CAF: Genetic Factors in Resistance

Genetic Factors in Resistance

The Role of Human Leukocyte Antigen-A2 and Subtypes

The idea of a "major histocompatability gene" was first proposed in 1956 to distinguish between a gene or genes causing acute rejection of homologous tissue graft and the gene or genes which are associated with chronic rejection of such tissue grafts. The latter were called "minor histocompatability genes". It is now well established that the "major histocompatability genes" are a group of closely linked genes whose products are responsible for the acute transplantation reactions. This genetic region, common to most vertebrates, is called the Major Histocompatability Complex (MHC) which in humans is known as the Human Leukocyte Antigen (HLA) system.

In the attempt to develop organ transplantation into a practical therapeutic methodology, a great deal of intensive research has been conducted in man, the mouse and other animals to define the genetic structure and functions of the MHC and its gene products. As a result it is now recognized to play a dominant role not only in transplantation biology but in immune responsiveness, disease susceptibility and "self" recognition.

The human MHC or HLA complex is located on the short arm of chromosome 6. The complex has more than 200 genes, over 40 of which encode leukocyte antigens. Three closely linked loci, HLA-A, B, and C are the sites of the so-called classic, or class I genes which are the principal performers on the immunologic stage. There are multiple alleles (alternative gene forms) for each class I locus which are designated by Arabic numbers e.g. HLA-A1, A2,..A11, B7, B27, C1, C7 etc. The antigens for the class I genes can be identified by serological reactions while the antigens of the class II genes, the D locus, formerly were identified by radioactive thymidine uptake in mixed lymphocyte cultures. Now the alleles of the latter antigens are defined by molecular methods. When the genes of the serologically defined Class 1 antigens were studied using molecular methods they were often found to be polymorphic, containing a number of alleles or subtypes for the serologically defined antigen. These molecularly defined alleles or subtypes are also designated by Arabic numbers but the numbers are preceded by an asterisk, for subtype alleles, four digits follow the asterisk. Subtypes for HLA-A*02 would be; HLA-A*0201, HLA-A*0205 etc. The term phenotype refers to the specific molecule or antigen produced by an HLA gene. Haplotype refers to a pair or group of closely linked genes usually inherited as a unit. Since each of the 23 human chromosomes is paired, the number of possible combinations among the genes of the six class I and class II loci on the sixth chromosome is extremely large.

Class I genes are expressed by most cells in the body, the level of expression depending on the type of cell. Class II genes are most often expressed by certain immune cells such as B lymphocytes, activated T lymphocytes, macrophages, dendritic cells of the lymph nodes, and cells of the thymic epithelium. The function of both class I and class II molecules is to carry short peptides to the cell surface for presentation to circulating T-cells. The recognition of pathogen derived peptides as non-self by the T-cell is the beginning of immune recognition and the cascade of immune responses which follow. Among these are the killing of infected cells by activated, cytotoxic T cells and the formation of specific antibodies.

During the 1970's investigators reported a number of associations between various class I HLA phenotypes and the response to infection. Among these were rapid clearance of Hepatitis B antigen from the blood, associated with the HLA-B8 phenotype which was also associated with chronic aggressive hepatitis. A negative relationship was shown for the frequency of the haplotype HLA-A2, BW17 in villages in the lowlands of Sardinia where Plasmodium falciparum , a frequently fatal form of malaria, was endemic (1). A low in vitro immune response to vaccinia virus was reported for individuals with the HLA-CW3 phenotype. Arthritis associated with Salmonella , Yersinia enterocolitica , and Shigella infections was associated with the HLA-B 27 phenotype. This phenotype also displays a strong association with ankylosing spondylitis, Reiter's syndrome, and acute anterior uveitis, conditions for which an infectious component has not been firmly established. These observations, although of great interest, were not considered conclusive in establishing cause and effect relationships. Most of the data on infectious diseases was derived from cross-sectional studies which compared the frequency with which given phenotypes or haplotypes were found in patients with the abnormal response to the infection with the frequency with which they were found in the general population instead of controls.

To consider these and other possible associations between HLA types and infectious diseases the National Institute of Allergy and Infectious Diseases (NIAID) convened a workshop to determine whether the reported associations were strong enough to warrant further study and, if so, what types of studies should receive priority (2). The workshop concluded that there was sufficient evidence to justify further studies of these relationships but that future studies should be conducted prospectively within a sound epidemiologic framework. The mechanisms involved in the association should be examined carefully and on a molecular basis wherever possible.

Although a great deal of work has been done since to determine the role of the HLA complex in resistance to or the expression of infectious diseases, until recently the principal results that have confirmed that role have been found in studies of malaria (3)(4). These have determined that certain HLA haplotypes provided protection against the more severe forms of malaria. The authors concluded that their data support the hypothesis that the extraordinary polymorphism of the major histocompatability complex genes has evolved primarily through natural selection by infectious pathogens.

The first cases of Human Immunodeficiency Virus type 1 infection (HIV-1) were reported little over a year following the NIAID workshop report. Since that time a number of prospective studies have been undertaken to define the relationship of HLA phenotypes to HIV-1 infections. In the majority of these, the studies were designed to determine the influence of HLA phenotypes or haplotypes on the rates of progression from sero-conversion to AIDS. These studies also measured the time from sero-conversion to specific endpoints such as the onset of opportunistic infections, the plasma levels of HIV-1 RNA, the decline in CD4+ T-cells, or the response to antiretroviral therapy among participants (5)(6). It was observed that maximum heterozygosity of class I alleles delayed the onset of AIDS.

A recent study has further investigated and extended this finding (7). The study incorporated findings from the Chicago component of the Multicenter AIDS Cohort Studies (MACS). The subjects of the study consisted of 996 men who had had sex with men whose blood samples were available for molecular HLA typing. Of these, 562 were HIV-1 positive. The peptide binding specificities of the class I HLA alleles identified in the study were used to group them into supertypes. The association of these discreet supertypes with disease progression rates in the HIV-1 infected men was then analyzed. It was found that the HLA supertypes alone and in combination showed differences in the response to infection independent of that provided by single HLA alleles. The close correlation seen between the frequencies of these HLA supertypes and viral load suggests that HIV-1 adapts to the most common HLA alleles in the population giving an advantage to individuals possessing rare or uncommon alleles. As had been noted previously black men in this study had significantly lower viral loads than white men. This could be due to the greater frequency of rare or uncommon alleles in African populations as a result of the large number of infectious diseases endemic in sub-Saharan Africa . The study further supports the concept that HLA molecules are essential for the generation of an effective immune response and not surrogate markers for other genes that might effect the progression of the disease.

The presence of the haplotype HLA-B*35, Cw*04 has been associated with a rapid progression from seroconversion to AIDS-defining conditions in Caucasians (8). It was found subsequently in a study which combined the gene frequencies in five separate cohorts of Caucasians that the most common subtype of HLA-B*35, HLA-B*3501 is not associated with rapid progression to AIDS. A less common subtype tentatively designated as HLA-B*35-Px was found to be primarily responsible for that effect (9). The product of B* 35-Px differs from that of B*3501 by a single amino acid in a critical location on its molecule. Another recent study determined the effect of HLA profiles on the course of HIV-1 disease following acute infection using multiple measures of progression.(10). One result was the finding that HLA profile, that is specific combinations of alleles, influences the time to AIDS through its effect on the rate of HIV-1 replication as measured by plasma viral load during the first year following acute infection. All these studies have provided strong evidence that phenotypes of both class I and class II genes are involved in the response to HIV-1 infection.

Within the recent past, three studies have been published all of which have indicated that certain HLA-A2 subtypes (HLA-A*02 subtypes) may be instrumental in preventing infection with HIV-1. The first of these had studied cohorts of female sex workers (prostitutes) in Nairobi , Kenya over a period of 13 years. A total of 498 women were followed in the cohort, of whom 274 seroconverted to HIV-1 during the study (11). An unselected sample of 232 women was HLA typed of whom 122 had seroconverted to HIV-1 positive. Known risk factors for HIV-1 seroconversion were compared between those who were HLA typed and the remainder, and were similar. The HLA supertype, HLA A2/6802, composed of HLA-A2 subtypes HLA-A*0202, HLA-A*0205, HLA-A*0214 plus a closely linked allele A*6802, was associated with a significantly lower risk of HIV-1 seroconversion among the 232 participants who had been HLA typed, P =.0003.

A second report from this group of investigators studied HLA types in HIV-1 infected mothers and their children who were enrolled in the Nairobi HIV-1 Perinatal Transmission Study (12). The study, conducted between 1993 and 1996, evaluated 171 children born to 135 infected mothers. Of the 171 children, 19 were infected perinataly and 152 were uninfected. Of the latter,21 subsequently seroconverted for HIV-1 during breastfeeding. Perinatal transmission occurred in 2 of 72 (2.8%) children with the HLA A2/6802 supertype versus 17 of 99 children (17.2%) not carrying this type p =.005. Possession of the supertype, however did not protect against HIV-1 transmission during breast feeding.

A third, more recent report, has found that several of these same alleles of the HLA-A2 supertype are associated with resistance to infection with HIV-1 among white males (13). The report described a case-control study of white homosexual men who had been followed in the MACS cohorts for more than 15 years. The case group consisted of 100 high-risk men who reported having engaged in anal-receptive intercourse with many partners in the 2.5 years before their second MACS visit and had remained seronegative for more than 15 years. The assumption was made that these men who had lived in areas where rates of HIV-1 among homosexual men were high were repeatedly exposed to HIV-1. The control group consisted of 184 low-risk men who had seroconverted to HIV-1 positive despite reporting relatively few exposures. All of the cases included in the study underwent typing for class I HLA and TAP, the HLA transporter genes associated with antigen processing. In all among the 284 men, 29 HLA-A alleles, 54 HLA-B alleles and 24 HLA-C alleles were identified. In addition 55 TAP 1 and 126 TAP 2 genes were identified.

Based upon variations in the nature of the different peptides bound and presented, HLA-A2 was divided functionally into two distinct subgroups represented by A*0201 and A*0205. The A*0205 subgroup including A*0205, A*0206, and A*6802 were significantly more frequent in the group resistant to HIV-1 than in the control group. However the A*0201 subgroup which included A*0201 and A*0212 demonstrated no such difference. In summary, HLA-A2 was identified in 49 (49%) high-risk, seronegative men and in 75 (40.76%) low-risk seropositive men. The A*0205 subgroup was identified in 9 (9%) high-risk seronegative men and in 3 (1.63%) low risk seropositive men, p= .005. In addition the TAP2 gene Ala665 was associated with HIV-1 resistance p=.005. The reason for the association of a transporter gene with resistance to HIV-1 is unclear but it may reflect more efficient transport of selective viral peptides or it may be due to linkage disequilibrium, that is, close linkage to an unidentified gene or genes responsible for the resistance. The study also confirmed the earlier finding that HLA-B*35-Px was associated with rapid progression of HIV disease and lower resistance to HIV-1 infection.

In this study of white homosexual men only a small proportion of the individuals possessing the HLA-A2 supertype had a subtype that could be associated statistically with resistance to infection with HIV-1, less than 10%. It should be added here that this supertype is known to have over 60 alleles.

Investigators at the National Cancer Institute (NCI) have published a report on a series of elegant in vitro experiments that may add to the evidence concerning the role of HLA-A2 in the HIV-1 pandemic (14). Peripheral blood mononuclear cells that had been HLA typed by molecular methods were mixed in cultures with similar but irradiated (inactivated) cells from other donors. After 7 days, the investigators examined the resulting supernatant fluids of the cultures for anti-HIV-1 activity. None of the cells were from donors who were infected with HIV-1. The responding (non-irradiated) cells in the mixed culture represented most class I and II HLA genotypes. The irradiated cells were of mixed types or, in one experiment, the same HLA type as the responding cells. In the latter, no difference was seen in the amount of anti-HIV activity in their supernatant fluids. The supernatant fluids of the mixed cultures from 19 of 20 HLA-A*02 (HLA-A2) positive donors exhibited strong inhibition of HIV-1 replication in culture. In comparison, 4 of 14 donors who were HLA-A*02 negative exhibited similar viral inhibition levels ( P <.0006). Statistical analysis did not reveal any significant association of anti-HIV-1 activity with supernatants from donors with any of the other class I or class II genes included in the study. The experiments also demonstrated that the anti-HIV-1 activity of the supernatant fluids from HLA-A*02 donors was independent of cytokine and chemokine activity. No difference was seen in the anti-HIV response from cells belonging to HLA-A*02 subtypes A*0201, A*0202 or A*0205.

A follow-up report by the authors has indicated that much of the anti HIV-1 activity of the supernatant fluids in these studies was probably due to ribonucleases belonging to the Rnase A superfamily (5). How this relates to the specificity of the HLA types (HLA-A2 supertype) of the donor cells in producing supernatants with high anti-HIV-1 activity remains to be seen. There is clearly much more to learn before these in-vitro study results can be reconciled with the findings of the cohort studies.

The report of the Sixth International Histocompatability Workshop Conference in 1976 provided frequencies of the various Class I phenotypes which may be applicable to the current directions of this pandemic (16). The phenotype frequency reported for HLA-A2 in Central European Caucasians was 44%, among Japanese it was 45% but in African black populations it was only 22%. Although the total number typed in each of the three population groups was small, <500 unselected individuals the figures for Caucasians and Japanese in that report approximate those of more recent reports. These figures represented frequencies at or just before the onset of the HIV-1 pandemic and thus should not have been influenced by it. Currently, the National Donor Registry gives a frequency of 18.88% for HLA-A2 among African Americans.

As mentioned previously, HLA-A2 was part of a haplotype reported to have had a negative relationship with endemic P. falciparum malaria in earlier studies reported from Sardinia (1). The authors discussed these findings further in a subsequent article (17). HLA frequencies should be examined in other population studies of malaria such as those studying the protective effect against P. falciparum of variations in a gene on the X chromosome coding for G6PD (glucose-6-phosphate dehydrogenase (18).

Although it is tempting to ascribe the fulminant course of the HIV-1 epidemic in sub-Saharan Africa to an interplay between two major pandemics, i.e. the relatively recent (several millennia) pandemic of P. falciparum malaria and the on-going HIV-1 pandemic, the evidence coming out of recent studies support this hypothesis only tangentially. While the frequency of the HLA-A2 supertype in Africans is much lower than in Caucasians, we don't know which among the many subtypes accounts for this. In addition, in the MACS study no class I HLA types were identified to account for the resistance demonstrated in the great majority of high-risk individuals. A larger population base may have indicated that other A*02 or class I alleles were involved. The case classifications used to define the high-risk and control populations were dependent largely on self-reported behavior, often of questionable reliability. Other factors, including behavioral or genetic, may have been involved in the apparent protection. Since the population base for this study consisted of Caucasian males the results may not reflect those that might have been found in African-American populations whose HLA frequencies more directly relate to those of their African forbearers.

It is apparent that all these studies are adding another dimension to our understanding of the HIV-1 pandemic and will play a significant role in our attempts to control it.. We will continue to provide updates on this unfolding story as new developments indicate.

References:

1) Piazza A, Belvedere M C, Bernoco D, Conight C, Contu L, et al. HLA variation in Four Sardinian villages under differential selective pressure by malaria. In: Histocompatability Testing 1972. Munksgaard, Copenhagen 1972: p 73-84 .

2) Grayston J T, Payne F J. Summary of a workshop on the major histocompatability complex in infectious disease epidemiology. J Infect Dis 1979; 139: 246-249

3) Hill AV, Allsop CE, Kwiatkowski D, Anstey NM, Twumasi P, et al. Common west African HLA antigens are associated with protection from severe malaria. Nature 1991; 352: 595-600

4) Hill AV, Elvin J, Willis AC, Aidoo M, Allsop CE, et al. Molecular analysis of HLA-B53 and resistance to severe malaria. Nature 1992; 360: 434-439

5) Kaslow RA, Carrington M, Apple R, Park L, Munro A, et al. Influence of combinations of human major histocompatability complex genes on the course of HIV-1 infection. Nature Med 1996; 2 (4): 405-411

6) Mann DL, Garner RP, Dayhoff KC, Fernandez MA, Davis C, et al. Major histocompatability complex genotype is associated with disease progression and virus load levels in a cohort of human immunodeficiency virus type 1-infected Caucasians and African Americans. J Infect Dis 1998;178: 1799-1802

7) Trachtenberg E, Korber B, Sollars C, Kepler TB, Hraber PT, et al. Advantage of rare HLA supertype in HIV disease progression. Nature Med 2003; 9: 928-935

8) Carrington M, Nelson GW, Martin MP, Kissner T, Vlahov D, et al. HLA and HIV-1; heterozygote advantage and B*35-Cw*04 disadvantage. Science 1999; 283: 1748-1752

9) Xiaojiang G, Nelson GW, Karacki BA, Martin MP, Phair J, et al. Effect of a single amino acid change in MHC Class I molecules on the rate of progression to AIDS. NEJM 2001; 344: 1668

10) Keet PM, Tang J, Klein MR, LeBlanc S, Enger C, et al. Consistent associations of HLA class I and II and transporter gene products with progression of human immunodeficiency virus type 1 infection in homosexual men. J Infect Dis 1999, 180: 299-309

11) MacDonald KS, Fowke KR, Kimani J, Dunand VA , Nagelkerke NJD, et al. Influence of HLA supertypes on susceptibility and resistance to human immunodeficiency virus type 1 infection. J Infect Dis 2000; 181: 1581-1589

12) MacDonald KS , Embree JE, Nice JD, Nagelkerke NJD, Castillo J, et al. The HLA A2/6802 supertype is associated with reduced risk of perinatal human immunodeficiency virus type 1 transmission. J Infect Dis 2001; 183: 503-506

13) Liu C, Carrington M, Kaslow RA, Gao X, Rinaldo CR, et al. Association of polymorphisms in Human Leukocyte Antigen class 1 and transporter associated with antigen processing genes with resistance to Human Immunodeficiency virus type 1 infection. J Infect Dis 2003; 187: 1404-1410;

14) Greene E, Pinto LA, Cohen SS, Trubey, CM, Trivett MT, et al. Generation of alloantigen-stimulated anti-human immunodeficiency virus activity is associated with HLA-A*02 expression. J Infect Dis 2001; 183: 409-416

15) Rugeles MT, Truby CM, Bedoya, VI, Pinto LA, Oppenheim JJ, et al. Ribonuclease is partly responsible for the HIV-1 inhibitory effect activated by HLA alloantigen recognition. AIDS 2003; 17 (4): 481-486

16) Joint report from the Sixth International Histocompatibility workshop and conference. In : Histocompatability Testing 1975 . Munksgaard, Copenhagen 1976: 414-458

17) Piazza A, Mayr WR, Contu L, Amoroso A, Borelli I, et al. Genetic and population structure of four Sardinian villages. Ann Hum Genet 1985; 49 (Pt 1): 47-63

18) Tishkoff S A, Varkonyl R, Cahinhinan N, Abbes S, Argyropoulos G, et al. Haplotype diversity and linkage disequilibrium at human G6PD; recent origin of alleles that confer malarial resistance Science 2001; 293: 455-462