator for understanding disease progression in an African setting and may be useful in the design of HIV-1 vaccine trials in southern Africa.

Inactivated- or killed-virus HIV/AIDS vaccines.

Inactivated or "killed" virus (KV) is a "classical" approach that has produced safe and effective human and veterinary vaccines but has received relatively little attention in the effort to develop an HIV/AIDS vaccine. Initially, KV and rgp120 subunit vaccines were the two most obvious approaches but, unfortunately, rgp120 has not been efficacious and the KV approach has been limited by a variety of scientific, technical, and sociological factors. For example, when responses to cellular antigens, present on SIV grown in human cells, proved to be largely responsible for efficacy, the KV approach was widely discounted. Similarly, when lab-adapted HIV-1 appeared to lose envelope glycoprotein during preparation (not the case for primary isolates), this was viewed as a fundamental barrier to the KV concept. Also, a preference for "safer", genetically-engineered vaccines, and emphasis on cellular immunity, have left KV low on the priority list for funding agencies and investigators. The recent suggestion that "native" trimeric gp120 displays conserved conformational neutralization epitopes, along with the failure of rgp120, and difficulties in raising strong cellular responses with DNA or vectored vaccines, has restored some interest in the KV concept. In the past 15 years, several groups have initiated pre-clinical development of KV candidates for SIV or HIV and promising, albeit limited, information has been produced. In this chapter we discuss the rationale (including pros and cons) for producing and testing killed-HIV vaccines, the prospects for success, the nature and scope of research needed to test the KV concept, what has been learned to date, and what remains undone.

Effects of CCR5-Delta32 and CCR2-64I alleles on HIV-1 disease progression: the protection varies with duration of infection.

OBJECTIVE: To examine temporal variation in the effects of CCR5-Delta32 and CCR2-64I chemokine receptor gene polymorphisms on HIV-1 disease progression. DESIGN: Pooled analysis of individual patient data from 10 cohorts of HIV-1 seroconverters from the United States, Europe, and Australia. METHODS: We studied HIV-1 seroconverters of European (n = 1635) or African (n = 215) ancestry who had been genotyped for CCR5-Delta32 and CCR2-64I. We used Cox proportional hazards models with time-varying coefficients to determine whether the genetic protection against AIDS (1987 case definition) and death varied with time since seroconversion. RESULTS: Protection against AIDS conferred by CCR5-Delta32 held constant at a 31% (RH 0.69, 95% CI 0.54, 0.88) reduction in risk over the course of HIV-1 infection, whereas protection against death held constant at a 39% reduction in risk (RH 0.61, 95% CI 0.45, 0.88). When the period from AIDS to death was isolated, the survival benefit of CCR5-Delta32 diminished 2 years after AIDS. Protection against AIDS conferred by CCR2-64I was greatest early in the disease course. Compared with individuals without CCR5-Delta32 or CCR2-64I, individuals with one or two copies of CCR2-64I had a 58% lower risk of AIDS during the first 4 years after seroconversion (RH 0.42, 95% CI 0.23, 0.76), a 19% lower risk during the subsequent 4 years (RH 0.81, 95% CI 0.59, 1.12), and no significant protection thereafter. CONCLUSION: The protection against AIDS provided by CCR5-Delta32 is continuous during the course of infection. In contrast, the protection provided by CCR2-64I is greatest early in the course of infection.

High incidence and prevalence of HIV-1 infection in high risk population in Calcutta, India.

HIV-1 infection in India has been increasing steadily over the last decade. In the absence of potent antiviral therapy, estimates of HIV infection are needed to monitor the epidemic, institute prevention strategies in target populations and determine the suitable populations for vaccine studies. In this report we present the HIV-1 seroprevalence and annual estimates of seroincidence in a high risk population from Calcutta, the most populous city in the eastern part of India. In 1206 high risk subjects tested over two years between February of 1999 and December 2000, we have determined an overall seroprevalence of 40.1% using enzyme-linked immunosorbent assay followed by a confirmatory Western blot testing. Furthermore, using a newly described Standardized Testing Algorithm for Recent HIV-1 Seroconversion (STARHS), we have estimated an annual seroincidence rate of about 7% in this population during this two-year study. Such a high annual seroincidence rate makes this population well suited for studies of HIV-1 prevention, including vaccine trials.

Genetic characteristics of HIV-1 subtype C envelopes inducing cross-neutralizing antibodies.

This study aimed to characterize genetic features of HIV-1 subtype C envelope glycoproteins capable of eliciting cross-reactive neutralizing antibodies during natural infections. The gp160 sequences were determined for 36 HIV-1 subtype C isolates (donor viruses) from infected individuals residing in Malawi, Zimbabwe, Zambia and South Africa, whose sera displayed a range of cross-neutralizing activities against a panel of 5 subtype C and 5 subtype B viruses (panel viruses). Hierarchical clustering analysis of neutralization data of the panel viruses predicted phylogenetic relationships between subtype B and C panel viruses, suggesting some subtype-specific neutralization determinants. A similar comparison of subtype C donor viruses showed no significant correlation; however of three donor sequence pairs resolvable by phylogenetic analysis, two were also associated within the neutralization clustering dendrogram, suggesting that closely related viruses may elicit antibodies targeting common neutralization determinants. Significantly, viruses that had shorter V1-V4 loops induced antibodies that showed more neutralization breadth against the subtype C panel viruses (p=0.0135). This study indicates that that some structural features of envelope, such as shorter variable loops, may facilitate the elicitation of cross-reactive neutralizing antibodies in natural infections. Collectively these data provide some insights into design features of an envelope immunogen aimed at inducing neutralizing antibodies.

Sequential turnover of human immunodeficiency virus type 1 env throughout the course of infection.

We examined the rates of variant population turnover of the V1-V2 and V4-V5 hypervariable domains of the human immunodeficiency virus type 1 (HIV-1) gp120 molecule in longitudinal plasma samples from 14 men with chronic HIV-1 infection using heteroduplex tracking assays (HTA). Six men had high rates of CD4+ T-cell loss, and eight men had low rates of CD4+ T-cell loss over 2.5 to 8 years of infection. We found that V1-V2 and V4-V5 env populations changed dramatically over time in all 14 subjects; the changes in these regions were significantly correlated with each another over time. The subjects with rapid CD4 loss had significantly less change in their env populations than the subjects with slow CD4 loss. The two subjects with rapid CD4 loss and sustained low CD4 counts (<150/microl for at least 2 years) showed stabilization of their V1-V2 and V4-V5 populations as reflected by low levels of total change in HTA pattern and low HTA indices (a novel measure of the emergence of new bands and band distribution); this stabilization was not observed in other subjects. The stabilization of env variant populations at low CD4 counts following periods of rapid viral evolution suggests that selective pressure on env, likely from new immune responses, is minimal when CD4 counts drop dramatically and remain low for extended periods of time.

Rate and incidence estimates of recent human immunodeficiency virus type 1 infections among pregnant women in Sao Paulo, Brazil, from 1991 to 2002.

The serological testing algorithm for recent human immunodeficiency virus (HIV) seroconversion (STARHS) was employed to estimate HIV incidence among pregnant women from Sao Paulo, Brazil. A cross-sectional study (1999 to 2002) showed an incidence of infection of 0.2 per 100 pregnant women per year (95% confidence interval, 0.041 to 0.608). Western blot profiles suggested an association between results of the STARHS analysis and gp41/gp31 bands.

High incidence of HIV-1 in South Africa using a standardized algorithm for recent HIV seroconversion.

Southern Africa has among the highest rates of HIV-1 infection in the world as judged by cross-sectional HIV-1 prevalence surveys carried out among women attending antenatal clinics. Incidence rates, which provide information on the number of new cases of infection, are more informative of the current state of the epidemic than estimates of prevalence, which provide information on the rates averaged over some previous time. Cohort studies to measure incidence rates are expensive and difficult to carry out, however, and few have been done in Africa. A recently developed standardized algorithm for recent HIV-1 seroconversion (STARHS) based on a sensitive/less-sensitive enzyme-linked immunosorbent assay was used to determine the incidence of HIV-1 subtype C infection among women attending public sector antenatal clinics in Hlabisa, a rural district in KwaZulu-Natal, South Africa. The STAHRS results were confirmed by using a mathematic model to obtain an independent estimate of the age-specific incidence rates from the age-specific prevalence data. The data reveal extraordinarily high HIV-1 incidence rates in South Africa. In 1999, the annual incidence of HIV-1 among susceptible women aged 15 to 49 years standardized to the age distribution of adult women in Hlabisa was 17%. Incidence peaked among 22-year-old women at 24% per year. The HIV-1 incidence rates provide valuable additional information indicating that new infections are continuing unabated and that the HIV-1 epidemic is still growing in rural South Africa.

Development of a new less-sensitive enzyme immunoassay for detection of early HIV-1 infection.

The sensitive/less-sensitive (S/LS) enzyme immunoassay (EIA) testing strategy for discriminating "early" from "longstanding" HIV infection has been widely applied for detecting recent seroconverters and estimating HIV incidence rates. The originally developed assay (3A11-LS EIA; Abbott Laboratories, Abbott Park, IL) involved performance of LS EIAs using a bead-based assay that required specialized equipment and reagents of limited availability. In contrast, 96-microwell-based EIAs are more universally applied for HIV serodiagnosis throughout the world. The authors report development and preliminary validation of an LS protocol using an EIA in a 96-well format: the Vironostika HIV-1 MicroElisa System (Vironostika-LS EIA; Bio Merieux, Raleigh, NC). The results with samples from recent HIV-1 seroconverters, persons with longstanding HIV-1 asymptomatic infection, patients on highly active antiretroviral therapy, and AIDS patients show a high degree of correlation between the Vironostika-LS EIA and 3A11-LS EIA. The authors also demonstrate that the Abbott 3A11-LS EIA and Vironostika-LS EIA performed comparably on HIV-1-positive samples from persons infected with non-B HIV-1 subtypes. These results support the potential use of the Vironostika-LS EIA for detection of recent HIV-1 infections for incidence projections and for other epidemiologic, clinical, and molecular surveillance applications.

HIV-1 infection in individuals with the CCR5-Delta32/Delta32 genotype: acquisition of syncytium-inducing virus at seroconversion.

Homozygosity for the 32 base-pair deletion (Delta32/Delta32) in the CCR5 coreceptor gene is associated with incomplete HIV-1 resistance. Six HIV-1-infected Delta32/Delta32 patients have been reported. We report 2 additional Delta32/Delta32-infected individuals, among 106 seroconverters in a vaccine preparedness study. Like the previous 6, these individuals experienced rapid CD4 decline. However, taken together, the 8 patients have neither uniformly high virus load nor rapid progression to AIDS. We obtained five virus isolates from 1 patient at 5, 6, 7, 10, and 12 months after the estimated time of infection. The earliest isolate exhibits the syncytium-inducing (SI) phenotype and exclusive use of the CXCR4 coreceptor, suggesting acquisition of HIV-1 through this coreceptor. Of the remaining 104 seroconverters, 8 were CCR5-Delta32/+ and 96 were CCR5-+/+. Three CCR5-+/+ seroconverters who showed the uncommon pattern of early SI virus and rapid CD4 decline had uniformly high viral load and more heterogeneous coreceptor usage. These results further support the conclusion that Delta32-mediated resistance is incomplete and is associated with acquisition of exclusively-X4 variants of HIV-1. The pathogenic potential of these viruses may be different from late-stage X4 virus or early X4 virus acquired by individuals with other CCR5 genotypes.

Phase I/II study of a candidate vaccine designed against the B and E subtypes of HIV-1.

A phase I/II trial of a candidate vaccine to prevent HIV infection was carried out in Bangkok, Thailand, testing AIDSVAX B/E (VaxGen, Inc., Brisbane, CA), a bivalent subunit vaccine prepared by combining recombinant gp120 from a subtype B virus (HIV-1MN) with gp120 from a subtype E virus (HIV-1A244) in alum adjuvant. The studies provide human data on the immunogenicity of various dose combination of non-subtype B vaccine antigens. The results suggest that AIDSVAX B/E is safe and immunogenic in humans. The optimal dose for humans in developing countries was 300 microg of each antigen (B and E). Clade E responses were measurably increased by immunizing with gp120 B/E over B alone. Using the B/E combination did not interfere with the response to either clade. Antibodies to AIDSVAX B/E were able to bind to oligomeric gp120 on the surface of cells infected with primary isolates of HIV-1.

Breakthrough infections during phase 1 and 2 prime-boost HIV-1 vaccine trials with canarypox vectors (ALVAC) and booster dose of recombinant gp120 or gp160.

Candidate human immunodeficiency virus (HIV)-1 vaccines that elicit cytotoxic T lymphocytes may modulate HIV infection, requiring a prototype evaluation to assess participants who become infected with HIV. Of 1497 participants in canarypox HIV-1 vaccine prime-boost trials, 28 (1.9%) acquired HIV-1 infection after vaccination. Median plasma HIV-1 RNA levels (vaccinees, 4.78 log10 copies/mL; placebo recipients, 4.27 log10 copies/mL) and CD4 cell counts (vaccinees, 552 cells/mm3; placebo recipients, 657 cells/mm3) before administration of antiretroviral therapy (ART) and time to a composite end point (plasma HIV-1 RNA level >55,000 copies/mL, CD4 cell count <350 cells/mm3, or initiation of ART) did not differ significantly between vaccinees and placebo recipients (P =.4, P =.1, and P =.7, respectively). Persons who acquire HIV-1 infection while enrolled in HIV-1 vaccine trials can be successfully followed after infection, to determine whether vaccines alter the course of HIV-1 infection.

Hierarchical targeting of subtype C human immunodeficiency virus type 1 proteins by CD8+ T cells: correlation with viral load.

An understanding of the relationship between the breadth and magnitude of T-cell epitope responses and viral loads is important for the design of effective vaccines. For this study, we screened a cohort of 46 subtype C human immunodeficiency virus type 1 (HIV-1)-infected individuals for T-cell responses against a panel of peptides corresponding to the complete subtype C genome. We used a gamma interferon ELISPOT assay to explore the hypothesis that patterns of T-cell responses across the expressed HIV-1 genome correlate with viral control. The estimated median time from seroconversion to response for the cohort was 13 months, and the order of cumulative T-cell responses against HIV proteins was as follows: Nef > Gag > Pol > Env > Vif > Rev > Vpr > Tat > Vpu. Nef was the most intensely targeted protein, with 97.5% of the epitopes being clustered within 119 amino acids, constituting almost one-third of the responses across the expressed genome. The second most targeted region was p24, comprising 17% of the responses. There was no correlation between viral load and the breadth of responses, but there was a weak positive correlation (r = 0.297; P = 0.034) between viral load and the total magnitude of responses, implying that the magnitude of T-cell recognition did not contribute to viral control. When hierarchical patterns of recognition were correlated with the viral load, preferential targeting of Gag was significantly (r = 0.445; P = 0.0025) associated with viral control. These data suggest that preferential targeting of Gag epitopes, rather than the breadth or magnitude of the response across the genome, may be an important marker of immune efficacy. These data have significance for the design of vaccines and for interpretation of vaccine-induced responses.

Novel and promiscuous CTL epitopes in conserved regions of Gag targeted by individuals with early subtype C HIV type 1 infection from southern Africa.

Characterization of optimal CTL epitopes in Gag can provide crucial information for evaluation of candidate vaccines in populations at the epicenter of the HIV-1 epidemic. We screened 38 individuals with recent subtype C HIV-1 infection using overlapping consensus C Gag peptides and hypothesized that unique HLA-restricting alleles in the southern African population would determine novel epitope identity. Seventy-four percent of individuals recognized at least one Gag peptide pool. Ten epitopic regions were identified across p17, p24, and p2p7p1p6, and greater than two-thirds of targeted regions were directed at: TGTEELRSLYNTVATLY (p17, 35%); GPKEPFRDYVDRFFKTLRAEQATQDV (p24, 19%); and RGGKLDKWEKIRLRPGGKKHYMLKHL (p17, 15%). After alignment of these epitopic regions with consensus M and a consensus subtype C sequence from the cohort, it was evident that the regions targeted were highly conserved. Fine epitope mapping revealed that five of nine identified optimal Gag epitopes were novel: HLVWASREL, LVWASRELERF, LYNTVATLY, PFRDYVDRFF, and TLRAEQATQD, and were restricted by unique HLA-Cw*08, HLA-A*30/B*57, HLA-A*29/B*44, and HLA-Cw*03 alleles, respectively. Notably, three of the mapped epitopes were restricted by more than one HLA allele. Although these epitopes were novel and restricted by unique HLA, they overlapped or were embedded within previously described CTL epitopes from subtype B HIV-1 infection. These data emphasize the promiscuous nature of epitope binding and support our hypothesis that HLA diversity between populations can shape fine epitope identity, but may not represent a constraint for universal recognition of Gag in highly conserved domains.