Effects of CCR5-Delta32, CCR2-64I, and SDF-1 3'A alleles on HIV-1 disease progression: An international meta-analysis of individual-patient data.

BACKGROUND: Studies relating certain chemokine and chemokine receptor gene alleles with the outcome of HIV-1 infection have yielded inconsistent results. OBJECTIVE: To examine postulated associations of genetic alleles with HIV-1 disease progression. DESIGN: Meta-analysis of individual-patient data. SETTING: 19 prospective cohort studies and case-control studies from the United States, Europe, and Australia. PATIENTS: Patients with HIV-1 infection who were of European or African descent. MEASUREMENTS: Time to AIDS, death, and death after AIDS and HIV-1 RNA level at study entry or soon after seroconversion. Data were combined with fixed-effects and random-effects models. RESULTS: Both the CCR5-Delta32 and CCR2-64I alleles were associated with a decreased risk for progression to AIDS (relative hazard among seroconverters, 0.74 and 0.76, respectively; P = 0.01 for both), a decreased risk for death (relative hazard among seroconverters, 0.64 and 0.74; P < 0.05 for both), and lower HIV-1 RNA levels after seroconversion (difference, -0.18 log(10) copies/mL and -0.14 log(10) copies/mL; P < 0.05 for both). Having the CCR5-Delta32 or CCR2-64I allele had no clear protective effect on the risk for death after development of AIDS. The results were consistent between seroconverters and seroprevalent patients. In contrast, SDF-1 3'A homozygotes showed no decreased risk for AIDS (relative hazard for seroconverters and seroprevalent patients, 0.99 and 1.03, respectively), death (relative hazard, 0.97 and 1.00), or death after development of AIDS (relative hazard, 0.81 and 0.97; P > 0.5 for all). CONCLUSIONS: The CCR5-Delta32 and CCR2-64I alleles had a strong protective effect on progression of HIV-1 infection, but SDF-1 3'A homozygosity carried no such protection.

Vaccine-induced antibodies to the native, oligomeric envelope glycoproteins of primary HIV-1 isolates.

A simple and sensitive method for measuring antibodies to primary human immunodeficiency virus type 1 (HIV-1) isolates has been developed. The flow cytometric immuno-fluorescence assay detects antibodies that bind to the native, oligomeric form of the envelope glycoprotein (gp120) expressed on the surface of PM-1 cells infected with primary isolates of HIV-1. Sera from people infected with HIV-1 or those immunized with recombinant gp120 vaccines were tested. Significant correlation was observed between neutralizing activity and oligomeric gp120 binding activity. Thirteen to 100% of individuals immunized with the subtype B bivalent vaccine AIDSVAX B/B developed oligomeric gp120 binding antibodies against a variety of subtype B primary isolates. For several isolates, AIDSVAX B/B sera reacted better than monovalent AIDSVAX B sera, suggesting that addition of the second immunogen improved the breadth of the antibody response. Cross-subtype binding activities, induced by AIDSVAX B/B, were lower than activities to subtype B isolates, suggesting that additional immunogen(s) may be desirable in vaccine(s) formulated for geographic regions where non-B subtypes are dominant.

Homozygous and heterozygous CCR5-Delta32 genotypes are associated with resistance to HIV infection.

OBJECTIVE: To investigate evidence for resistance to HIV-1 infection associated with the heterozygous genotype CCR5-+/Delta32 and with the homozygous genotype CCR5-Delta32/Delta32, which results in a nonfunctional CCR5 receptor. DESIGN: Cohort study of initially HIV-seronegative high-risk individuals from eight different cities. Enrollment data were analyzed to investigate the association of demographic factors and risk behaviors with CCR5 genotypes on the assumption that increased genotype prevalence among persons with histories of longer or more intensive exposure to HIV would indicate HIV resistance associated with that genotype. Longitudinal data were analyzed to investigate the association of HIV seroincidence with CCR5 genotypes. The cohort of 2996 individuals included 1892 men who have sex with men (MSM), 474 male injection drug users (IDUs), 347 women at heterosexual risk, and 283 female IDUs. MEASUREMENTS: CCR5 genotype, HIV serostatus, demographic factors, and risk behaviors during the 6 months before enrollment, followed by measurement of HIV seroincidence during the subsequent 18 months (for men) and 24 months (for women). RESULTS: Forty (1.3%) subjects were homozygous CCR5-Delta32/Delta32 and 387 (12.9%) were heterozygous CCR5-+/Delta32. All but 1 CCR5-Delta32/Delta32 individuals and 51 CCR5-+/Delta32 individuals were Caucasian. Among 1531 Caucasian MSM, CCR5-+/Delta32 individuals were present more frequently (22.3%) among those reporting unprotected receptive anal intercourse than among those not reporting this risk (15.9%) (p =.002), suggesting a selective advantage of the heterozygous genotype. CCR5-+/Delta32 individuals also had a significantly reduced relative risk of HIV seroconversion adjusted for unprotected receptive anal intercourse compared with CCR5-/+ individuals (relative risk = 0.30, 95% confidence interval [CI]: 0.08-0.97). CCR5-Delta32/Delta32 prevalence among Caucasian MSM was significantly associated with age among subjects recruited from high HIV seroprevalence cities (New York City and San Francisco) (odds ratio [OR] for each decade increase in age = 2.57, CI: 1.56-4.21) but not among those recruited from lower HIV prevalence sites (Boston, Chicago, Philadelphia, Seattle, and Providence/Pawtucket, Rhode Island) (OR = 1.20, CI: 0.75-1.89). CONCLUSIONS: Cross-sectional and longitudinal analyses indicated that among high-risk HIV seronegative MSM, CCR5-+/Delta32 and CCR5-Delta32/Delta32 are associated with protection against HIV infection. These findings imply that strategies aimed at reducing susceptibility to HIV infection by blocking CCR5 receptor sites need not seek blockage of all receptor sites to achieve an imperfect but substantial degree of protection.

Early human immunodeficiency virus (HIV) infection in the HIV Network for Prevention Trials Vaccine Preparedness Cohort: risk behaviors, symptoms, and early plasma and genital tract virus load.

Risk behaviors, symptoms, and virologic characteristics were studied among 103 human immunodeficiency virus (HIV) seroconverters in vaccine preparedness cohorts during 1995-1998. Overall, 83% of subjects were men who had sex with men; most reported multiple risk episodes and symptoms (84%, > or =1 symptom) during seroconversion. Acute HIV was diagnosed in only 8 of 50 who sought medical care. Median initial pretreatment plasma virus load was 25,800 copies/mL (range, undetectable-262,000 copies/mL) a mean of 4 months after seroconversion, and 9.7% had nucleoside-associated mutations; none had multidrug resistance. Semen virus load was more variable, 1.3 log(10) lower and modestly correlated (r=.28; 95% confidence interval, 0.16-0.42) with plasma among untreated men. When the plasma RNA level was <5000 copies/mL, 32% of untreated men, 13% on nucleoside regimens, and 7% on protease inhibitor-containing regimens had detectable seminal RNA. Acute HIV was seldom diagnosed, representing missed opportunities for early treatment and prevention. Most subjects had several relatively stable virus loads before initiation of antiretrovirals, indicating feasibility of assessing HIV vaccines on virus set point in efficacy trials.

Identification of a high-risk heterosexual population for HIV prevention trials in Rio de Janeiro, Brazil. Projeto Praço Onze Study Group.

The study of interventions to prevent HIV transmission requires access to populations with a high rate of HIV transmission. We estimated HIV incidence among heterosexual males and females who were seen at an HIV testing site in Rio de Janeiro, Brazil. Stored sera from individuals who visited the site between March and December 1998 were analyzed using the sensitive/less sensitive (S/LS) assay and a chart abstraction was performed. During the study period, 6353 serum samples were tested. Of those tested, 1203 were found to be HIV-seropositive or indeterminate, of which 1050 (87%) remained available for further testing. In addition, 84 serum samples, representing 63 adults, were found to produce results suggesting early HIV infection. Of these, 14 were heterosexual and female (median age, 38 years), and 19 were heterosexual and male (median age, 25 years). The estimated HIV seroincidence was 1.9 (95% confidence limits (CL), 0.9%-3.9%) and 2.8 (95% CL, 1.4%-5.3%) per 100 person-years among heterosexual women and men, respectively. A survey on willingness to participate in future placebo-controlled HIV vaccine trials in this population indicated that 54.5% and 53.9% of heterosexual women and men, respectively, indicated that they would definitely be willing to participate. We have identified a heterosexual population in Rio de Janeiro with a high rate of HIV transmission willing to participate in placebo-controlled vaccine trials. This study demonstrates the usefulness of the newly described S/LS assay, which allows one to estimate HIV incidence from single serum specimens.

Shorter survival of SDF1-3'A/3'A homozygotes linked to CD4+ T cell decrease in advanced human immunodeficiency virus type 1 infection.

The SDF-1 3'A allelic polymorphism has been reported to influence either positively or negatively the progression of human immunodeficiency virus type 1 (HIV-1) disease. Therefore, the SDF-1 genotype of 729 HIV-1-infected individuals pooled from 3 distinct cohorts was determined. A statistically nonsignificant association between the SDF1-3'A/3'A genotype and accelerated disease progression was evident among seroconverters (n=319), but a striking correlation of decreased survival after either diagnosis of AIDS according to the 1993 definition or loss of CD4(+) T cell counts <200 was observed. The relative hazards for SDF1-3'A/3'A homozygotes, compared with heterozygotes and wild-type homozygotes were 2.16 (P=.0047), for time from diagnosis according to the 1993 Centers for Disease Control and Prevention AIDS case definition (AIDS-'93) to death, and 3.43 (P=.0001), for time from CD4(+) T cells <200 to death. Because no difference in survival was observed after diagnosis according to AIDS-'87, the association of the SDF1-3'A/3'A genotype with the accelerated progression of late-stage HIV-1 disease appears to be explained for the most part by the loss of CD4(+) T lymphocytes.

Dissociation of immunologic and virologic responses to highly active antiretroviral therapy.

OBJECTIVE: Immunologic markers, levels of HIV DNA, and infectious HIV were compared in partial responders (PR) to HAART who had high plasma HIV RNA levels but stable or increasing levels of CD4+ peripheral blood mononuclear cells (PBMC), and patients with complete failure (CF) who had very low or decreasing levels of CD4+ PBMC and high plasma HIV RNA levels. DESIGN AND METHODS: CD4 and CD8 levels were monitored by flow cytometry. Beta2-microglobulin (beta2M) and neopterin levels were measured by quantitative enzyme immunoassays. Plasma and PBMC from 11 PR and 13 CF were analyzed for infectious HIV levels in limiting dilution cultures. Polymerase chain reaction (PCR) assays were used to quantify cellular HIV DNA and plasma HIV RNA. RESULTS: In comparison with CF, PR had little or no CD4+ cell loss, a substantial increase in CD8+ cells, significantly fewer positive plasma HIV cultures (p = .03), lower frequencies of infectious HIV in total PBMC (p = .005) and in CD4+ PBMC (p < .001), and lower frequencies of HIV DNA in CD4+ PBMC (p = .007). CONCLUSIONS: Lower levels of infectious HIV and a lower frequency of CD4+ PBMC that contain "productive" HIV DNA in PR as compared with CF may contribute to the stable or increasing CD4+ PBMC levels of the PR. However, HAART may also have effects on lymphocyte homeostasis independent of its antiviral activity.

Full-length human immunodeficiency virus type 1 genomes from subtype C-infected seroconverters in India, with evidence of intersubtype recombination.

The development of an effective human immunodeficiency virus type 1 (HIV-1) vaccine is likely to depend on knowledge of circulating variants of genes other than the commonly sequenced gag and env genes. In addition, full-genome data are particularly limited for HIV-1 subtype C, currently the most commonly transmitted subtype in India and worldwide. Likewise, little is known about sequence variation of HIV-1 in India, the country facing the largest burden of HIV worldwide. Therefore, the objective of this study was to clone and characterize the complete genome of HIV-1 from seroconverters infected with subtype C variants in India. Cocultured HIV-1 isolates were obtained from six seroincident individuals from Pune, India, and virtually full-length HIV-1 genomes were amplified, cloned, and sequenced from each. Sequence analysis revealed that five of the six genomes were of subtype C, while one was a mosaic of subtypes A and C, with multiple breakpoints in env, nef, and the 3' long terminal repeat as determined by both maximal chi2 analysis and phylogenetic bootstrapping. Sequences were compared for preservation of known cytotoxic T lymphocyte (CTL) epitopes. Compared with those of the HIV-1LAI sequence, 38% of well-defined CTL epitopes were identical. The proportion of nonconservative substitutions for Env, at 61%, was higher (P < 0.001) than those for Gag (24%), Pol (18%), and Nef (32%). Therefore, characterized CTL epitopes demonstrated substantial differences from subtype B laboratory strains, which were most pronounced in Env. Because these clones were obtained from Indian seroconverters, they are likely to facilitate vaccine-related efforts in India by providing potential antigens for vaccine candidates as well as for assays of vaccine responsiveness.

Correlation between humoral responses to human immunodeficiency virus type 1 envelope and disease progression in early-stage infection.

Human immunodeficiency virus (HIV)-1-infected rapid and slow progressors showed differential humoral responses against HIV envelope peptides and proteins early in infection. Sera from slow progressors reacted more strongly with short envelope peptides modeling gp160NL4-3, predominantly in gp41. Reactivity to six peptides (in constant regions C3, C4, and C5 of gp120 and in gp41) correlated with slower progression. In a novel association, reactivity to three peptides (in constant regions C1 and C3 and variable region V3 of gp120) correlated with faster progression. Envelope peptide reactivity correlated with subsequent course of disease progression as strongly as did reactivity to gag p24. Patients heterozygous for 32-bp deletions in the CCR5 coreceptor reacted more frequently to an epitope in gp41. Rapid progressors had greater gp120 native-to-denatured binding ratios than did slow progressors. While antibody-dependent cellular cytotoxicity against gp120 did not strongly differentiate the groups, slow progressors showed a broader neutralization pattern against 5 primary virus isolates.

Cross-clade envelope glycoprotein 160-specific CD8+ cytotoxic T lymphocyte responses in early HIV type 1 clade B infection.

A major objective of current HIV-1 vaccination strategies is the induction of HIV-1-specific CD8+ MHC class I-restricted CTL responses, which are suggested to play a pivotal role in viral clearance and protection against HIV-1 disease progression. However, the marked genetic diversity of HIV-1 and existence of distinct viral subtypes or clades could potentially hinder the development of a universally efficacious HIV-1 vaccine. In this study we examined HIV-1 intraclade (B(LAI) versus B(MN)) Env gp160-specific CTL reactivity in recently HIV-1 clade B-infected individuals. We further evaluated the extent of interclade CTL cross-recognition of the divergent A and C Env gp160 subtypes, that are highly prevalent in the global pandemic. Freshly isolated PBMCs were stimulated in vitro with autologous PBMCs infected with recombinant vaccinia vectors expressing HIV-1 env, gag, pol, and nef genes derived from HIV-1 clade B. All 13 of the 19 HIV-1-seropositive subjects who elicited significant clade B Env gp160LAI CD8+ CTL responses also demonstrated comparable levels of CTL cross-reactivity against clade C92BR025 Env gp160. Nine of these individuals also showed extensive interclade CTL cross-recognition of clade A92UG037 Env gp160. Two HLA class I B7 donors had nondetectable intraclade CTL response against B Env gp160MN, while generating significant intraclade B(LAI) and interclade (A and C) Env gp160 CTL cross-reactivity. These observations serve to underscore the central importance of the HLA background of individuals in determining the pattern of immune reactivity to natural HIV-1 infection and presumably vaccines. Five donors studied also demonstrated broad CTL cross-reactivity against clade A92UG037 Gag p55, Pol, and/or Nef antigens. In conclusion, this present study indicates that there is a considerable degree of CD8+ CTL cross-recognition of the highly divergent HIV-1 Env gp160 subtypes during early phases of HIV-1 infection. Such findings suggest that HIV-1 vaccines based on a single clade that can induce extensive cross-clade immunity may demonstrate utility in diverse geographical regions.

Transmission of genetically diverse strains of HIV-1 in Pune, India.

Surveillance of the different HIV-1 subtypes has important implications for developing candidate vaccines and understanding the dynamics of HIV transmission in various populations. In this study, HIV-1 viral subtypes were determined for homologies in the V3-V5 region by heteroduplex mobility assay (HMA) in 46 patients with sexually transmitted diseases (STD) in Pune, India. Proviral DNA from peripheral blood mononuclear cells (PBMCs) from 20 recent sero-coverters and 26 HIV seropositive individuals were analyzed. Of the 46 samples analyzed, 44 (96%) were HIV-1 subtype C and one each of subtypes A and B. Further analyses revealed that 29 (66%) of the C subtype samples had maximum homology to the C3-Indian reference strain, while 15 (34%) were most homologous to the C2-Zambian strain. The C3 genotype prevailed in the majority (80%) of the seropositive individuals. Most of the C3 (Indian) strains were closely homologous to each other, while more nucleotide sequence divergence was seen in C2 samples. A higher quasispecies complexity was observed in the samples collected from seropositive individuals. These findings may have important implications for the design and testing of effective candidate HIV-1 vaccines for India.

PIP: HIV-1 viral subtypes were determined for homologies in the V3-V5 region by heteroduplex mobility assay (HMA) in 46 patients with sexually transmitted diseases (STDs) in Pune, India. Proviral DNA from the peripheral blood mononuclear cells (PBMCs) of 20 recent seroconverters and 26 HIV-seropositive individuals were analyzed. 44 of the samples analyzed were HIV-1 subtype C, 1 sample was subtype A, and 1 sample was subtype B. 29 of the subtype C samples had maximum homology to the C3-Indian reference strain, while 15 were most homologous to the C2-Zambian strain. 80% of the seropositive subjects were infected with the C3 genotype. Most C3 strains observed were closely homologous to each other, while greater nucleotide sequence divergence was seen in the C2 samples. A higher quasi-species complexity was observed in the samples collected from seropositive individuals. These study findings may have important implications for the design and testing of effective candidate HIV-1 vaccines for India.

Direct HIV cytopathicity cannot account for CD4 decline in AIDS in the presence of homeostasis: a worst-case dynamic analysis.

The central paradox of HIV pathogenesis is that the viral burden, either free or cellular, seems too low to deplete the CD4 population by direct killing. Until recently, little data could be used to compare direct and indirect pathogenic theories critically. Clinical trials with potent new antiviral agents have measured important kinetic parameters of HIV infection, including viral and infected cell half-lives. This has led to the construction of explicit models of direct killing. Using a worst-case dynamic analysis, we show that such cytopathic models are untenable. Rates of infected cell removal are orders of magnitude too low to suppress steady state CD4 counts significantly in the face of lymphocyte replenishment, especially in early infection. Furthermore, the direct cytopathic models, as proposed, predict an extremely variable disease course across the broad range of observed viral burdens (five orders of magnitude), which is inconsistent with the relatively small differences in disease progression observed between patients. In contrast, immunologic theories of pathogenesis, such as homeostatic dysregulation based on immune activation, do not suffer from these difficulties and are more consistent with the natural history of HIV infection.

Lack of associations of chemotactic cytokines with viral burden, disease progression, or lymphocyte subsets in HIV-infected individuals.

Plasma samples from HIV-infected (HIV+) rapid progressors (RP) and nonprogressors (NP) in the San Francisco Men's Health Study showed significantly elevated levels of RANTES but not macrophage inflammatory protein 1 (MIP1) alpha or MIP1 beta in comparison to HIV-seronegative (HIV-) controls. In 32 individuals who became infected with HIV during the course of this study, RANTES levels were significantly higher in plasma samples collected at the time antibodies to HIV were first detected than in pre-seroconversion plasma samples. Both RP and NP showed significant temporal increases in plasma RANTES concentrations. No significant associations were observed, however, between plasma levels of these chemotactic cytokines and progression or known predictors of progression to AIDS including viral burden, levels of beta 2-microglobulin or neopterin, and levels of activated CD8+ lymphocytes. These findings are consistent with the results of a number of recent reports which suggest that these chemokines do not play a major systemic role in the long-term control of viremia or protection against the progression of HIV disease. It remains possible that chemotactic cytokines may contribute locally to control HIV in lymph nodes or other organs but it is also possible that they may be mediators of potentially harmful inflammatory responses.

Slower evolution of human immunodeficiency virus type 1 quasispecies during progression to AIDS.

The evolution of human immunodeficiency virus type 1 (HIV-1) quasispecies at the envelope gene was studied from the time of infection in 11 men who experienced different rates of CD4+ cell count decline and 6 men with unknown dates of infection by using DNA heteroduplex mobility assays. Quasispecies were genetically homogeneous near the time of seroconversion. Subsequently, slower proviral genetic diversification and higher plasma viremia correlated with rapid CD4+ cell count decline. Except for the fastest progressors to AIDS, highly diverse quasispecies developed in all subjects within 3 to 4 years. High quasispecies diversity was then maintained for years until again becoming more homogeneous in a subset of late-stage AIDS patients. Individuals who maintained high CD4+ cell counts showed continuous genetic turnover of their complex proviral quasispecies, while more closely related sets of variants were found in longitudinal samples of severely immunocompromised patients. The limited number of variants that grew out in short-term PBMC cocultures were rare in the uncultured proviral quasispecies of healthy, long-term infected individuals but more common in vivo in patients with low CD4+ cell counts. The slower evolution of HIV-1 observed during rapid progression to AIDS and in advanced patients may reflect ineffective host-mediated selection pressures on replicating quasispecies.

The role of CCR5 and CCR2 polymorphisms in HIV-1 transmission and disease progression.

Entry of human immunodeficiency virus type 1 (HIV-1) into target cells requires both CD4 (ref. 1, 2) and one of a growing number of G-protein-coupled seven-transmembrane receptors. Viruses predominantly use one, or occasionally both, of the major co-receptors CCR5 or CXCR4, although other receptors, including CCR2B and CCR3, function as minor co-receptors. CCR3 appears critical in central nervous system infection. A 32-base pair inactivating deletion in CCR5 (delta 32) common to Northern European populations has been associated with reduced, but not absolute, HIV-1 transmission risk and delayed disease progression. A more commonly distributed transition causing a valine to isoleucine switch in transmembrane domain I of CCR2B (64I) with unknown functional consequences was recently shown to delay disease progression but not reduce infection risk. Although we confirm the lack of association of CCR2B 64I with transmission, we cannot confirm the association with delayed progression. Although subjects with CCR5 delta 32 defects had significantly reduced median viral load at study entry, providing a plausible explanation for the association with delayed progression, this association was not seen with CCR2B 64I. Further studies are needed to define the role of CCR2B64I in HIV pathogenesis.

The role of viral phenotype and CCR-5 gene defects in HIV-1 transmission and disease progression.

Cellular entry of human immunodeficiency virus type 1 (HIV-1) requires binding to both CD4 (ref, 1, 2) and to one of the chemokine receptors recently discovered to act as coreceptors. Viruses that infect T-cell lines to form syncytia (syncytium-inducing, SI) are frequently found in late-stage HIV disease and utilize the chemokine receptor CXCR-4; macrophage-tropic viruses are non-syncytium-inducing (NSI), found throughout disease and utilize CCR-5 (ref. 3-11). We postulated that CCR-5 gene defects might reduce infection risk in seronegative subjects and prolong AIDS-free survival in seropositive subjects with NSI but not SI virus. Homozygous (delta ccr5/delta ccr5) and heterozygous (CCR5/delta ccr5) CCR-5 deletions (delta ccr5) were found in 7 (2.7%) and 51 (19.5%), respectively, of 261 seronegative subjects from the San Francisco Men's Health Study. CCR-5/delta ccr5 genotype was identified in 33 of 172 (19.2%) nonprogressors and 25 of 234 (10.7%) progressors from the seropositive arm of this cohort. The delta ccr5 allele conferred a significant protective effect against HIV-1 infection (P = 0.001) and a survival advantage against disease progression (P = 0.02). Although both progressing and nonprogressing CCR5/delta ccr5 subjects were identified, a distinct survival advantage was shown for those with NSI virus (P < 0.0001). Thus, the protective effect of delta ccr5 against disease progression is lost when the infecting virus uses CXCR-4 as a coreceptor.