1st 6 pages 3/4/97

ROLE OF MUTANT

CKR-5 ALLELE IN CONFERRING GENETIC

RESISTANCE TO HIV

by Catherine Liu

The Vaccine Revolution

Dr. Robert Siegel

March 19, 1997

ABSTRACT

Chemokine receptor 5 (CKR-5) was recently characterized as a major co-receptor for entry of macrophage (M)-tropic HIV-1 strains. A 32-base pair deletion allele (CKR-5∆32) was also identified and found to encode a non-functional receptor that does not support membrane fusion or infection by M-tropic HIV-1 strains. Combined results from 3 independent studies provide strong evidence to suggest that individuals homozygous for CKR-5∆32 are protected from HIV infection. Out of a total of 1058 high risk for HIV exposure, seronegative individuals, there were 33 (3.1%) deletion homozygotes. In contrast, there was not a single homozygote among 2741 seropositive subjects. There is somewhat less of a consensus about the effect of heterozygosity on HIV infection and disease progression, but it appears that heterozygotes are not resistant to infection; however, they may have some limited protection against disease progression. Although other genetic and environmental cofactors may play a role in HIV infection, these findings have significant implications for the development of new HIV therapies, as drugs can be designed to target the HIV-CKR5 interaction and block viral entry.

Key Words: chemokine receptor 5 (CKR-5), CKR-5∆32, macrophage (M)-tropic, T cell (T)-tropic, HIV resistance

INTRODUCTION

The existence of HIV exposed, uninfected individuals and long-term survivors of the disease is an intriguing subject for both researchers and the general public. An understanding of the biological basis for this apparent "resistance" to HIV may provide significant insight for the development of new HIV drugs and therapies. Possible explanations for HIV disease resistance suggest that long-term survivors and high-risk uninfected individuals may have a stronger immune response to the virus, be exposed to a less pathogenic strain of the virus, or have some form of genetic predisposition to be protected against HIV (17). The issue of genetic resistance to HIV has received much scientific press over the last year with the discovery of a mutant allele of the recently identified chemokine receptor 5 (CKR-5).

It has been known for several years now that HIV cannot infect a cell with CD4 alone and that the virus requires a second receptor for successful fusion and entry into its target cell. Over this past year, two co-receptors were discovered and recognized as members of the seven-transmembrane domain, G-protein-coupled chemokine receptor family. The first one identified was named CXCR4 (also called fusin, LESTR, or HUMSTR) and was shown to act as a co-receptor for syncytium-inducing (SI), T-cell tropic (T-tropic) HIV-1 strains. The second one identified was named CKR-5 (also called CC CKR-5 or CCR-5) and was found to be the co-receptor for non-syncytium inducing (NSI), macrophage-tropic (M-tropic) HIV-1 strains. M-tropic strains appear to be responsible for HIV-1 transmission by sexual contact and the transfer of infected blood, and predominate in newly infected individuals and throughout the asymptomatic phase. Over time and usually before an increase in disease severity or the onset of AIDS symptoms, a "phenotypic switch" occurs as T-tropic viruses emerge. This switch reflects a change in predominant co-receptor usage from CKR-5 to CXCR4 throughout the course of HIV disease, and further demonstrates the elusive nature of HIV and the complexity of the HIV infection process (7). This paper will focus specifically on CKR-5, its discovery and potential for conferring resistance to HIV as well as raise questions about additional genetic and environmental factors involved in HIV infection and resistance.

BACKGROUND

Discovery of CKR-5

The road to the discovery of CKR-5 began in 1986 after Jay Levy recognized that CD8+ T cells not only had a cytotoxic effect against HIV-infected cells, but also could strongly inhibit HIV replication. In 1989, he found that CD8+ T cells secreted a soluble factor that inhibited HIV replication, which he termed cell antiviral factor (CAF) (2). A group led by Gallo and Lusso identified the ß-chemokines RANTES, MIP-1 alpha, and MIP-1ß as the major HIV suppressive factors secreted by CD8+ T cells. ß-chemokines are chemotactic cytokines that attract macrophages, T-cells, eosinophils, and basophils to sites of inflammation. These chemokines were found to block infection by macrophage-tropic HIV-1 strains, but not by T-tropic virus. The suppressive effect of these chemokines was confirmed by the fact that specific antibodies to the chemokines blocked the inhibitory effect of CD8+ cell supernatants (5). A study by Paxton et al. (1996) found that levels of these ß-chemokines were unusually high in HIV seronegative individuals with multiple high-risk sexual exposures to HIV. The CD4+ T cells of these individuals were also found to be highly resistant to infection by primary and M-tropic HIV-1 strains, but not to T-tropic strains (15). Another study observed that HIV-infected people who are long-term non-progressors have higher levels of ß-chemokines in their blood (19). These above findings implicated the role of a ß-chemokine receptor as a possible accessory factor for macrophage-tropic HIV-1 entry. The ß-chemokine receptor CKR-5 was independently identified by 5 separate research groups as the major co-receptor for infection by M-tropic HIV-1 strains (1; 4; 7; 8; 9).

Mechanism of M-tropic HIV-1 entry: CD4 and CKR-5

Wu et al. (1996) and Trkola et al. (1996) recently proposed a mechanism by which CKR-5 could mediate entry of M-tropic HIV-1 into its target cells. First the HIV surface protein, gp120, binds to the CD4 receptor on T cells. This interaction results in a conformational change in gp120, creating a new recognition site on the glycoprotein that allows it to bind to CKR-5 (18; 19; 21). The discovery of this "second structure" in HIV-receptor interactions should facilitate the development of drugs targeting HIV-1 entry. Former drug strategies that targeted the CD4-viral envelope complex were ineffective probably because of their failure to account for the two-step viral entry process (12). However, it is still unknown how the HIV transmembrane protein, gp41, interacts with the host cell membrane to allow viral penetration and final fusion with the host cell (19).

Figure 1. Proposed mechanism of HIV entry (Source: Wain-Hobson, 1996)

GENETIC MUTATION IN CKR-5 AND ITS IMPLICATIONS FOR HIV-1 RESISTANCE

The significance of CKR-5 as a major HIV-1 co-receptor has been further supported by the discovery of a 32 base pair CKR-5 deletion allele which has been found to occur in a homozygous state in some HIV seronegative individuals at high risk for exposure to the virus, but not in any HIV exposed, seropositive people (6; 13; 16). The CKR-5∆32 deletion causes a frame shift at amino acid 185, encoding a truncated protein which lacks the last three transmembrane segments of the normal CKR-5 receptor, as well as regions involved in G-protein-coupling.

CKR-5∆32

TTT CCA TAC Agt cag tat caa ttc tgg aag aat ttc cag aca TTA AAG ATA GTC

[ 32-bp deletion ________]

This mutant protein was determined to be a nonfunctional ß-chemokine receptor as well as a nonfunctional M-tropic HIV-1 co-receptor, accounting for the uninfected, seronegative status of homozygotes at high risk for HIV exposure; homozygotes for the deletion allele avoid infection because they lack a functional CKR-5 receptor (6; 13; 14; 16). Interestingly enough, there appears to be no negative phenotype associated with the CKR-5 defect, probably because of the redundant nature of the chemokine system.

So far, three studies have been published that collectively examined CKR-5 genotype frequencies in cohorts of HIV infected people, at-risk, HIV seronegative individuals, and normal, uninfected populations. The first study conducted by Samson et al. (1996) genotyped DNA samples from 704 healthy, Northern European Caucasian individuals, 124 Western and Central African individuals, and 248 Japanese people for CKR-5 (16). The allele frequencies were found to be .908 for wild-type CKR-5 and .092 for the mutant in the sample of Caucasian individuals. The CKR-5 genotype frequencies translate to about 83% of the Caucasian population being homozygous for wild-type, 16% being heterozygous, and 1% being homozygous for the deletion allele, which is not significantly different from the expected Hardy-Weinberg distribution. However, there were no CKR-5∆32 alleles found in the cohorts from Africa and Japan, suggesting that the mutant allele is either absent or extremely rare in these regions. Samson et al. then compared the CKR-5 allele and genotype frequencies of 723 HIV seropositive Caucasian individuals from Belgium and France with the seronegative cohort of healthy Caucasian people. This comparison revealed that the frequency of the CKR-5∆32 allele was significantly less in the HIV-infected population (0.054) versus the uninfected population (0.092) [p<0.0005]. There was a smaller percentage of heterozygotes in the infected group (10.8%) versus the uninfected group (16.2%), and there were no homozygotes for the deletion allele among the 723 seropositive individuals compared to 1.1% among the seronegative population. The authors of this study also found that the truncated CKR-5∆32 protein cannot function effectively as an HIV-1 co-receptor. From their results, the Samson group confirmed that CKR-5 is the major co-receptor used by M-tropic HIV-1 strains and concluded that homozygotes will demonstrate strong resistance to HIV-1 infection, and that heterozygotes may also have some degree of protection. They suggested that the partial resistance of heterozygotes may be due to a decrease in the number of functional CKR-5 receptors or a dominant-negative effect of CKR-5∆32.

The second study of this kind was conducted by Dean et al. (1996) and examined CKR-5 genotype frequencies in high-risk for HIV exposure populations in the United States (6). This study was more extensive than the previous one in that it included 6 different long-term AIDS cohort studies and a total of 1955 subjects. The participants in this study included HIV-1 seronegative individuals at high risk for exposure, HIV-1 infected AIDS patients, and HIV-1 infected patients who have not progressed to AIDS. While the previous study compared allele and genotype frequencies of HIV seropositive people with healthy, uninfected individuals, this study made comparisons within high risk groups for HIV-1 infection (homosexual men, intravenous drug users, and hemophiliacs) who differed in their seronegative and seropositive status. Unlike the Samson study, these authors did not find any significant difference in the allele frequencies of CKR5 between HIV-1 infected and seronegative individuals in any of the cohorts. However, they did find a significant difference when they compared genotype frequencies between seropositive and seronegative subjects. Out of the 612 seronegative individuals, 17 (3%) were homozygous for the CKR-5∆32 deletion allele. As in the Samson study, there was not a single deletion homozygote among the 1343 seropositive individuals. However, unlike the Samson study, the Dean group did not find any evidence showing that heterozygotes are protected against HIV-1 infection; there was approximately the same proportion of heterozygotes in both the seropositive (15%) and seronegative (14%) populations. But, they did find some indications to suggest that heterozygotes may have a delayed progression to AIDS compared to wild-type homozygotes. In the homosexual cohorts, there were more than twice as many heterozygotes among the long-term nonprogressors compared with rapid progressors; however, there was no significant difference in the heterozygote frequencies in long-term nonprogressors versus rapid progressors in the hemophiliac populations studied. Kaplan-Meier survival analyses also demonstrated that heterozygotes have a significantly delayed progression to AIDS compared with wild-type homozygotes. These authors also confirmed the Samson group's observations that the CKR-5∆32 allele is rare in populations of African descent (allele frequency of 0.017) compared to the Caucasian population (allele frequency of 0.115).

The third paper that examined CKR-5 genotype frequencies was published by Huang et. al (1996) and studied a combination of the types of populations looked at in the previous two papers (13). These authors genotyped 637 normal platelet donors to the New York Blood Center, 191 selected Asian populations, 137 African populations, and 1252 homosexual men from the Chicago MACS cohort studies. Once again, these researchers did not find any homozygotes for the CKR-5∆32 allele among 675 HIV infected participants. In addition, they did not find a single deletion allele in the Asian and African groups. The protective effect of the CKR-5∆32 homozygous state was further confirmed by follow-up of Caucasian MACS participants who had anal receptive intercourse with over 6 different partners in the 6 months prior to enrollment. After being followed for 8 years or longer, these subjects were observed to remain seronegative. 33.3% of these individuals were homozygous for CKR-5∆32. However, one should be a bit cautious when analyzing this percentage, since only 12 cases were followed for the 8+ year period. Unlike the Samson study, but like the Dean study, this group did not find any evidence to support that heterozygosity protects against HIV-1 infection. The percentage of heterozygotes in normal platelet donors, at-risk seronegative subjects, and infected people were found to be comparable. In order to examine the role of the heterozygous genotype on disease progression, they compared CD4 decline and viral load in heterozygotes and wild-type homozygotes, and found that heterozygotes had a slower CD4 decline and lower mean plasma viremia. However, the authors of this study were more skeptical than the Dean group about the protective effect of heterozygosity on disease progression as they did not note any significant differences between wild-type homozygotes and heterozygotes in Kaplan Meier survival analyses of time to AIDS or death in seroconverters.

When the data is combined from these 3 studies, there is not a single CKR-5∆32 homozygote among 2741 HIV-1 infected individuals. When data on high risk, seronegative individuals from the Dean and Huang studies are combined, there is a total of 33 (3.1%) CKR-5∆32 homozygotes out of 1058 at-risk, uninfected people. Combining the data from normal, uninfected Caucasian individuals in the Samson and Huang studies results in a total of 17 (1.3%) CKR-5∆32 homozygotes out of 1341 people (Table 1). The absence of any deletion homozygotes among the HIV-1 infected subjects compared to the prevalences of 1.3% in normal, Caucasian individuals and 3.1% in high risk, uninfected subjects is highly significant, with p<0.0001 in both cases (z-test). Furthermore, when compared to normal, Caucasian individuals, there is a significantly greater proportion of homozygotes among the high risk, seronegative subjects (1.3% vs. 3.1%, respectively), with a p-value of 0.0027. Collectively, these results support the hypothesis that individuals homozygous for the CKR-5∆32 allele are strongly protected against HIV-1 infection.

Table 1. Percentage of CKR-5∆32 homozygotes by HIV-1 status.

HIV-1 status	Total number of subjects	CKR-5∆32/ CKR-5∆32
seropositive§	2741	0 (0%)
high-risk, seronegative†	1058	33 (3.1%)
normal, seronegative*	1341	17 (1.3%)

§ Samson et al., 1996; Dean et al., 1996; Huang et. al, 1996

† Dean et al., 1996; Huang et. al, 1996

* Samson et al., 1996; Huang et al., 1996. The majority of these subjects were Caucasian individuals

The proportion of deletion heterozygotes out of all seropositive individuals studied (Samson, Dean, and Huang papers) was 366/ 2527 (14.5%); the proportion of deletion heterozygotes out of all at-risk seronegative individuals (Dean and Huang papers) studied was 169/ 1058 (16%). This combined data indicates that there was no significant difference between the frequency of heterozygotes in seropositive and at-risk, seronegative individuals, enhancing the arguments made by Dean et al. and Huang et al. that heterozygotes are not protected against HIV-1 infection. Furthermore, there were 199 (14.8%) heterozygotes in the population of 1341 healthy, Caucasian individuals (Samson and Huang papers), not significantly different from the proportion of heterozygotes in the cohorts of seropositive and at-risk, seronegative people (Table 2). Therefore, the combined data contradicts the observation made by the Samson group that there are fewer heterozygotes in infected groups than in uninfected populations and their conclusion that heterozygotes may also be somewhat resistant or protected from HIV.

Table 2. Percentage of CKR-5∆32 heterozygotes by HIV-1 status.

HIV-1 status	Total number of subjects	CKR-5/ CKR-5∆32
seropositive§	2527	366 (14.5%)
high-risk, seronegative†	1058	169 (16.0%)
normal, seronegative*	1341	199 (14.8%)

§ Samson et al., 1996; Dean et al., 1996; Huang et. al, 1996. The total number of subjects is less than that in Table 1 because the Huang study only included numerical data on heterozygotes for Caucasian participants of the MACS study.

† Dean et al., 1996; Huang et. al, 1996

* Samson et al., 1996; Huang et al., 1996. The majority of these subjects were Caucasian individuals

All three studies also found that the CKR-5∆32 allele is much rarer in cohorts of African and Asian ancestry than in Caucasian populations. Dean et al. found a very low frequency of the allele in African Americans (0.017) compared to Caucasians (0.11) in his cohorts of high-risk individuals. Samson et al. and Huang et al. did not find a single deletion allele in a total of 261 African and 439 Asian samples. However, it was unclear in both studies how these samples were selected and whether or not these individuals were infected with HIV or at risk for infection. This information would have been useful because the frequency of the deletion allele may vary among individuals with different HIV status, as observed in Caucasian populations. Though it is probably unlikely, if the majority of these African and Asian samples were HIV positive, this could account for the fact that there were no CKR-5∆32 alleles in the samples. If the CKR-5∆32 allele plays an essential role in protection against HIV infection (primarily in deletion homozygotes) or delaying disease progression in infected individuals, the apparent discrepancy between the frequency of the allele in Caucasian versus African and Asian groups suggests that the latter populations are less likely to demonstrate HIV resistance or delayed disease progression. Future research should examine larger populations of non-Caucasians to determine whether or not this is true.

CONCLUSION

Questions for Future Research

Overall, the combined results from the Samson, Dean, and Huang studies provide fairly compelling evidence to support the conclusion that individuals homozygous for CKR-5∆32 are protected from HIV infection. There is somewhat less of a consensus about the effect of heterozygosity on HIV infection and disease progression, but it appears that heterozygotes are not resistant to infection; however, they may have some limited protection against disease progression. More long-term follow-ups of heterozygotes need to be done to determine the actual value of having one copy of the mutant allele.

There were several limitations with these studies that should be considered when conducting future research in this area. First, the true HIV-exposure status of the at-risk, seronegative individuals in the Dean and Huang studies was not clear. Although these people were in high risk groups for HIV exposure, some of these individuals may never have been exposed to the virus. Perhaps, more detailed questions could be asked regarding the subjects' knowledge of their partners' HIV status as well as their own sexual behavior (number of partners, use of condoms and other methods of protection) and infection with other STDs. These factors can play a significant role in determining whether or not an individual becomes infected. Secondly, none of these studies control for the effects of anti-viral therapy on disease progression. This is critical particularly when Kaplan-Meier survival analyses are being done, since anti-viral drugs can affect disease and survival outcome.

The knowledge that has been gained through these studies raises several interesting questions for future research: Since CKR-5 is the co-receptor for M-tropic HIV strains, but not T- tropic strains, are deletion homozygotes susceptible to infection by the latter types of virus? Is the resistance of deletion homozygotes to M-tropic HIV absolute or relative? If it is relative, is the level of resistance affected by the viral inoculum size or route of transmission? Only 3.1% of the high-risk, seronegative individuals in the combined studies were homozygous for CKR-5∆32; what other genetic, environmental, or behavioral factors could have contributed to protection from disease in the other 97%, particularly in those who were homozygous for wild-type?

Are There Other Factors Involved?

A few studies have examined the role of other factors in contributing to HIV disease resistance or susceptibility. One study noted that a small cohort of prostitutes in Nairobi, Kenya remained persistently seronegative by PCR after follow-up for 3-10 years (10). Furthermore, it found that this persistent seronegativity is not due to differences in sexual behavior or in the incidence of other sexually transmitted diseases. Because the CKR-5∆32 allele is extremely rare in African populations, it is highly unlikely that the mutation contributed to resistance in this group. The authors argue that other factors besides this mutant allele play a role in protection; in particular, they suggest that HIV-1 specific cellular immune responses may be important and should be further examined.

A study by Garred et al. (1997) recognized the influence of another genetic cofactor on HIV susceptibility and disease progression (11). The authors found that homozygotes for variant mannose-binding lectin (MBL) alleles have an increased risk for HIV infection. MBL functions as part of the complement system and therefore is particularly important in the first line of host defense prior to establishment of the adaptive immune response. None of the high risk, HIV negative subjects were homozygous for variant MBL alleles in comparison to 8% of the HIV-infected individuals. Furthermore, both heterozygosity and homozygosity for the variant MBL alleles were correlated with a significantly shorter survival time after AIDS diagnosis. Therefore, the association between CKR-5 genotype and rate of disease progression in subjects in the Dean and Huang studies may have been confounded by the effects of MBL genotype.

The existence of other co-receptors for HIV entry is also a likely possibility. A study by Cheng-Mayer et al. (1997) showed that there was no strict correlation between macrophage tropism and use of the CKR-5 co-receptor as well as T-cell tropism and use of the CXCR4 co-receptor (3). The authors found that several T-tropic and M-tropic viral strains were able to use both CXCR4 and CKR-5 for entry, despite their specific tropism for either T-cells or macrophages. These findings suggest that other co-receptors play a role in determining tissue-specific HIV entry.

Implications for Therapy

Despite the fact that other cofactors may determine susceptibility to HIV infection and rate of disease progression, the recent identification of CKR-5 and its mutant allele represents a tremendous breakthrough in HIV research and opens another door to the development of new HIV drugs. In fact, LeukoSite, a biotechnology company in Cambridge, Massachusetts, has already formed a collaboration with Warner-Lambert, a pharmaceutical company, to design drugs that target the HIV-CKR5 interaction. According to Christopher Mirabelli, Ph.D., chairman of LeukoSite, "We plan to develop small molecule drug antagonists to CKR-5 that make the receptor invisible to the virus." (12). Because there is no known negative phenotype associated with the homozygous CKR-5∆32 genotype, there should be no major side effects expected from CKR-5 antagonists. Our current knowledge about CKR-5 as well as further research to determine other genetic and environmental HIV cofactors will help us develop a whole new array of therapies that target the critical point of viral entry. Together with nucleoside analogs and protease inhibitors, these new forms of treatment will contribute significantly to the fight against HIV disease.

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