Ireneus P. M. Keet

Association between CCR5 genotype and the clinical course of HIV-1 infection.

Viral, immune, and host genetic factors may influence the clinical course of HIV-1 infection. High viral load [1, 2], presence of syncytium-inducing HIV-1 [3-5], low T-lymphocyte function [6], and certain HLA types [7, 8] have been associated with rapid disease progression [9]. Several coreceptors for HIV-1 have recently been identified. Syncytium-inducing, T-cell line-adapted HIV-1 variants use the C-X-C chemokine receptor 4, macrophagetropic variants use the C-C chemokine receptor 5 (CCR5), and primary syncytium-inducing viruses can use both [10-16]. Persons who have been exposed to HIV-1 on multiple occasions but remain uninfected seem to be homozygous for a 32-nucleotide deletion (delta32) in the CCR5 gene [17, 18]; this concurs with the idea that macrophage-tropic HIV-1 variants establish new infections [19, 20]. In vitro, HIV-1 replication in cells that were heterozygous for CCR5 delta32 was reduced compared with the level of HIV-1 replication in wild-type cells [18]. Several cohort studies [17, 21-24] have shown a substantial correlation between CCR5 delta32 heterozygosity and delayed disease progression. To further substantiate this finding and to examine the biological principle underlying the protection offered by CCR5 delta32 heterozygosity, we analyzed the role of CCR5 genotype alone and in relation to established progression markers in the clinical course of HIV-1 infection in participants from the Amsterdam Cohort Studies. Methods Study Sample Between October 1984 and March 1986, 961 asymptomatic men who were living in the Amsterdam area and who reported having had at least two homosexual contacts in the preceding 6 months were enrolled in a prospective study on the prevalence and incidence of HIV-1 infection and risk factors for AIDS [25]. In the first serum sample taken, 728 men tested negative for HIV-1 antibodies; 131 of these men underwent seroconversion during the study. The remaining 238 men were positive for HIV antibodies; 5 of these men refused to participate further. Enrollment of seropositive persons was stopped after 6 months (in April 1985). Epidemiologic studies on the incidence of HIV-1 infection [26] showed that infection in seroprevalent homosexual men must have occurred an average of 1.5 years before entry into the Amsterdam Cohort Studies. Therefore, the time of seroconversion for seroprevalent men was set at 1.5 years before study entry. No differences in AIDS-free survival were found between persons who underwent seroconversion during the study and seroprevalent persons by using Kaplan-Meier (P > 0.2) and Cox proportional-hazard analyses in which the development of AIDS was the end point criterion (relative hazard, 1.17 for persons who had seroconversion compared with seroprevalent persons [95% CI, 0.84 to 1.63]). This result suggests a good estimation of the seroconversion date in the latter group. When we restricted our analyses to persons who had seroconversion, relative hazards were similar but less precise than estimates for the group as a whole. Therefore, we used 131 persons who had seroconversion and 233 seroprevalent persons as one study sample. Every 3 months, clinical and epidemiologic data were collected and serum and peripheral blood mononuclear cells were cryopreserved. Most seropositive men (n = 242 [66%]) did not receive early treatment. The remaining 122 men (34%) received zidovudine (70 [19%]), didanosine 10 [3%]), or other antiretroviral therapy (42 [12%]) before AIDS was diagnosed. None of the men received a combination of more than two antiretroviral drugs during our study. The mean age of participants at the time of seroconversion was 34.5 years (range, 19.5 to 57.7 years). By 1 January 1996 (the censor date), 189 men had developed AIDS according to the 1987 definition of AIDS [27] (median follow-up, 5.9 years [range, 0.6 to 12.3 years]), 94 men had not developed AIDS (median follow-up, 10.1 years [range, 0.3 to 13.7 years]), and 81 men were lost to follow-up (median follow-up, 2.0 years [range, 0.6 to 12.5 years]). A nested casecontrol study done using the same group of participants from the Amsterdam Cohort Studies was designed to identify factors that may be correlated with long-term survival. Long-term survivors (n = 23) remained free of clinical diseases for at least 9 years, with a mean CD4+ T-lymphocyte count of more than 400 cells/mm3 in the eighth and ninth year of HIV-1-positive follow-up (median follow-up, 10.8 years [range, 9.1 to 11.1 years]; mean CD4+ T-lymphocyte counts in the ninth year of follow-up, 534 cells/mm3 [range, 408 to 953 cells/mm3]). Each long-term survivor was matched with two progressors (men who developed AIDS after 2 to 7 years of HIV-1-positive follow-up). Matching was based on mean CD4+ T-lymphocyte count ( 250 cells/mm3) in year 2 of HIV-positive follow-up, HIV-1 serostatus at entry in the cohort study, and age ( 10 years). Use of Polymerase Chain Reaction for CCR5 Genotyping Samples of DNA were available for CCR5 genotyping for 343 of 364 men (94%). Genomic DNA was isolated from cryopreserved peripheral blood mononuclear cells (Qiagen blood kit, Qiagen, Hilden, Germany) and 100 mg of DNA was analyzed by using polymerase chain reaction (PCR) with primers (sense, position 612 to 635 in CCR5, 5-GATAGGTACCTGGCTGTCGTCCAT-3; antisense, position 829 to 850 in CCR5, 5-AGATAGTCATCTTGGGGCTGGT-3) flanking the described 32-nucleotide deletion in the CCR5 gene [17, 18]. Samples were amplified with 1 unit of Taq polymerase (Promega, Madison, Wisconsin) in the provided buffer with a final MgCl2 concentration of 3 mmol/L. Conditions of PCR comprised 5 minutes of denaturation at 95C; 30 cycles of 1 minute at 95C, 1 minute at 56C, and 2 minutes at 72C; and 5 minutes of elongation at 72C in a Perkin Elmer Cetus DNA thermal cycler 480 (Perkin Elmer, Foster City, California). Products of PCR were analyzed by using 2% agarose gel electrophoresis and ethidium bromide staining. Five randomly chosen samples with a reduced product size revealed the described 32-base pair deletion on automatic DNA sequencing (data not shown) [17, 18]. Virologic Assays Cocultivation of HIV-1-positive peripheral blood mononuclear cells with MT2 cells was performed every 3 months to detect syncytium-inducing HIV-1 variants [28, 29]. Serum viral load was measured by using a quantitative HIV-1 RNA nucleic acid-based sequence amplification (Organon Teknika, Boxtel, the Netherlands) with electrochemiluminescent labeled probes [30]. Serum samples obtained approximately 2 years after seroconversion (1 year after seroconversion; mean time point, 2.3 years [range, 1.5 to 3.0 years]) were available for measurement of HIV-1 RNA viral load for 335 of 364 participants (92%). Serum levels of HIV-1 RNA were analyzed after log10 transformation. Numbers of RNA copies that were below the test threshold of quantification were arbitrarily set at 10 (3).0 copies/mL. Immunologic Assays Antibodies to HIV-1 were detected in serum by using a commercial recombinant HIV-1/-2 enzyme immunoassay (Abbott, Chicago, Illinois) and were confirmed with an HIV-1 Western blot IgG assay (version 1.2, Diagnostic Biotechnology Ltd., Singapore, Thailand). Enumeration of CD4+ and CD8+ T lymphocytes was done by using flow cytofluorometry. For seroprevalent persons for whom we estimated the time of seroconversion to have been 18 months before entry into the cohort study, CD4+ T-lymphocyte count was first measured 18 months after the estimated time of seroconversion. Beginning in January 1988, reactivity of T lymphocytes in response to stimulation with CD3 monoclonal antibodies in vitro was routinely determined in whole-blood cultures [31]. The proliferative response measured after 4 days of culture by incorporation of [3H] thymidine was expressed as a percentage of the median values of the responses measured in two to five healthy controls tested on the same day. Statistical Analysis The Fisher exact test was used to compare HIV-1-seronegative participants with HIV-1-seropositive participants for CCR5 genotype distributions. In the casecontrol study, conditional logistic regression was performed to estimate the chance that a CCR5 delta32 heterozygote would be a long-term survivor. The Mann-Whitney U test was used to compare CCR5 delta32 heterozygotes and CCR5 wild-type homozygotes. For each participant, the slope of the decrease in CD4+ T lymphocytes was determined separately by fitting a simple regression line to his CD4+ T-lymphocyte count. At least three CD4+ T-lymphocyte counts had to be available for analysis; this was the case for 66 (97%) of the 68 CCR5 delta32 heterozygotes and 250 (91%) of the 275 CCR5 wild-type homozygotes. A Kaplan-Meier analysis was used to estimate the cumulative incidence of conversion to syncytium-inducing HIV-1 variants in relation to CCR5 genotype. We also estimated the duration of AIDS-free survival in relation to CCR5 genotype for the period during which only non-syncytium-inducing variants were present (conversion to syncytium-inducing HIV-1 was used as a censor criterion) or for the period after conversion to syncytium-inducing HIV-1 variants. A Kaplan-Meier analysis and a Cox proportional-hazards analysis were used to study the predictive value of CCR5 genotype alone or in combination with serum viral RNA load, CD4+ T-lymphocyte count, T-lymphocyte function, and syncytium-inducing phenotype. We evaluated the predictive value of the markers by fitting separate Cox models at 2, 4, 6, and 8 years after seroconversion. Participants were at risk from each specific time point; this method excluded participants who had previously developed AIDS. Because data on HIV-1 RNA load were available approximately 2 years after seroconversion only, data on viral load were not included in the models at 4, 6, and 8 years after seroconversion. All markers were also analyzed as time-dependent covariates. Participants who did not have AIDS were censored at 1 January 1996. Significance in

HLA class I homozygosity accelerates disease progression in human immunodeficiency virus type 1 infection

Polymorphic products of HLA class I genes restrict cytotoxic T lymphocyte responses to the constantly evolving spectrum of HIV-1 antigens. Accordingly, homozygosity at class I loci can reduce the repertoire for such HLA-dependent interactions, leading to accelerated disease progression. To test this hypothesis we studied subjects from two distinct HIV/AIDS cohorts: 140 Dutch homosexual men and 202 Rwandan heterosexual women followed up to 13 years from HIV-1 seroconversion. We performed intermediate- and selective high-resolution molecular typing at HLA class I (A, B, and C) and high-resolution typing at HLA class II DRB1 and DQB1. Homozygosity at the HLA-A or -B locus or both was found at increasingly high frequency among individuals with successively more rapid progression to late-stage HIV-1-related conditions. In the combined cohorts (n = 342) the odds ratio (OR) due to HLA-A or -B antigen homozygosity in rapid versus slow progressors was 3.8 (p = 0.003); for Dutch men alone the OR was 3.5 (p = 0.102), and for Rwandan women the OR was 4.1 (p = 0.009). In contrast, homozygous genotypes at either HLA-C, DRB1, or DQB1 alone, or DRB1-DQB1 haplotypes, did not exert any deleterious effect on HIV-1 disease progression. These findings suggest strongly that diversity in addition to sequence specificity at HLA-A and -B loci can influence the rate of disease progression following HIV-1 infection.

Cerebrospinal-fluid HIV-1 RNA and drug concentrations after treatment with lamivudine plus zidovudine or stavudine

BACKGROUND Treatment and prevention of HIV-1-related central-nervous-system disease may be dependent on penetration of antiretroviral drugs into the central nervous system. Few data are available about cerebrospinal-fluid penetration and concomitant changes of HIV-1-RNA concentrations during treatment with antiretroviral agents. We investigated these effects in HIV-1-infected people. METHODS 28 antiretroviral-naive individuals with CD4 cell counts of 200/microL or more and plasma HIV-1-RNA concentrations of 10,000 or more copies/mL who were free of neurological symptoms were randomly assigned lamivudine plus either stavudine (n = 17) or zidovudine (n = 11). We did lumbar punctures on 28 individuals before and 22 individuals after 12 weeks of treatment to assess HIV-1-RNA and drug concentrations in cerebrospinal fluid. FINDINGS All 28 individuals had detectable HIV-1-RNA concentrations in the cerebrospinal fluid (median 4.64 log10 copies/mL and 4.20 log10 copies/mL in the lamivudine plus zidovudine and lamivudine plus stavudine groups, respectively). There was no correlation between plasma and cerebrospinal-fluid HIV-1-RNA concentrations (r = 0.18, p = 0.35). After 12 weeks of treatment none of the individuals had detectable HIV-1-RNA concentrations in the cerebrospinal fluid. The highest drug concentration in the cerebrospinal fluid was for lamivudine followed by stavudine and zidovudine. Concentrations were consistent over time, unlike plasma concentrations. Therefore, we found time-dependent cerebrospinal-fluid to plasma drug-penetration ratios, which were highest for zidovudine followed by stavudine and lamivudine. INTERPRETATION The two drug combinations were equally effective in the decrease of cerebrospinal fluid HIV-1-RNA concentrations. All drugs penetrated the cerebrospinal fluid. Antiretroviral drugs other than zidovudine might be useful in the prevention of AIDS dementia complex.

Predictors of rapid progression to AIDS in HIV-1 seroconverters.

OBJECTIVE To determine whether at the time of HIV-1 seroconversion rapid progressors to AIDS and a low CD4+ count can be distinguished by the clinical presentation of primary HIV-1 infection and serological and immunological characteristics. DESIGN Prospective cohort study on HIV-1 infection in homosexual men. SETTING The Municipal Health Service, Amsterdam, The Netherlands. SUBJECTS One hundred and eight men who seroconverted for HIV-1 during follow-up. MAIN OUTCOME MEASURES Progression to AIDS and progression to a CD4+ lymphocyte count < 200 x 10(6)/l. RESULTS Symptomatic primary HIV infection with fever and skin rash, absence of anti-HIV core and transient HIV p24 antigenemia were independent predictors of progression to AIDS at the time of HIV-1 seroconversion. A low CD4+ count immediately after seroconversion and the calendar year were independent predictors of progression to a low CD4+ count at the time of HIV-1 seroconversion. CONCLUSIONS Even in the earliest stage of HIV-1 infection a small group of individuals at high risk for rapid progression to AIDS can be recognized by the clinical presentation of primary HIV infection, the presence of HIV p24 antigenaemia and the absence of a serological response to HIV core protein.

Conversion Rate towards a Syncytium-Inducing (SI) Phenotype during Different Stages of Human Immunodeficiency Virus Type 1 Infection and Prognostic Value of SI Phenotype for Survival after AIDS Diagnosis

The presence of syncytium-inducing (SI) human immunodeficiency virus type 1 (HIV-1) variants is predictive for accelerated progression to AIDS. This study showed that a 4-year survival with AIDS also occurred significantly more often for patients who lacked SI variants. However, multivariate Cox analysis excluded the predictive value of SI viruses for rapid death as being independent from low CD4+ T cell counts. Incidence of appearance of SI variants was increased in persons with CD4+ T cell counts <500/microliter but remained constant in the strata of CD4+ T cell counts <500/microliter, excluding the possibility that loss of immune control is the only prerequisite for the development of SI HIV-1 variants.

Consistent associations of HLA class I and II and transporter gene products with progression of human immunodeficiency virus type 1 infection in homosexual men

Polymorphic products of genes in the HLA region contributing to variability in the course of human immunodeficiency virus type 1 (HIV-1) infection were identified by screening 375 Caucasian seroconverters who were aggregated from 3 cohorts. AIDS-free time was related to numerous (15) class I alleles, alone or in conjunction with transporter protein variants, to homozygosity at the A or B locus, and to alleles of two class II haplotypes. A prognostic scoring algorithm derived from the 3 cohorts captured multiple HLA contributions to protection or to risk (relative hazard=0.57-60 per unit increase in score, all P<<.001). The impact of HLA was strong and appeared independent of the effects of chemokine receptor/ligand polymorphisms and antiretroviral treatment. The algorithm also predicted divergent rates of CD4+ cell decline in 2 other groups, totaling 227 seropositive persons (P=.06 - <.001). Confirmation of these relationships should encourage investigation of HIV-1 antigen processing and presentation mediated by polymorphisms in the HLA region.

Presence of the variant mannose-binding lectin alleles associated with slower progression to Aids

Objective:To examine the association between mannose-binding lectin (MBL) polymorphism and progression to AIDS and death in HIV-1 infection. Design and methods:In 131 HIV-1-infected homosexual seroconverters, survival analyses were performed to determine both the association between MBL genotype and time from HIV-1 seroconversion to AIDS and death, and time from AIDS to death. Results:Of the 131 seroconverters, of whom 61 developed AIDS, 76 were typed as homozygous wild-type and 55 as carriers of variant alleles (52 heterozygous and three homozygous variant alleles). A Survival analyses suggested that HIV-1-infected men with the variant alleles progressed somewhat slower to AIDS [relative hazard (RH), 0.62; 95% confidence interval (CI), 0.36–1.10] and death (RH, 0.73; 95% CI, 0.42–1.25). Interestingly, CD4+ T-cell count determined at the moment of AIDS was found to be significantly lower among persons with the mutation (97 × 106/l versus 204 × 106/l; P = 0.03). Furthermore, when AIDS-free times before the diagnosis of an opportunistic infection were compared with those preceding a diagnosis of Kaposis sarcoma, Kaposis sarcoma diagnosis was more postponed than that of an opportunistic infection (RH, 0.21; 95% CI, 0.05–0.95; versus RH, 0.67; 95% CI, 0.35–1.27). Conclusion:Indications for a weak pre-AIDS protective effect of variant MBL alleles were demonstrated.

Herpes simplex virus type 2 and other genital ulcerative infections as a risk factor for HIV-1 acquisition.

We studied the role of genital ulcerative infections for acquisition of human immunodeficiency virus type 1 (HIV-1) infection in a cohort of 989 homosexual men in Amsterdam between October 1984 and December 1988. Among 53 HIV-1 seroconverters serological and anamnestic data were gathered regarding herpes simplex virus type 2 (HSV-2) and syphilis in the 6 months before seroconversion. For statistical analysis a control who remained seronegative during the same interval was selected at random for each HIV-1 seroconverter. A significant difference between the prevalence of HSV-2 antibodies among HIV-1 seroconverters and controls was found (72% vs 38%). HSV-2 seroconversions among men initially seronegative for HSV-2 were found among three of 18 HIV-1 seroconverters and among three of 36 controls. (O.R. = 2.2, 95% C.I. 0.4-12.1). Self-reported cases of anogenital herpes were found more frequently among HIV-1 seroconverters (8) than among controls (4). One case of syphilis was diagnosed among HIV-1 seroconverters, and one among controls. Summing up these cases we assessed the total number of genital ulcerative infections: 12 among HIV-1 seroconverters and eight among controls (23 vs 15%, O.R. 1.7, C.I. 0.6-4.62). These data suggest little evidence for genital ulcerative infections being an important independent risk factor for HIV-1 acquisition among homosexual men in Amsterdam during the time period studied.

Lymphoproliferative Response to HIV Type 1 p24 in Long-Term Survivors of HIV Type 1 Infection Is Predictive of Persistent AIDS-Free Infection

To establish immunologic correlates of progression to AIDS in long-term survivors of HIV-1 infection, HIV-1-specific T cell-mediated responses, together with T cell reactivity to recall antigens, were studied in frozen samples collected after 5 and 8 years of documented HIV-1 infection. Eight of 21 homosexual men, who remained asymptomatic and maintained CD4+ T cell numbers >400 cells/microl for 9 years of HIV-1 infection, progressed to AIDS (CDC 1993 definition) within 12.5 years of infection (late progressors, LPs). The remainders showed minimal deterioration of immune parameters (long-term nonprogressors, LTNPs). CD4+ T cell numbers and T cell function measured at years 5 and 8 of follow-up were comparable in the two groups. At both time points responses to recall antigens did not significantly differ between the two groups, although a significant decline of lymphoproliferative responses to Candida and tetanus toxoid was observed in LPs. Circulating HIV-1-specific cytotoxic T lymphocyte precursors were found in broad frequency ranges in both LPs and LTNPs and, similarly, no significant differences were found in comparing the breadth of serum neutralizing activity against heterologous HIV-1 primary isolates. In contrast, lymphoproliferative responses to p24gag, but not p17gag or gp160env, were detected only in LTNPs and were totally absent in LPs at both time points (p < 0.01). Our data suggest that the presence of circulating p24-specific CD4+ T cells may reflect effective viral control and be predictive of subsequent favorable clinical course in long-term asymptomatic individuals.

Differences in progression to AIDS between injection drug users and homosexual men with documented dates of seroconversion

&NA; We compared rates of progression to AIDS for 99 injection drug users and 120 homosexual men with documented dates of HIV‐1 seroconversion. The crude risk of developing AIDS was higher among homosexual men than injection drug users [relative hazard (RH) = 2.4; 95% confidence interval (CI) = 1.3‐4‐4]. The relative hazard was slightly smaller among participants with a seroconversion interval of ≤1 year (RH = 2.2; 95% CI = 1.0‐5.2). The effect was partially explained by the inclusion of Kaposis sarcoma in the AIDS case definition. Excluding those with Kaposis sarcoma, the relative hazard was 2.0 (95% CI = 1.1‐3.8). Using the 1993 AIDS case definition decreased the effect (RH = 1.9; 95% CI = 1.1‐3.4). Finally, the high pre‐AIDS mortality among injection drug users could partially explain the difference in progression rate between injection drug users and homosexual men. Combining the effect of the above‐mentioned factors resulted in a relative hazard of 1.3 (95% CI = 0.7‐2.6). Thus, the slower progression to AIDS among injection drug users compared with homosexual men was largely explained by differences in the spectrum of AIDS‐defining illnesses, pre‐AIDS mortality, and length of seroconversion interval. (Epidemiology 1996;7:571‐577)