Silvia Broersen

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

Differential coreceptor expression allows for independent evolution of non–syncytium-inducing and syncytium-inducing HIV-1

We demonstrated previously that CD45RA(+) CD4(+) T cells are infected primarily by syncytium-inducing (SI) HIV-1 variants, whereas CD45RO(+) CD4(+) T cells harbor both non-SI (NSI) and SI HIV-1 variants. Here, we studied evolution of tropism for CD45RA(+) and CD45RO(+) CD4(+) cells, coreceptor usage, and molecular phylogeny of coexisting NSI and SI HIV-1 clones that were isolated from four patients in the period spanning SI conversion. NSI variants were CCR5-restricted and could be isolated throughout infection from CD45RO(+) CD4(+) cells. SI variants seemed to evolve in CD45RO(+) CD4(+) cells, but, in time, SI HIV-1 infection of CD45RA(+) CD4(+) cells equaled infection of CD45RO(+) CD4(+) cells. In parallel with this shift, SI HIV-1 variants first used both coreceptors CCR5 and CXCR4, but eventually lost the ability to use CCR5. Phylogenetically, NSI and SI HIV-1 populations diverged over time. We observed a differential expression of HIV-1 coreceptors within CD45RA(+) and CD45RO(+) cells, which allowed us to isolate virus from purified CCR5(+) CXCR4(-) and CCR5(-) CXCR4(+) CD4(+) cells. The CCR5(+) subset was exclusively infected by CCR5-dependent HIV-1 clones, whereas SI clones were preferentially isolated from the CXCR4(+) subset. The differential expression of HIV-1 coreceptors provides distinct cellular niches for NSI and SI HIV-1, contributing to their coexistence and independent evolutionary pathways.

Adaptation to Promiscuous Usage of Chemokine Receptors Is Not a Prerequisite for Human Immunodeficiency Virus Type 1 Disease Progression

Fifty percent of individuals infected with human immunodeficiency virus type 1 (HIV-1) progress to AIDS in the presence of only non-syncytium-inducing (NSI) variants. These rapidly replicating NSI isolates are associated with a high viral load. The question of whether disease progression in the absence of syncytium-inducing (SI) HIV-1 variants is associated with an expansion of the coreceptor repertoire of NSI HIV-1 variants was studied. Biological HIV-1 clones were isolated both early and late in infection from progressors and long-term survivors with wild-type or mutant CCR5 or CCR2b genotypes and analyzed for their capacity to use CCR1, CCR2b, CCR3, CCR5, and CXCR4 on U87 cells coexpressing CD4. All HIV-1 clones were restricted to the use of CCR5. Absent replication of all HIV-1 clones in peripheral blood mononuclear cells from a CCR5 Delta32 homozygous blood donor confirmed this result. These findings indicate that an expanded coreceptor repertoire of HIV-1 is not a prerequisite for a progressive clinical course of HIV-1 infection.

Cytopathic Effects of Non-Syncytium-Inducing and Syncytium-Inducing Human Immunodeficiency Virus Type 1 Variants on Different CD4+-T-Cell Subsets Are Determined Only by Coreceptor Expression

ABSTRACT In peripheral blood mononuclear cells, syncytium-inducing (SI) human immunodeficiency virus type 1 (HIV-1) infected and depleted all CD4+ T cells, including naive T cells. Non-SI HIV-1 infected and depleted only the CCR5-expressing T-cell subset. This may explain the accelerated CD4 cell loss after SI conversion in vivo.

Increased neutralization sensitivity and reduced replicative capacity of human immunodeficiency virus type 1 after short-term in vivo or in vitro passage through chimpanzees.

ABSTRACT Development of disease is extremely rare in chimpanzees when inoculated with either T-cell-line-adapted neutralization-sensitive or primary human immunodeficiency virus type 1 (HIV-1), at first excluding a role for HIV-1 neutralization sensitivity in the clinical course of infection. Interestingly, we observed that short-term in vivo and in vitro passage of primary HIV-1 isolates through chimpanzee peripheral blood mononuclear cells (PBMC) resulted in a neutralization-sensitive phenotype. Furthermore, an HIV-1 variant reisolated from a chimpanzee 10 years after experimental infection was still sensitive to neutralization by soluble CD4, the CD4 binding site recognizing antibody IgG1b12 and autologous chimpanzee serum samples, but had become relatively resistant to neutralization by polyclonal human sera and neutralizing monoclonal antibodies. The initial adaptation of HIV-1 to replicate in chimpanzee PBMC seemed to coincide with a selection for viruses with low replicative kinetics. Neither coreceptor usage nor the expression level of CD4, CCR5, or CXCR4 on chimpanzee PBMC compared to human cells could explain the phenotypic changes observed in these chimpanzee-passaged viruses. Our data suggest that the increased neutralization sensitivity of HIV-1 after replication in chimpanzee cells may in part contribute to the long-term asymptomatic HIV-1 infection in experimentally infected chimpanzees.

New distal marker closely linked to the fragile X locus

SummaryWe have isolated II-10, a new X-chromosomal probe that identifies a highly informative two-allele TaqI restriction fragment length polymorphism at locus DXS466. Using somatic cell hybrids containing distinct portions of the long arm of the X chromosome, we could localize DXS466 between DXS296 and DXS304, both of which are closely linked distal markers for fragile X. This regional localization was supported by the analysis, in fragile X families, of recombination events between these three loci, the fragile X locus and locus DXS52, the latter being located at a more distal position. DXS466 is closely linked to the fragile X locus with a peak lod score of 7.79 at a recombination fraction of 0.02. Heterozygosity of DXS466 is approximately 50%. Its close proximity and relatively high informativity make DXS466 a valuable new diagnostic DNA marker for fragile X.