Haynes W. Sheppard

Hierarchical Targeting of Subtype C Human Immunodeficiency Virus Type 1 Proteins by CD8+ T Cells: Correlation with Viral Load

ABSTRACT 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.

Safety and Immunogenicity of a Canarypox-Vectored Human Immunodeficiency Virus Type 1 Vaccine with or without gp120: A Phase 2 Study in Higher- and Lower-Risk Volunteers

Live attenuated viral vectors that express human immunodeficiency virus (HIV) antigens are being developed as potential vaccines to prevent HIV infection. The first phase 2 trial with a canarypox vector (vCP205, which expresses gp120, p55, and protease) was conducted in 435 volunteers with and without gp120 boosting, to expand the safety database and to compare the immunogenicity of the vector in volunteers who were at higher risk with that in volunteers at lower risk for HIV infection. Neutralizing antibodies to the MN strain were stimulated in 94% of volunteers given vCP205 plus gp120 and in 56% of volunteers given vCP205 alone. CD8(+) cytotoxic T lymphocyte cells developed at some time point in 33% of volunteers given vCP205, with or without gp120. Phase 3 field trials with these or similar vaccines are needed, to determine whether efficacy in preventing HIV infection or in slowing disease progression among vaccinees who become infected is associated with the level and types of immune responses that were induced by the vaccines in this study.

Human immunodeficiency virus type 1-specific cytotoxic T lymphocyte activity is inversely correlated with HIV type 1 viral load in HIV type 1- infected long-term survivors

HIV-1-specific cytotoxic T cell (CTL) activity has been suggested to correlate with protection from progression to AIDS. We have examined the relationship between HIV-specific CTL activity and maintenance of peripheral blood CD4+ T lymphocyte counts and control of viral load in 17 long-term survivors (LTSs) of HIV-1 infection. Longitudinal analysis indicated that the LTS cohort demonstrated a decreased rate of CD4+ T cell loss (18 cells/mm3/year) compared with typical normal progressors (approximately 60 cells/mm3/year). The majority of the LTSs had detectable, variable, and in some individuals, quite high (>10(4) RNA copies/ml) plasma viral load during the study period. In a cross-sectional analysis, HIV-specific CTL activity to HIV Gag, Pol, and Env proteins was detectable in all 17 LTSs. Simultaneous analysis of HIV-1 Gag-Pol, and Env-specific CTLs and virus load in protease inhibitor-naive individuals showed a significant inverse correlation between Pol-specific CTL activity and plasma HIV-1 RNA levels (p = 0.001). Furthermore, using a mixed linear effects model the combined effects of HIV-1 Pol- and Env-specific CTL activity on the viral load were significantly stronger than the effects of HIV-1 Pol-specific CTL activity alone on predicted virus load. These data suggest that the presence of HIV-1-specific CTL activity in HIV-1-infected long-term survivors is an important component in the effective control of HIV-1 replication.

Homozygous and heterozygous CCR5-Δ32 genotypes are associated with resistance to HIV infection

Objective: To investigate evidence for resistance to HIV‐1 infection associated with the heterozygous genotype CCR5‐+/&Dgr;32 and with the homozygous genotype CCR5‐ &Dgr;32/&Dgr;32, 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‐&Dgr;32/&Dgr;32 and 387 (12.9%) were heterozygous CCR5‐+/&Dgr;32. All but 1 CCR5‐&Dgr;32/&Dgr;32 individuals and 51 CCR5‐ +/&Dgr;32 individuals were Caucasian. Among 1531 Caucasian MSM, CCR5‐+/&Dgr;32 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‐+/&Dgr;32 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‐&Dgr;32/&Dgr;32 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‐+/&Dgr;32 and CCR5‐&Dgr;32/&Dgr;32 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.

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-Δ32 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-Δ32 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-Δ32 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-Δ32 diminished 2 years after AIDS. Protection against AIDS conferred by CCR2-64I was greatest early in the disease course. Compared with individuals without CCR5-Δ32 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-Δ32 is continuous during the course of infection. In contrast, the protection provided by CCR2-64I is greatest early in the course of infection.

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.

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.

Viral, Nutritional, and Bacterial Safety of Flash-Heated and Pretoria-Pasteurized Breast Milk to Prevent Mother-to-Child Transmission of HIV in Resource-Poor Countries A Pilot Study

Background:Heat-treated breast milk of HIV-positive mothers has potential to reduce vertical transmission. This study compared the impact of flash-heating (FH) and Pretoria pasteurization (PP) on HIV, nutrients, and antimicrobial properties in human milk. Methods:Milk samples were spiked with 1 × 108 copies/mL of clade C HIV-1 and treated with FH and PP. We measured HIV reverse transcriptase (RT) activity before and after heating (n = 5). Heat impact on vitamins A, B6, B12, and C; folate, riboflavin, thiamin, and antimicrobial proteins (lactoferrin and lysozyme) was assessed. Storage safety was evaluated by spiking with Escherichia coli or Staphylococcus aureus. Results:Both methods inactivated ≥3 logs of HIV-1. FH resulted in undetectable RT activity. Neither method caused significant decrease in any vitamin, although reductions in vitamins C and E were noted. Heat decreased immunoreactive lactoferrin (P < 0.05) but not the proportions of lactoferrin and lysozyme surviving digestion. FH seems to retain more antibacterial activity. Both treatments eliminated spiked bacteria. Conclusions:FH may be superior to PP in eliminating all viral activity; both methods retained nutrients and destroyed bacterial contamination. Heat-treated breast milk merits further study as a safe and practical infant feeding option for HIV-positive mothers in developing countries.

Immunogenicity of a Recombinant Human Immunodeficiency Virus (HIV)–Canarypox Vaccine in HIV-Seronegative Ugandan Volunteers: Results of the HIV Network for Prevention Trials 007 Vaccine Study

In the first preventative human immunodeficiency virus (HIV) vaccine study to be carried out in Africa, 40 HIV-seronegative Ugandan volunteers were randomly assigned to receive a canarypox vector containing HIV-1 clade B (env and gag-pro) antigens (ALVAC-HIV; n = 20), control vector containing the rabies virus glycoprotein G gene (n = 10), or saline placebo (n = 10). Cytotoxic T lymphocyte activity against target cells expressing clade A, B, and D antigens was assessed using standard chromium-release and confirmatory interferon-gamma enzyme-linked immunospot (ELISPOT) assays. Neutralizing antibody responses to cell line-adapted strains and primary isolates in all 3 clades were also tested. Twenty percent of vaccine recipients generated detectable cytolytic responses to either Gag or Env, and 45% had vaccine-induced HIV-specific CD8(+) T cell responses, as measured by the ELISPOT assay. In contrast, only 5% of the control group had vaccine-specific responses. Neutralizing antibodies against primary and laboratory-adapted HIV-1 clade B strains were seen in 10% and 15% of vaccine recipients, respectively, but responses against clades A and D were not detected. Although the immunogenicity of this clade B-based vaccine was low, ALVAC-HIV elicited CD8(+) T cell responses with detectable cross-activity against clade A and D antigens in a significant proportion of vaccine recipients.

Home collection for frequent HIV testing: Acceptability of oral fluids, dried blood spots and telephone results

ObjectiveTo assess the feasibility and acceptability of bimonthly home oral fluid (OF) and dried blood spot (DBS) collection for HIV testing among high-risk individuals. DesignA total of 241 participants [including men who have sex with men (MSM), injecting drug users (IDU), and women at heterosexual risk] were recruited from a randomly selected subset of study participants enrolled at four sites in the HIV Network for Prevention Trials (HIVNET) cohort, and assigned at random to bimonthly home collection of OF or DBS specimens over a 6 month interval. Participants could select telephone calls or clinic visits to receive HIV test results. MethodsBimonthly specimens were tracked for adherence to the schedule, were evaluated for adequacy for testing, and tested using antibody assays and polymerase chain reaction (PCR) for DBS. The acceptability of bimonthly home OF and DBS collection and telephone counseling was assessed in an end-of-study questionnaire. ResultsThe laboratory received 96 and 90% of expected OF and DBS specimens, respectively; 99% of each specimen type was adequate for testing. Almost all (95%) participants chose results disclosure by telephone. The majority of participants (85%) reported that bimonthly testing did not make them worry more about HIV, and almost all (98%) judged that with bimonthly testing their risk behavior remained the same (77%) or became less risky (21%). ConclusionBimonthly home specimen collection of both OF and DBS with telephone counseling is acceptable and feasible among study participants at high risk. These methods will be useful for the early detection of HIV infection and remote follow-up of research cohort participants in HIV vaccine and prevention trials.