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Home医源资料库在线期刊传染病学杂志2003年第187卷第2期

Limited Protective Effect of the CCR532/CCR532 Genotype on Human Immunodeficiency Virus Infection Incidence in a Cohort of Patients with Hemophilia and Select

来源:传染病学杂志
摘要:6%)hadtheCCR532/CCR532genotype(whichoccursin2%oftheScandinavianpopulation)andarapiddiseasecourse。Surprisingly,noprotectiveeffectoftheCCR5/CCR532genotypeondiseaseprogressionorsurvivalwasseenforchildrenbutwasevidentforadults。Agegrouprelatedimmunologicdif......

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1Department of Medical Microbiology and Immunology, Århus University School of Medicine, 2Department of Biostatistics, Århus University, and 3Department of Clinical Immunology, Skejby Hospital, Århus, and Departments of 4Infectious Diseases, 5Pediatric Diseases (Hemophilia Center), and 6Clinical Immunology and 7Blood Bank, Rigshospitalet, 8Department of Virology, Statens Serum Institute, and 9Department of Infectious Diseases, Hvidovre Hospital, Copenhagen, Denmark; Departments of Coagulation Disorders, 10Karolinska Hospital, Stockholm, 11Malmø University Hospital, Malmø, and 12Sahlgrenska Hospital, Göteborg, Sweden

Received 22 July 2002; revised 20 September 2002; electronically published 6 January 2003.

The relationship among CCR5 genotype, cytomegalovirus infection, and disease progression and death was studied among 159 human immunodeficiency virus (HIV)infected patients with hemophilia. One patient (0.6%) had the CCR532/CCR532 genotype (which occurs in 2% of the Scandinavian population) and a rapid disease course. His HIV V3 region contained genotypic features attributable to X4 virus and resembled functionally verified X4 virus and virus from patients treated with a CD4 cellstimulating drug, tucaresol. Age-related differences in disease progression rate and survival time were seen for CCR5/CCR5 patients. Surprisingly, no protective effect of the CCR5/CCR532 genotype on disease progression or survival was seen for children but was evident for adults. Age grouprelated immunologic differences might explain this variation, and transmission route and/or viral phenotype variation within donor virus may be related to the limited protection of the CCR532/CCR532 genotype. Sequence comparisons indicate that X4 virus can be selected in vivo due to either absence of CCR5 receptors or relative increase of CXCR4 receptors.

 


    Informed consent was obtained from all patients, and human experimentation guidelines required by Danish and Swedish boards of medical ethics were followed.
     Financial support: Alfred Benzon Foundation; Novo Nordisk Foundation; AIDS Foundation, Denmark; and Danish Medical Research Council.

      Present affiliation: Weatherall Institute of Molecular Medicine, The John Radcliffe Hospital, Oxford, United Kingdom.
     Reprints or correspondence: Dr. Astrid K. N. Iversen, Weatherall Institute of Molecular Medicine, The John Radcliffe Hospital, Headington, 0X3 9D5 Oxford, United Kingdom (.


     Other than CD4, several receptors among the group of 7-membrane G proteincoupled chemokine receptors have been demonstrated to be essential to human immunodeficiency virus (HIV) type 1 entry into cells [14]. Most viruses isolated during the initial phase of HIV infection exclusively use the CCR5 receptor on macrophages, dendritic cells, and CD4+ T cells (R5 or macrophage-tropic virus). However, during disease progression, highly cytopathic virus variants that use the CXCR4 receptor, in addition to or instead of the CCR5 receptor, evolve in 50% of patients 1 year before diagnosis of AIDS (X4 or T cell linetropic virus) [58]. Moreover, viral variants sometimes evolve to use one of several other coreceptors (e.g., CCR1 to CCR3 and GPR15), in addition to or instead of CCR5 and/or CXCR4 [9, 10].

     The molecular basis for HIV tropism is complicated because many HIV isolates that primarily use 1 coreceptor in vitro can be made to use another if the other coreceptor, or CD4, is overexpressed in a given cell [8, 11]. Thus, the relative affinity for CD4 and coreceptor seems to be a major rate-limiting step for the efficiency of viral replication. Therefore, selection pressures in vivo might select not only for the ability to use other coreceptors but also for HIV sequence variants able to infect target cell subsets that express suboptimal levels of CD4 and/or coreceptor. The viral determinant most important to tropism is the envelope gene, especially the V3 loop, in which the amino acid (aa) charge in a few specific aa positions to a large extent determines in vitro tropism and syncytium-inducing (SI) capacity [1216].

     An allelic variant of the CCR5 gene (CCR532) is found primarily in white populations, and persons homozygous for this allele seem to be highly resistant to HIV-1 infection [1719] although protection is not absolute [2027]. Worldwide, 15,000 HIV-infected persons have been genotyped for CCR5; of these, only 8 were homozygous for the CCR532 allele, 1 of whom was infected through contaminated factor VIII concentrates [21, 22, 27]. The frequency of the CCR532 allele in different white populations varies with a north-to-south gradient across Europe, with Scandinavians having the highest frequency [28]. In Denmark and Sweden, 2% and 21% of the population are homozygous or heterozygous, respectively, for the CCR532 allele [28, 29].

     The frequency of CCR532 heterozygosity is increased in white homosexual men who have been infected with HIV for >10 years and is thus associated with slower HIV disease progression and prolonged survival in this patient group [17, 29, 30]. The effect may be mediated through a general decrease in virus load, compared with that of CCR5 homozygous HIV-infected persons, which, in turn, may be related to decreased cell surface expression of CCR5 and differences in the production of -chemokines and other soluble antiviral factors [31]. Similarly, other, less frequently found mutated coreceptor genes affect disease progression in HIV-infected persons [32, 33].

     Cytomegalovirus (CMV) is a common infection of both HIV-infected and -uninfected persons. The prevalence of CMV infection, as indicated by the presence of serum antibodies, varies in HIV-infected persons by risk group. CMV seroprevalence is 100% in homosexual men but, in HIV-infected patients with hemophilia, is similar to that of the general population (40%85% in Scandinavia, depending on age) [34, 35]. CMV belongs to the herpesvirus family and encodes a chemokine receptor homologue, US28, which have been reported to function as an HIV coreceptor [36, 37]. However, studies of the HIV coreceptor activity of US28 have shown conflicting results [3639]. Furthermore, some epidemiologic studies show that HIV- and CMV-positive patients progress more rapidly to AIDS and have reduced survival times, compared with HIV-positive CMV-negative subjects [4046]. Others studies have found no effect of CMV status on HIV disease [4749].

     Our study was undertaken to examine the influence of CCR5 genotype and CMV infection status on disease progression and mortality in HIV-infected patients with hemophilia who were followed-up for 1318 years. We also analyzed HIV V3 loop sequences from a patient with severe hemophilia A, who was homozygous for the CCR532 allele.

SUBJECTS, MATERIALS, AND METHODS

     Patient population.     All eligible patients of Swedish or Danish origin with hemophilia A, hemophilia B, or von Willebrand disease and HIV infection that was acquired during 19791985 were included in the study. Samples were available from 159 patients (158 men and 1 woman). Eighty-two patients (52%) attended hospital-based hemophilia centers in Sweden, and 77 patients (48%) attended similar centers in Denmark. The genetic background of the patients was quite homogeneous since the populations in Sweden and Denmark are closely related [50]. Because health care systems in Sweden and Denmark are comparable, the patients can be presumed to have received the same level of medical attention and care. Patients were reviewed regularly and were assessed for the presence of signs and symptoms attributable to HIV infection. AIDS refers to symptoms and signs classifiable as CDC group IV disease [51].

     Patient samples.     Most serum samples were derived as part of the routine management of these patients; others originated from cohorts of patients with hemophilia established in 1980. For most patients, HIV seroconversion was determined by testing of several old serum samples. Seroconversion was estimated as the midpoint between the last HIV antibody-negative and the first HIV antibody-positive sample. However, for 44 of the 77 Danish samples, seroconversion date was determined by testing of first available blood sample from 1983, 1984, or 1985. The time to a CD4 cell count 200 cells/L was estimated as the time between infection and the first sample with CD4 cell count 200 cells/L. Information regarding CMV infection was obtained, in part, from patient files and in part from analysis of the earliest available serum sample. For the purpose of CCR5 genotyping, 1 serum sample was obtained from each patient: 54 originated from Rigshospitalet, Copenhagen, Denmark; 25 from Skejby Hospital, Århus, Denmark; 44 from Karolinska Hospital, Stockholm, Sweden; 27 from Malmö University Hospital, Malmö, Sweden; and 9 from Sahlgrenska Hospital, Göteborg, Sweden.

     CCR5 genotyping and CMV analysis.     Analysis of the CCR5 genotype was performed on serum samples by use of the nested polymerase chain reaction (PCR) and were visualized by gel electrophoresis. Of the samples, 38 were analyzed as described elsewhere [29]; the remainders were analyzed as described below, because the amount of material available often was quite limited. Nested PCR was performed over the CCR5 coding sequence by use of the first-round primers CCR5-1 (5-AAATACAATGTGTCAACTCTTGA, aa 531554) and CCR5-3 (5-ATTTCGACACCGAAGCAGAG, aa 928908). The second-round primers were CCR5-2 (5-CTTGACAGGGCTCTATTTTA, aa 550570) and CCR5-4 (5-TTTTTAGGATTCCCGAGTAG, aa 878898).

     First- and second-round PCR were in a 50 L mixture that contained 5 L serum (round 1 [R1]) or 5 L R1 product (round 2 [R2]), 25 (R1) or 50 (R2) pmol of each primer, 40 mM Tricine-KOH (pH 8.7 at 25°C), 15 mM KOAc, 3.5 mM Mg (Oac)2, 3.75 g/mL bovine serum albumin (BSA), 0.005% Tween 20, 0.005% Nonidet-P40, 0.2 mM each of all 4 dNTPs, and Advantage 2 polymerase mix (AdvanTaq DNA polymerase, proofreading polymerase, and TaqStart antibody; Advantage 2 PCR kit; Clontech). Samples were first denatured at 95°C for 1 min, subjected to 35 cycles of PCR amplification (95°C for 30 s and 68°C for 1 min [R1] or 95°C for 20 s, 65°C for 10 s, and 68°C for 45 s [R2]), followed by 2 min at 68°C. All amplifications were done in a Perkin-Elmer Cetus GeneAmp PCR System 9600 (R1) or 2400 (R2).

     Care was taken to avoid contamination of PCR samples. All reactions were done in triplicate at different time points. Blank reactions were interspersed with the samples, and different rooms were used to set up for the first and second PCR reactions and for gel electrophoresis.

     Amplification of the wild-type CCR5 gene resulted in the generation of a 367-base pair (bp) DNA fragment, whereas amplification of the CCR532 allele resulted in the generation of a 335-bp DNA fragment. PCR amplicons were run in 4% agarose gels, 1× TBE (agarose for the separation of GeneAmp PCR products; Perkin-Elmer Cetus). Lanes containing samples homozygous for the wild-type allele contained 1 367-bp band, lanes with heterozygous samples contained 2 bands of 367 and 335 bp, respectively, whereas lanes containing samples homozygous for the CCR532 mutation contained 1 335-bp band. Antibodies to CMV were obtained by use of ELISAs, according to the manufacturer' recommendations (CMV IgG, Axsym; Abbott).

     Sequencing of CCR5 gene from patient with CCR532/CCR532.     Nucleic acids were extracted from serum derived from the CCR532 homozygous patient by use of the QIAamp UltraSens virus kit as recommended by the manufacturer (Qiagen), and PCR reactions were performed over the CCR5 coding sequence, as described above. The PCR amplicons were purified with the QIAquick PCR purification kit (Qiagen) and sequenced on an ABI 377A sequencer with Big Dye terminator technology, according to the manufacturer's recommendations (Big Dye terminator cycle sequencing kit; Applied Biosystems).

     Sequencing of HIV RNA virus from patient with CCR532/CCR532.     Recombinant ribonuclease inhibitor (RNaseOUT; Invitrogen) was added to the extracted nucleic acids from the CCR532 homozygous patient. To remove DNA from part of the sample, an aliquot of the material was subjected to DNase digestion, as recommended by the manufacturer (DNase I, Amp grade; Invitrogen). Reverse-transcriptase (RT) and first-round PCR was performed over the V3 env coding sequence by use of the Qiagen OneStep RT-PCR kit and primers E5 and E6 (E5, 5-ACAGTACAATGTACACATGG-3, positions 69546973 in HIVHXB2; E6, 5-ATGGGAGGGGCATACATTG-3, positions 75227540 in HIVHXB2). The RT step was done at 50°C for 35 min, whereas the subsequent PCR consisted of an initial PCR activation step at 95°C for 15 min, followed by 40 PCR cycles (94°C for 30 s, 61°C for 30 s, and 72°C for 1 min) and a final extension step (72°C for 10 min).

     Second-round PCR was performed with primers E2 and E4 (E2, 5-TTAGGCCAGTAGTATCAACTCA-3, positions 69766997 in HIVHXB2; E4, 5-TTGCTTTTCCTACTTCCTGCC-3, positions 75047524 in HIVHXB2). The PCR was in a 50 L mixture that contained 5 L of R1 product, 50 pmol of each primer, 40 mM Tricine-KOH (pH 8.7 at 25°C), 15 mM KOAc, 3.5 mM Mg (Oac)2, 3.75 g/mL BSA, 0.005% Tween 20, 0.005% Nonidet-P40, 0.2 mM each of all 4 dNTPs, and Advantage 2 polymerase mix (AdvanTaq DNA polymerase, proofreading polymerase, and TaqStart antibody; Advantage 2 PCR kit; Clontech). Samples were first denatured at 95°C for 1 min and then subjected to 35 cycles of PCR amplification (95°C for 20 s and 68°C for 45 s), followed by a final extension step (68°C for 2 min). All amplifications were done in a Perkin-Elmer Cetus GeneAmp PCR System 9600 (R1) or 2400 (R2). The PCR reactions were subsequently analyzed by electrophoresis on a 2% agarose gel (agarose for the separation of GeneAmp PCR products; Perkin-Elmer Cetus). A 548-bp fragment was seen in the reactions in which the DNase-treated nucleic acid had been amplified. Both RNase-treated and -untreated sample aliquots were subjected directly to nested PCR, but no HIV DNA could be amplified.

     The PCR amplicons were ligated to a PCR-TOPO vector and were transformed into TOP10 cells by TOPO TA cloning, as recommended by the manufacturer (Invitrogen). The transformation mixture was plated on kanamycin-containing agar plates and were incubated overnight at 37°C. Colonies were analyzed by use of PCR, as described above (R2 only for 20 cycles). PCRs were analyzed by use of gel electrophoresis. Positive reactions were purified with the QIAquick PCR purification kit (Qiagen) and were sequenced on an ABI 377A sequencer, as described above. Sequences were analyzed with Factura and Sequence Navigator (Applied Biosystems) and were compared to sequences in the Los Alamos HIV database [52]. The sequences have been assigned  accession numbers AY150664AY150672.

     Statistical analysis.     Data were analyzed with respect to 3 end points, 2 of which are only known to lie within certain intervals (the time elapsed between HIV seroconversion and achievement of CD4 cell count 200 cells/L, AIDS, or death). However, since these intervals are relatively small, compared with the elapsed time periods, the end points were treated as single values and thus could be subjected to a simple survival analysis. Moreover, the measured CMV infection status was assumed to be equal to the CMV infection status at the time of HIV infection and to be constant during the observation period.

     Statistics.     Patients were divided into 3 age groups. Medians and upper quartiles of each end point were estimated by the Kaplan-Meier method. The effects of CMV infection and CCR5 genotype were analyzed in a Cox regression model, stratified either by age group or for each age group separately, with the terms CMV status and CCR5 genotype, and the interaction between the 2. If the interaction was insignificant at the 5% level, the interaction term was removed from the model. The relative risk (RR) was, in most cases, estimated as the ratio between hazards. Medians and upper quartiles of each end point were estimated by the Kaplan-Meier method.

     Potential differences among the 3 age groups in time to a CD4 cell count of 200 cells/L, AIDS, or death were examined by use of the log-rank test. A necessary precondition for reporting differences in effects between groups as RRs is that the proportional hazards assumption is fulfilled. Otherwise the RR varies with time. Proportionality of the hazards in the various age groups were examined graphically and/or tested in a Cox regression model by including the logarithmic time in interaction with the explanatory variables. Furthermore, the proportional hazards assumption between age groups was tested to reveal age-dependent time-varying hazards.

RESULTS

     Of the 159 patients, 56 were children or young adults <20 years old (age group 1), 69 were 2040 years old (age group 2), and 34 were >40 years old (age group 3). Most were infected during 1982 (mean infection time point, May 1982; SD, ±1.2 years). Of these patients, 54 were observed until September 1997, and 105 were followed until December 1997. Both CCR5 genotype and CMV infection data were obtained for 157; only CCR5 genotype or CMV infection data were obtainable for 2 patients. Clinical data regarding decrease to CD4 cell count 200 cells/L was obtained from 139 patients, and time of AIDS diagnosis for 141 as some old medical records were incomplete.

     CCR5 genotype distribution.      Of the 157 HIV-positive patients with hemophilia, 39 (25%) were heterozygous for the CCR532 allele, which is nearly equivalent to the percentage found in the general Scandinavian population (21%) [28, 29]; 117 (75%) were homozygous for the CCR5 wild-type gene. One patient (0.6%) was homozygous for the CCR532 allele (which equals one-fourth of the percentage of CCR532 homozygous persons found in healthy Danish and Swedish control subjects) [28, 29]. That patient was excluded from cohort data analysis.

     Clinical course of CCR532 homozygous patient.     This patient was born in 1971, had severe hemophilia A, and had been hospitalized >200 times before age 14 because of hemorrhages. Since his early childhood, he had been treated with locally produced clotting factor preparations generated from only a few donors, but he started to receive imported, highly concentrated factor VIII preparations in 1982. He tested positive for HIV in 1985 but is thought to have been infected in 1982. He was diagnosed with AIDS in 1986 after an episode of Pneumocystis carinii pneumonia. His first CD4 cell count was obtained 2 months after this episode and showed 100 cells/L. His CD4 cell count never increased above this level. Simultaneously, a test of his phytohemagglutinin and pokeweed mitogen responses showed responses equal to 20%30% of healthy control samples, whereas his responses to various antigens (tuberculin, Candida albicans, Staphylococcus aureus, Escherichia coli, and allogenic cells) were very low, indicative of a combined functional T and B cell defect. When the responses were tested again a year later, the only positive response was a weak reaction to allogenic cells.

     From 1988 and until his death in 1990, he had CD4 cell count of 110 cells/L. He was hospitalized >70 times after the AIDS diagnosis, primarily due to high fevers of unknown cause, pneumonia, diarrhea due to various opportunistic infections, and esophagitis due to C. albicans and/or CMV. He developed various allergic reactions to many of the antibiotics used to treat the infections and had several episodes with unexplained rashes. In 1987 and 1988, he was treated for 17 months with azidothymidine, but treatment was terminated due to leukopenia. Treatment was started again several times at various doses but was terminated after short intervals due to adverse effects on his bone marrow.

     Clinical course of HIV infection in relation to CCR5 genotype.     Among CCR532 heterozygous patients, 21 (60%) of 35 had CD4 cell count 200 cells/L, 11 (31%) of 36 were diagnosed with AIDS, and 18 (46%) of 39 died. Among CCR5 homozygous patients, 81 (78%) of 104 reached CD4 cell counts 200 cells/L, 54 (51%) of 105 were diagnosed with AIDS, and 67 (57%) of 117 died.

     Effect of CCR5 genotype varies with age group.     The effect of CCR5 genotype on HIV disease progression and mortality was estimated by using 2 surrogate markers (200 CD4 cells/L and AIDS) and 1 clinical end point (death). Cox regression analysis of all patients revealed a significant protective effect of the CCR532 mutation in heterozygous patients, with respect to disease progression. Adjusted for age groups, the relative risk (RR) of reaching CD4 cell count 200 cells/L for CCR5 homozygous versus heterozygous patients was 1.71 (95% confidence interval [CI] 1.032.83; P = .04), and the RR of reaching AIDS was 2.08 (95% CI, 1.074.02; P = .03). In contrast, no effect on survival time was observed.

     When we analyzed the patients by age group, we found a variation in the effect of the CCR532 mutation. Cox analysis revealed no protective effect of the CCR532 mutation in those 20 years old with respect of time to CD4 cell count 200 cells/L, an AIDS diagnosis, or death (). In patients 2040 years old, no effect of the CCR532 mutation was seen with respect to reaching a CD4 cell count 200 cells/L. Cox regression analysis could not be used to analyze the impact of the CCR532 mutation with respect to AIDS and death, since there were too few events in the heterozygous group. However, by use of the Fisher's exact test, CCR532 heterozygous patients seemed less likely than age-matched CCR5 homozygous patients to develop symptoms of AIDS (RR, 2.85; P = .05) and die (RR, 2.43; P = .03) within the observation time ().

fig.ommitted

Table 1.          Estimates of relative risks (RRs) of an adverse effect of the CCR5/CCR5 genotype, cytomegalovirus (CMV) infection, and an interaction between CCR5 and CMV for reaching CD4 cell count 200 cells/L, diagnosis of AIDS, or death in a cohort of human immunodeficiency virusinfected patients with hemophilia.

     A slower disease progression was seen in CCR532 heterozygous versus CCR5 homogeneous patients 40 years old. The RR of reaching a CD4 cell count 200 cells/L for homozygous versus heterozygous subjects was 2.83 (95% CI, 1.037.8; P = .04), and the RR of reaching AIDS was 2.6 (95% CI, 0.867.86; P = .09). No effect on survival time was found ().

     CMV infection, CCR5 genotype, and age group.     Cox regression analysis stratified by age group showed no significant effect of CMV infection on any of the 3 end points. No effect of CMV serostatus was seen with respect to having a CD4 cell count 200 cells/L (P = .26), AIDS (P = .61), or death (P = .37), and no interaction with CCR5 could be observed (P = .07, .40, and .98 for a CD4 cell count 200 cells/L, AIDS, and death, respectively; ). The patients were analyzed further with respect to CMV infection, CCR5 genotype, and age group. The analysis was performed separately for CCR5 homozygous and CCR532 heterozygous patients to examine potential differences in the effect of CMV infection in the 2 groups (e.g., due to potential threshold effects of number of chemokine and/or US28 receptors on cells), but no significant correlations were observed.

     Age group and genotype-dependent effect on disease progression and survival rate.     Potential differences in disease progression and mortality rates in the 3 age groups were examined for all patients adjusted for genotype and separately for patients carrying the CCR5 homozygous or CCR532 heterozygous genotype. Disease progression was described in terms of time to the end points of CD4 cell count 200 cells/L and AIDS.

     When we examined all patients adjusted for genotype, the age groups varied significantly in time to reaching CD4 cell count 200 cells/L (P = .0017), AIDS (P = .0012), and death (P = .0001). For CCR5 homozygous patients, age had a highly significant effect on disease progression (time to CD4 cell count 200 cells/L, P = .0006; time to AIDS, P = .0006; and death, P = .0001), such that the time to event decreased with increasing age (). In contrast, for CCR532 heterozygous patients, there was no effect of age on time to any of the 3 end points (), but the nonsignificance may at least in part be due to the small number of heterozygous patients.

fig.ommitted

Figure 1.        Kaplan-Meier plot of cumulative time (months) to a CD4 cell count 200 cells/L (A), AIDS (C), or death (D) in CCR5 homozygous patients divided by age group and time to a CD4 cell count 200 cells/L in CCR5/CCR532 patients (B). There were too few events to allow for Cox regression analysis of time to AIDS and death in the CCR532 heterozygous group. Solid lines, subjects <20 years old; dotted lines, subjects 2040 years old; dashed lines, subjects >40 years old.

     The survival curve of age group 1 differed from those of age groups 2 and 3, because it initially was horizontal due to few deaths. The RR increased rapidly after 130 months, which suggests either a smaller effect of earlier protective factors or changes in infection dynamics.

     Sequencing results.     The CCR532/CCR532 genotype was confirmed by sequencing (data not shown). No other changes were found relative to the CCR5 consensus sequence. Nine clones containing HIV RNAderived envelope fragments were sequenced ( patient 1). The V3 loops were quite similar with variation only at aa position 11. A positively charged aa (R) at position 32 and a relatively high charge of the whole V3 loop indicated that these sequences belonged to T cell line/CXCR4 tropic, SI HIV variants [1216]. Sequence changes associated with the ability to grow in T cell lines and to induce syncytia (i.e., positively charged aa at positions 11, 13, 25, and/or 32 have been found in 6 other CCR532/CCR532 HIV-infected patients) [2027]. However, only sequences from 2 of these patients have been published and/or deposited in the HIV database (; patients 2 and 3) [2022].

fig.ommitted

Figure 2.        Sequence comparisons between CCR532/CCR532 patient (patient 1) and human immunodeficiency virus (HIV) subtype B consensus sequence and various V3 loop sequences with a GPGRVY crown sequence. The V3 loop is divided into 3 sections by the central V3 crown sequence (dots). Changes relative to the patient 1 sequence are shown in boldface type; similar amino acids (aa) are shown by dashes. Four aa positions associated with tissue tropism and coreceptor usage (aa 11, 13, 25, and 32) are indicated. A, All available sequences from CCR532/CCR532 patients. Patient 1 (1.11.9), patient 2 (2.12.8; dates of sequences are not available but the sequences are from samples obtained during 19851992) [21, 22], and patient 3 (3.1) [20]. B-CONS, HIV subtype B consensus sequence [52]. B, In vitrogenerated HIV clones c238 and c146 [14, 16]. Both infect HeLa-CD4+ cells and induce syncytium formation in MT2 leukemia cells. Sequences derived from patients treated with an immunomodulating drug, tucaresol, which enhances T helper cell activity and the production of interleukin-2 and interferon- [53]. BK132 is an HIV isolate from Thailand that exclusively uses the CXCR4 receptor [54]. DK is an HIV isolate derived from patient bulk virus grown on U87MG-transformed CD4+ cells transfected with CXCR4. The DK/RM virus dominated the virus population in mixed virus experiments in which CCR5 blocking drugs were used (AOP-RANTES) [55]. C, Patient-derived HIV V3 loop sequences were obtained from HIV database [52]. Most sequences are generated by polymerase chain reaction amplification and none are tested functionally for coreceptor usage or tissue tropism. No information was available on ethnic origin, CCR5 genotype, or disease course of the patients. ARG, Argentina; Biol iso., biological isolate; CH, Switzerland; CZ, Czechoslovakia; NL, The Netherlands; UK, United Kingdom; US, United States.

     Sequence comparisons showed that the V3 sequences from patients 1 and 2 diverged from those of patient 3 and the HIV group B consensus sequence that carries a GPGRAF crown. Both patients 1 and 2 had severe hemophilia A and were infected by contaminated coagulation factor concentrates administered intravenously, whereas patient 3 was infected via homosexual contact. Virus from patients 1 and 2 [21, 22] have different V3 crown sequences (GPGRVY and GPGRVL, respectively) and diverge from the HIV B consensus sequence at 4 and 47 of the remaining 29 aa positions, respectively. The greater viral variation in patient 2 is caused by the sequences being derived from samples spanning 7 years. The sequence changes between patients 1 and 2 might result from structural consequences of the Y/L variation in the crown sequence and from variation in the array of coreceptors, other than the CXCR4 receptor that these viruses can use. Virus from patient 3 retained the HIV Bconsensus crown sequence while displaying variation from the consensus sequence in 11 of the remaining 29 aa positions [20].

     In vitrogenerated HIV clones 146 and 238 [14, 16] diverged from the sequences of patient 1 at 3 and 2 positions, respectively (positions 11, 25, and 32; ). Functional studies showed the viruses to have the same growth patterns, since both infected HeLa-CD4 cells induced syncytia in MT2 cells and were unable to grow in macrophages but had different in vitro growth potentials (clone 146 grew >60 times more efficiently than clone 238). The patient-derived Thai HIV isolate, BK 132, diverged from patient 1 by 3 aa (positions 7, 11, and 25) and used the CXCR4 receptor [54]. We are currently substituting the V3 loop of a well-characterized HIV clone with the patient 1derived V3 loop to functionally test the tissue tropism of the recombinant (A.K.N.I. and B. Chesebro, unpublished data).

     The V3 sequences of patient 1 were identical to those of virus derived from 2 patients treated with the immunomodulating, CD4 T cellstimulating drug, tucaresol, in 3132 of 35 aa positions, including the V3 crown sequence [53, 5658] . Tucaresol is a small Schiff baseforming molecule that can substitute for the physiologic donor of carbonyl groups and provide a costimulatory signal to CD4+ T cells, thus supplementing stimulation provided by receptors on antigen-presenting cells, which results in production of interleukin-2 and interferon-. The tucaresol-selected V3 sequences were strikingly similar to the T cell line tropic, SI HIV clone c146, though only 1 aa potentially differed between the V3 loops of the viruses (X in position 34). No information regarding the nature of aa 34 was available in the HIV database or in the literature [53]. Furthermore, the V3 loop of patient 1 resembled an in vitrogenerated X4 virus isolate (DK) in 28 of 35 aa positions, with a cluster of 6 changes left of the V3 crown and an identical V3 crown sequence [55]. This virus was derived from patient bulk virus grown in U87MG-transformed CD4+ cells transfected with CXCR4 and dominated the virus population in mixed virus experiments in which CCR5-blocking drugs were used (AOP-RANTES).

     When we compared the patient 1 V3 sequence with the entire patient-derived V3 sequences in the HIV database [52], it was evident that the sequence is quite rare . No V3 sequence was completely identical to that of our patient's virus, although sequences of the Czech virus and of one from the United States diverged at only 1 aa (position 2 or 11, respectively). No pattern of HIV transmission was evident, since no transmission mode predominated; the patients had been infected by heterosexual or homosexual contact or through injection drug use. Detailed information regarding ethnic origin, CCR5 genotype, or disease progression rate was not available for any of the patients.

DISCUSSION

     The frequency of the CCR532 homozygous genotype in our study was about one-fourth that of the general population in Denmark and Sweden [28, 29]. Thus, this genotype seems to offer only limited protection against HIV infection transmitted via contaminated coagulation factor concentrates administered intravenously. Delineating the precise extent of protection would require analysis of a larger number of patients. This result contrasts with the observed high resistance of CCR532/CCR532 carriers to HIV infection transmitted though sexual contact [1719] but is in accordance with 8 independent reports of infection of persons carrying this genotype [2027]: 1 person had hemophilia A and was infected through contaminated coagulation factor concentrates similar to our patient, and 1 person and 6 people were infected by heterosexual and homosexual contact, respectively.

     The discrepancy between the protective effect of the CCR532/CCR532 genotype found in cohort studies of HIV-positive homosexuals and our finding may in part be due to early differences in infection dynamics. These differences could result from variation in the size of the initial infective inoculum, in viral phenotypes, and in the primary coreceptor usage by the cells first exposed to the virus. After blood transfusion of contaminated factor concentrates, the initial viral replication is located in lymph nodes carrying both CXCR4 and CCR5 CD4+ T cells, as well as monocyte and/or macrophages expressing high levels of the CCR5 receptor and low levels of the CXCR4 receptor. In contrast, the initial replication after sexual transmission is probably in mucosal macrophages and/or dendritic cells carrying primarily CCR5 receptors.

     Therefore, the effect of mutations inactivating the CCR5 gene might be less in persons infected parenterally than sexually, as indicated by some studies of patients with hemophilia and injection drug users (IDUs) [17, 59] but not by others [60]. It has been demonstrated elsewhere that HIV infection can be sustained by CXCR4 tropic viruses over many years in a CCR532/CCR532 patient [21, 22]. A functional CXCR4 tropic virus has analogously been found in another 32 homozygous HIV-infected patient [27] and genotypic viral changes associated with the ability to grow in T cell lines and to induce syncytia in 4 other HIV-infected persons [20, 23, 24, 26].

     Only 1 HIV-infected patient with hemophilia was previously reported to carry the CCR532/CCR532 genotype [21, 22]. However, although the CCR532/CCR532 genotype is relatively common in Scandinavian populations, it is rare elsewhere in Europe and among US white persons and is almost absent in other ethnic groups [17, 28, 29]. Because most cohorts of infected patients with hemophilia are small and often consist of persons of diverse ethnic backgrounds, absence of the CCR532/CCR532 genotype is not surprising.

     Patients were divided into 3 age groups after graph examination of the data revealed that the proportional hazards assumption was not acceptable with respect to age. This could be caused in part by variation in the general function of the immune system in children, adults, and older adults, as reflected by HIV survival time [61, 62]. Furthermore, with respect to the CMV analysis, dividing patients by age group seemed reasonable as a potential interaction between CMV and HIV seems to differ according to which virus the patient acquires first [48].

     Highly significant differences were seen among the 3 age groups with respect to disease progression and survival time, and the differences were due to a pronounced effect of age in patients homozygous for the CCR5 wild-type allele. We were not able to detect any age group variations in patients heterozygous for the CCR532 mutation, but this result might be due to the small sample size. Alternatively, the protective effect of the CCR532 mutation may lessen the influence of age dependent factors.

     In persons <20 years old, no difference was apparent between CCR532 heterozygous and CCR5 homozygous patients in disease progression rate and survival; however, these parameters differed by genotype in adults. Although no protective effect on time to a CD4 cell count 200 cells/L was seen in CCR532 heterozygous adults (2040 years old), they progressed more slowly to AIDS and survived longer than adults homozygous for the CCR5 wild-type allele. The latter results are in accordance with most previously published studies, which primarily included adult homosexual men and cohorts of patients with hemophilia [19, 29, 30, 60] but are in contrast to others [17, 59]. The lack of effect on time to a CD4 cell count 200 cells/L is analogous to results in a study of IDU [59].

     CCR532 heterozygous adults >40 years old progressed more slowly to CD4 cell count 200 cells/L than age-matched CCR5 homozygous patients, but they showed only a trend toward slower development of AIDS. No effect on survival was discernible in the older age group. The difference in effect on disease progression versus death rate indicates that the mortality rate of the older age group was heavily influenced by other, possibly age-related, factors.

     Why the protective effect of the CCR532 mutation is not apparent in HIV-infected children is puzzling. One explanation could be that the effect of differences in CCR5 genotype is outweighed by age-related factors that may delay disease progression and increase survival. One such factor could be a highly active immune system with high levels of -chemokines, possibly resulting in CCR5 receptor down-regulation and consequently a relative diminished importance of receptor genotype with respect to expression of functional CCR5 receptors [63, 64]. More studies of both healthy and HIV-infected children of different CCR5 genotypes are needed to elucidate these questions.

     We did not observe an effect of CMV infection status in disease progression or survival time in any age or genotype group. However, as patients were designated as CMV positive or negative on the basis of the first available blood sample or information on CMV serostatus from medical files, initially CMV-negative patients may acquire a CMV infection during the observation time. This may lead to an underestimation of the difference in time to events between CMV-negative and -positive patients.

     Our study indicates that the CCR532/CCR532 genotype in patients with hemophilia results in limited protection with regard to acquisition of HIV infection but has no positive effect on disease progression and survival time. In contrast, our data suggest that the genotype may result in selection of quite pathogenic HIV variants, since our CCR532/CCR532 patient carried virus with genotypic features indicative of X4 virus and died within 8 years of HIV infection. He had an unusually long period with extremely low CD4 cell counts and survived 4 years with various AIDS symptoms, which were primarily different opportunistic infections. Of the 8 previously reported cases of HIV infection in CCR532 homozygous patients, most patients had initial rapid reductions in CD4 cell count and, in general, low CD4 cell counts over the years, but otherwise differed in disease progression rate and survival time [27]. One had an average disease course, 2 were infected too recently to evaluate their disease course, and 5 had rapid disease progression. The data suggest that novel antiretroviral strategies targeting the CCR5 receptor [65] should be designed not to completely block the receptor, since this might drive viral selection toward more pathogenic variants, or should be used only in conjunction with other antiretroviral drugs.

     Surprisingly, we found a high degree of similarity between the viral sequences from our CCR532/CCR532 patient, functionally verified X4 viruses, and sequences derived from 2 patients treated with an immunomodulating, CD4 cellstimulating drug, tucaresol. The similarity between the sequences indicate that drugs selectively stimulating and expanding the CD4-positive T cell population, and, therefore, increase the number of available CXCR4 receptors relative to that of CCR5 receptors, might select for X4 virus variants. Such evolved viruses might be predicted to be more fit than the initially infecting virus and hence, potentially more pathogenic. Thus, our observation warns that immunomodulating treatment of HIV-infected patients should be done in combination with highly active antiretroviral drugs, while monitoring the coreceptor usage of patient virus, enabling treatment to be terminated if the patient develops X4 virus. Our study highlights the need to better understand the in vivo selection pressures driving development of various forms of HIV tropism to improve drug treatment strategies.

Acknowledgments

     We thank the patients who donated blood and the cohorts for participation and patience.

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