The chemokine receptor CKR-5 has recently been shown to be a major coreceptor for entry of HIV-1 [1,2]. Certain individuals have a deletion in the gene encoding this receptor , which appears to be inherited in a Mendelian fashion . Individuals who are not homo- or heterozygous for the deleted gene are prone to disease . In contrast, individuals who are homozygous for this defect appear to be resistant to HIV-1 infection [3,4], and it has therefore been assumed that individuals who are heterozygous for this deletion are partially resistant .
In 1984, Dagleish et al.  showed that the CD4 T-cell receptor was essential for entry of HIV-1 into human T cells and macrophages. However, transfection studies with CD4 showed that expression of the CD4 molecule alone was not sufficient to render non-permissive cells infectable  and the existence of a coreceptor was postulated. Recently, two such coreceptors have been identified [1,2,8]. One of these was the fusin receptor found on T cells. This receptor allows T-cell-tropic HIV-1 strains entry into otherwise non-permissive cells transfected with both a CD4 and a fusin-vaccinia construct. However, the receptor has no effect on permissiveness of macrophage-tropic HIV-1 strains, which are the strains usually transferred by sexual intercourse .
A second major coreceptor, which belongs to the chemokine receptor family and allows entrance of macrophage-tropic HIV-1 strains, has been identified . This CKR-5 receptor (also known as CCR5 and CC-CKR5) is from the same seven transmembrane G-protein-coupled receptor family as the fusin receptor. The CKR-5 molecule is a receptor for the chemotactic CC-chemokines, RANTES, macrophage inflammatory protein (MIP)-1α and MIP-1β. In vitro experiments have shown that addition of these chemokines prevents infection of primary T cells and some cell lines with macrophage-tropic HIV-1 isolates , possibly by blocking or downregulating the chemokine receptors [2,11,12].
The importance of a functional CKR-5 receptor for the entry of macrophage-tropic virus was shown by Liu et al. . They found that two individuals with T cells resistant to the entry of macrophage-tropic viruses in vitro were both homozygous for a 32 base-pair deletion in CKR-5. This deletion results in the production of a truncated protein that fails to reach the cell surface. In this study we show that the heterozygous genotype does not prevent infection but delays the progression of HIV-1-induced immune deficiency.
Subjects and methods
Two cohorts of Danish Caucasian homosexual men were established between 1984 and 1986 with the majority of study participants enrolled in 1985. Both cohorts were approved by the Medical Ethics Committee of Copenhagen County. The first cohort comprised HIV-1-seropositive homosexual men without AIDS attending clinics for HIV-1 screening in Copenhagen. The majority of these individuals were probably infected in 1983–1985, because the incidence of HIV-1 infection in Denmark was very low before 1983 . At enrolment, all study participants had a physical examination and had blood drawn for lymphocyte storage and determination of lymphocyte subsets. Ninety-nine cohort participants from whom frozen lymphocytes and serum were available were included in the study. Follow-up was performed in the spring of 1996 by checking the National Death Register, the Central AIDS Register and records in the two principal AIDS clinics in Copenhagen. Eighty-one individuals were followed with regular intervals and yearly CD4 counts until development of AIDS. The distribution of CKR-5 heterozygotes in these 81 (20%) individuals was similar to the distribution in the whole group. During the 11 years of follow-up, 652 CD4 counts were made with mean ± SEM number of samples of 54 ± 8 per year. The mean initial CD4 T-cell count was 594 ± 31×106/l and the final count was 145 ±19×106/l. The group that developed AIDS had similar initial CD4 T-cell counts (572 ± 30×106/l) to the rest of the group. The other cohort consisted of 35 HIV-seronegative homosexual men defined as having previous high-risk behaviour either by having unprotected sexual intercourse with more than 10 partners per year in the period 1983–1985 and having a history of at least one episode of a sexual transmitted disease (n = 31) or by having a history with regular unprotected sexual intercourse with a known HIV-seropositive partner over a period of 1 year (n = 4). Long-term non-progressors are defined as asymptomatic patients with a CD4 T-cell count above 500×106/l after 10 years of known infection. Fast progressors are defined by development of AIDS within 3 years from the first visit to the clinic. Controls were HIV-seronegative healthy blood donors (n = 37). All individuals were unrelated.
Lymphocytes were separated by Histopaque gradient centrifugation (Sigma, St Louis, Missouri, USA) and frozen in RPMI medium supplemented 10% fetal calf serum and dimethyl sulphoxide. Cells were kept in liquid nitrogen until thawing. DNA was extracted using guanidinum-phenol-chloroform extraction (pH 8) and alcohol precipitation.
Polymerase chain reaction
Genomic DNA from 100 000 cells were used as template for the polymerase chain reaction (PCR). PCR was performed in volumes of 50 μl in a thermal cycler. Primers for PCR were based on the published CKR-5 sequence (Gen Bank accession number U57840); the sequences are as follows: CKR5 sense primer, 5′-CAA TGT GTC AAC TCT TGA CAG G-3′; CKR5 antisense primer, 5′-ACC TGC ATA GCT TGG TCC AAC C-3′. These primers amplify a 547 base-pair fragment on homozygous wild-type DNA, two fragments of 547 and 515 base pairs on heterozygous CKR-5 DNA and one fragment of 515 base pairs on homozygous deletion CKR-5 DNA. Genomic DNA was amplified by 35 cycles at 94°C for 30 sec, 55°C for 30 sec and 72°C for 30 sec according to the protocol of Perkin Elmer (Norwark, Connecticut, USA). Controls for DNA contamination were performed. The specificity of the reaction products was analysed by restriction mapping (data not shown). Amplified products were separated on 2% agarose gels.
The hypothesis is that HIV-seropositive subjects that are heterozygous for the CKR-5 deletion (ΔCKR-5) have a better prognosis than those who do not possess the deletion. Comparisons between groups were performed using the Fisher's exact probability test (one-tailed), and the χ2 test. Comparisons between groups were performed using the Mann-Whitney U test. Survival until AIDS was compared using Kaplan-Meier estimates and the log-rank test.
Frequency of ΔCKR-5 in normal donors, HIV-seronegative and HIV-seropositive individuals
A total of 171 samples were examined for homo- and heterozygosity for the 32 base-pair deletion in CKR-5 by PCR. PCR amplification products of wild-type, hetero- and homozygous DNA are shown in Fig. 1.
Nine (24%) out of 37 randomly selected blood donors were heterozygous and one (3%) was homozygous for ΔCKR-5. Two (6%) out of 35 HIV-seronegative individuals at high risk of HIV-1 infection were homozygous and seven (20%) were heterozygous for ΔCKR-5 (Table 1). None of the 99 HIV-seropositive subjects were homozygous and 22 (22%) were heterozygous (Table 1). There was no significant differences between the genotype distribution in these groups.
Comparison of the frequency of ΔCKR-5 in HIV-seropositive long-term non-progressors versus fast progressors
Of the nine individuals who were AIDS-free after 11 years of known HIV-1 infection and had mean CD4 T-cell counts above 500×106/l, six were heterozygous for the deletion and three had the wild-type gene (Table 1). This is significantly different from the normal had a decrease in CD4 T-cell counts of more than 100×106/l per year. This fall is significantly higher than in the group of 16 HIV-seropositive subjects with the CKR-5 deletion. In this group, only one had a decrease of more than 100×106/l in CD4 T-cell counts (P < 0.05; Table 2). The mean decrease in CD4 T-cell counts per year also differed between the two groups, although this difference did not reach significance (Table 2).
Homozygosity for a 32 base-pair deletion in the CKR-5 gene seems to confer resistance to infection with HIV-1 [3,5]. In this study we have suggested that heterozygosity for this deletion does not confer resistance to infection but leads to slower CD4 T-cell decline and longer AIDS-free survival in HIV-seropositive subjects.
It is well known that changes in membrane receptors prevent infection in certain diseases. For example, individuals who are negative for the Duffy glycoprotein (the erythrocyte chemokine receptor ) on erythrocytes are resistant to infection with Plasmodium vivax strains of malaria. CKR-5 seems to play a similar role in HIV-1 infection. If heterozygosity for the CKR-5 deletion confers partial resistance to infection with HIV, we would expect a lower frequency of heterozygotes in the group of HIV-infected individuals than in controls. In our study, we find a similar frequency of heterozygotes in these two groups. This is supported by data from Dean et al. , who in three different cohorts found similar frequencies of heterozygotes in HIV-infected homosexual individuals and controls. Also, in a recent study, Huang et al.  found no evidence for a protective role of heterozygosity. In contrast, some groups [3–5] have found a lower frequency of heterozygotes in HIV-infected individuals than in controls. The suggestion by these groups that heterozygosity for the CKR-5 deletion leads to lower rates of infection is, therefore, not supported by our study or the studies by Dean et al.  or Huang et al. .
CKR-5 heterozygosity may lead to a reduced number of CKR-5 receptors and thereby decrease the ability of donors. None of the nine individuals who progressed to AIDS within 3 years of follow-up were heterozygous for the deletion (Table 1). The genotype distribution differs significantly between the two groups who had the extreme course of the disease (Table 1).
Development of AIDS is slower in HIV-seropositive subjects who are heterozygous for the gene encoding CKR-5
Fifty-six (57%) of the 99 HIV-seropositive individuals without AIDS at the first visit and followed in the AIDS clinics developed AIDS within the follow-up period of a mean ± SEM of 10.8 ± 0.4 years. It appears from the Kaplan-Meier plot in Fig. 2 that there is a significant difference in survival during the first 7 years of infection (log-rank, P = 0.03). After 7 years from the first visit, the two curves approach each other, and after 11 years there is no longer a significant difference between the groups (log-rank for the whole observation period, P = 0.16). Fifty of the 77 HIV-seropositive subjects with wild-type CKR-5 developed AIDS within an average of 5.5 years. Twelve of the 22 HIV-seropositive subjects with the heterozygous CKR-5 genotype developed AIDS in an average of 8.4 years (P < 0.01).
Decrease in CD4 T-cell counts is slower in HIV-seropositive subjects who are heterozygous for the gene encoding CKR-5
Eighty-one HIV-seropositive subjects without AIDS at the first visit were followed for a mean ± SEM of 8 ± 0.2 years (Table 2). Eighteen of the 65 HIV-seropositive subjects with the wild-type CKR-5 gene HIV-1 to infect new cells. This could result in a slower decrease in CD4 T-cell counts. We found a significantly slower rate of decrease in CD4 T cells in those HIV-seropositive subjects who were heterozygous for the deletion compared with individuals with the wild-type gene. In addition, almost all individuals with a fast decrease in CD4 T-cell counts of more than 100×106/l per year had two normal alleles. Thus, heterozygosity for the CKR-5 also leads to a slower progress in the development of the immune deficiency. Accordingly, the CKR-5 deletion must also be expected by have an influence on disease progression. In HIV-seropositive long-term survivors who had normal CD4 T-cell counts for up to 11 years of infection, we found a significantly higher frequency of the heterozygous deletion of 66% compared with 0% in fast progressors who developed AIDS within 3 years from the first visit. Although the first visit was not at the time of serocon-version, we know that the HIV-1 infection was first introduced in Denmark a few years before this time-point .
However, heterozygosity for the deletion in CKR-5 does not prevent progression to AIDS. The study shows that the mean time to AIDS in those with the CKR-5 defective allele was longer than in those with wild-type alleles and indicates that the deletion leads to prolonged survival. Both Dean et al.  and Huang et al.  have found a similar protective effect of heterozygosity on progression to AIDS. However, the effect of the deletion on survival is only significant during the first 7 years of follow-up after which the Kaplan-Meier plot of those patients with and without the defective allele starts to converge. This indicates that the deletion mostly has an effect in the initial years of infection. The reason for the change after 7 years may be a change in the virus envelope allowing the virus to utilize receptors other than CKR-5, as has been suggested by Doranz et al. . HIV-1 isolates can be divided into two groups based on cellular tropism. Macrophage-tropic, non-syncytium-inducing viruses dominate early in infection [9,18,19]. As patients progress to AIDS, most viruses will change to the T-cell-tropic rapidly replicating type [18,20]. Doranz et al.  has suggested that the fusin receptor becomes important at this timepoint. This is supported by in vitro experiments showing that blocking the fusin receptor only affects T-cell-tropic laboratory isolates and not macrophage-tropic isolates.
Our cohort group consists of a local homogeneous group of 99 Danish Caucasian homosexual men living in Copenhagen. There is practically no migration or loss of follow-up in this group, which has been followed for up to 11 years. If the genetic defect has clinical importance for the affected individuals, it should be evident from a cohort of this size. Other surrogate markers such as CD4, poke-weed mitogen and
β2-microglobulin have been shown to have prognostic values in cohorts of similar size. In addition, it is of great importance that a European cohort shows a similar AIDS-free survival to the much more genetically diverse American cohorts described by Dean et al. .
Factors other than the 32 base-pair deletion in the CKR-5 receptor may influence disease progression. Three long-time non-progressors in our study do not have the 32 base-pair deletion in CKR-5. These individuals may have insertions, mutations or small deletions in CKR-5 gene that does not appear in our analysis. Mutations in fusin or other coreceptor genes could also contribute to prolonged survival. For example, a mutation in the fusin gene could make it impossible for the lymphocytotropic virus, which emerges late in infection, to disseminate. Factors other than defects in the receptors may play a role for the progression of the infection. Increased levels of CC-chemokines that normally bind to the receptor may prevent entrance of the virus.
Our results show the importance of CKR-5 in the first years of HIV-1 infection and urge the need for pharmacological agents that block the CKR-5 receptor. Such agents may prevent HIV-1 infection or clear infection in recently infected individuals. Studies aiming at further characterization of the HIV coreceptors may help to identify such reagents.
We thank T. Axen and S. Hjelmblink for excellent technical assistance. P. Platz and J. Gaub have contributed to this work through the Copenhagen HIV Cohort Group.
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