Ramifications of HLA class I polymorphism and population genetics for vaccine development.

HLA polymorphism can complicate the design and development of vaccines, especially those that contain a selected number of epitopes and are directed at pathogens prevalent worldwide. Because of HLA class I restricted antigen recognition and ethnic variation in HLA distribution, such vaccines may not be uniformly effective across populations. We, therefore, considered whether it is possible to assemble a panel of HLA-A and/or HLA-B alleles that would allow the formulation of a single vaccine for a set of Caucasian, Black, or Asian populations. In applying an algorithm to predict levels of favorable response, we identified predominant alleles in 15 representative populations. Approximately 80% of the individuals in one African Black population and five Asian populations were positive for at least one of three HLA-A alleles. Eighty percent coverage was also theoretically possible in five Caucasian populations with only five HLA-A alleles. Four of five Black populations analyzed also required five alleles, but the allelic combinations differed. Our findings suggest that HLA-A alleles may be preferred targets because of the increased heterogeneity at HLA-B, although addition of a single HLA-B allele to a set of HLA-A alleles improved coverage. This approach provides for the identification of combinations of alleles that represent a desired percentage of a population and that could be targeted in designing vaccines. For vaccines with known HLA-restricted epitopes, it allows a prediction of theoretical levels of "responder" and "non-responder" status. Finally, these results might be used in the analysis of protein sequences to identify potential CD8+ T-cell epitopes in populations of interest. Biologic variables that may have further relevance are discussed.

Identification of highly conserved and broadly cross-reactive HIV type 1 cytotoxic T lymphocyte epitopes as candidate immunogens for inclusion in Mycobacterium bovis BCG-vectored HIV vaccines.

One of the fundamental goals of current strategies to develop an efficacious vaccine for AIDS is the elicitation of cytotoxic T lymphocyte (CTL) reactivities capable of recognizing cells infected with different subtypes of the human immunodeficiency virus type 1 (HIV-1). In efforts to explore new vaccine candidates by the UNAIDS/WHO Vaccine Committee, we review the most recent data concerning CTL epitopes that are conserved among the different HIV-1 subtypes. Moreover, we examine HLA allelic frequencies in several different populations, to determine those that could contribute to the goal of a cumulative phenotype frequency (CP) of at least 80%. By analyzing conserved epitopes in the context of HLA restricting alleles, we define a set of HIV-1 gene regions that may have the greatest potential to induce cross-clade reactive CTLs. The absence of well-defined correlates of immune protection that link CTL epitopes to delayed disease progression and/or prevention of infection does not permit an assignment of rank order of the most relevant component of a candidate vaccine. Thus far, most of the studies conducted in clade B-infected patients to define conserved and immunodominant epitopes indicate gag and pol gene products to be the most conserved among the HIV-1 subtypes. Moreover, anti-Pol and -Gag CTL responses appear to correlate inversely with disease progression, suggesting that they should be among the first choice of antigens to be included in a candidate vaccine construct aimed at induction of broad CTL responses. The impact of a clade B-based vaccine as a worldwide candidate capable of inducing protective immune responses can be determined only after "in vivo" studies. Meanwhile, extensive parallel studies in populations infected with non-clade B HIV-1 subtypes should define the patterns of immunodominant epitopes and HLA for comparison with the data already collected in clade B-infected subjects.

Transplantation of thymus tissue in complete DiGeorge syndrome.

BACKGROUND: The DiGeorge syndrome is a congenital disorder that affects the heart, parathyroid glands, and thymus. In complete DiGeorge syndrome, patients have severely reduced T-cell function. METHODS: We treated five infants (age, one to four months) with complete DiGeorge syndrome by transplantation of cultured postnatal thymus tissue. Follow-up evaluations included immune phenotyping and proliferative studies of peripheral-blood mononuclear cells plus biopsy of the thymus allograft. Thymic production of new T cells was assessed in peripheral blood by tests for T-cell-receptor recombination excision circles, which are formed from excised DNA during the rearrangement of T-cell-receptor genes. RESULTS: After the transplantation of thymus tissue, T-cell proliferative responses to mitogens developed in four of the five patients. Two of the patients survived with restoration of immune function; three patients died from infection or abnormalities unrelated to transplantation. Biopsies of grafted thymus in the surviving patients showed normal morphologic features and active T-cell production. In three patients, donor T cells could be detected about four weeks after transplantation, although there was no evidence of graft-versus-host disease on biopsy or at autopsy. In one patient, the T-cell development within the graft was demonstrated to accompany the appearance of recently developed T cells in the periphery and coincided with the onset of normal T-cell function. In one patient, there was evidence of thymus function and CD45RA+CD62L+ T cells more than five years after transplantation. CONCLUSIONS: In some infants with profound immunodeficiency and complete DiGeorge syndrome, the transplantation of thymus tissue can restore normal immune function. Early thymus transplantation - before the development of infectious complications - may promote successful immune reconstitution.

Familial antiphospholipid antibody syndrome: criteria for disease and evidence for autosomal dominant inheritance.

OBJECTIVE: To develop diagnostic criteria for a familial form of antiphospholipid antibody syndrome (APS), identify families with >1 affected member, examine possible modes of inheritance, and determine linkage to potential candidate genes. METHODS: Family members of probands with primary APS were analyzed for clinical and laboratory abnormalities associated with APS. Families with > or =2 affected members were analyzed by segregation analysis and typed for candidate genetic markers. RESULTS: Seven families were identified. Thirty of 101 family members met diagnostic criteria for APS. Segregation studies rejected both environmental and autosomal recessive models, and the data were best fit by either a dominant or codominant model. Linkage analysis showed independent segregation of APS and several candidate genes. CONCLUSION: Clinical and laboratory criteria are essential to identify the spectrum of disease associated with APS. We believe a set of criteria was developed that can precisely define affected family members with APS. Modeling studies utilizing these criteria strongly support a genetic basis for disease in families with APS and suggest that a susceptibility gene is inherited in an autosomal dominant pattern. However, in these families, APS was not linked with HLA, Fas, or other candidate genes, including beta2-glycoprotein 1, HLA, T cell receptor beta chain, Ig heavy chain, antithrombin III, Fas ligand, factor V, complement factor H, IgK, and Fas.

Genetic analysis of multiplex rheumatoid arthritis families.

To examine the genetic contribution of HLA and non-HLA genes in the etiopathogenesis of rheumatoid arthritis (RA), 60 Caucasian multiplex families were identified and DNA analyzed for over 52 markers including DRB1, DQA1 and DQB1 alleles. Many of the markers were chosen because of close proximity to candidate genes suggested by previous studies or models of pathogenesis. Sibling pair analysis (SIBPAL), relative pair analysis (RELPAL) and linkage studies using two different models of inheritance suggested linkage for the MHC and two additional chromosomal regions: chromosome 2 (D2S443 near CD8 and IGk; 2p13-2p11.1), and chromosome 15 (CYP19-estrogen synthase; 15q15). No support was found for two chromosomal regions, 1p36 and 3q13, recently suggested by other studies. We used transmission disequilibrium testing (TDT), conditional logistic regression, and segregation analysis to study the contributions that the shared epitope and TNF-c have in contributing to risk for RA. These studies provide additional evidence that the association of HLA alleles in RA patients from multiplex families is similar to that observed in sporadic disease, suggest candidate regions for further analysis and find additional support for an association of TNF-c alleles with RA susceptibility.

HLA and mate choice in humans.

Evidence from studies in rodents suggests that mate selection is influenced by major-histocompatibility-complex haplotypes, with preferences for dissimilar partners. This study was initiated to determine whether avoidance of a mate with the same HLA haplotype as one's own might be occurring in the Hutterites, a North American reproductive isolate of European ancestry, notable for their large sibships, communal lifestyle, and limited number of five-locus HLA haplotypes (HLA-A, -B, -C, -DR, and -DQ). HLA haplotypes were known for 411 Hutterite couples. The number of couples expected to match for a haplotype was calculated in two ways: first, from population genotype frequencies, with account being taken of the nonrandom mating pattern with respect to colony lineages, and, second, from computer simulations using conservative founder assumptions and the exact genealogy of the 411 couples. We observed fewer matches for HLA haplotypes between spouses than expected (first method, P = .005; second method, P = .020-.067). Among couples who did match for a haplotype, the matched haplotype was inherited from the mother in 29 cases and from the father in 50 cases (P = .018). These results are consistent with the conclusion that Hutterite mate choice is influenced by HLA haplotypes, with an avoidance of spouses with haplotypes that are the same as one's own.

Successful formation of a chimeric human thymus allograft following transplantation of cultured postnatal human thymus.

Transplantation of cultured postnatal human thymus was performed in a patient with complete DiGeorge syndrome. Biopsy of the graft 3 mo after implantation revealed normal CD1+ thymocytes in thymic cortical epithelial regions and CD1- thymocytes in thymic medullary epithelial regions, respectively. HLA analysis of graft thymocyte and thymic microenvironment components demonstrated that developing thymocytes and thymic macrophages were recipient derived, while thymic epithelial components were of donor origin. The patient, who initially had no T cells and had profoundly defective T cell function, developed normal T cell responses to mitogens and Ags, tolerance to donor in a mixed lymphocyte reaction, and normal Ab titers after tetanus toxoid and pneumovax immunization. Thus, transplantation of cultured postnatal human thymic tissue in humans can form functional chimeric thymic tissue, and may provide a strategy to reconstitute the peripheral T cell pool in select congenital and acquired immune deficiency syndromes.

HLA class I polymorphism: structure and function and still questions.

The HLA-A, HLA-B and HLA-C molecules have turned out to be highly polymorphic and functionally complex. They not only serve as peptide receptors, but also interact with beta 2-microglobulin, an alpha beta T cell receptor, CD8 and NK inhibitory molecules, all at different sites. The fact that more than 300 class I alleles have now been defined prompted us to ask the question of where polymorphism really occurs in a class I molecule. We have used a database of 275 HLA-A, HLA-B and HLA-C alleles to illustrate how extensive the polymorphism is. The data is presented here for comparison of alleles and allele families and to facilitate studies of class I structure and function.

Extended HLA profile of an inbred isolate: the Schmiedeleut Hutterites of South Dakota.

HLA-A, -B, -C, -DR, and -DQ typings of the Schmiedeleut Hutterites of South Dakota were collected as part of an ongoing genetic-epidemiologic study of HLA and fertility. A total of 1,082 individuals, including 852 married adults representative of the reproductive population of this isolate, were characterized for five-locus HLA haplotypes. HLA-A1, A2, A3, and A24 accounted for 75% of observed HLA-A alleles and HLA-B27, B35, B51, and B62 accounted for 55% of observed HLA-B alleles. S-leut Hutterites are derived from 68 or fewer ancestors. However, only 48 ancestral HLA haplotypes were observed and nine of these accounted for over 52% of the observed haplotypes. Measures of two-locus linkage disequilibrium derived from these haplotypes indicated that one-third to half of the observed HLA-A/B, B/DR, and A/DR allele combinations exhibited highly statistically significant linkage disequilibrium. Allele and haplotype frequencies did not differ between males and females. Recombination rates of 0.004% and 0.005% between HLA-A and -C and between HLA-B and -DR, respectively, were observed. This HLA profile points out a paucity of HLA alleles and haplotypes in this population and marked linkage disequilibrium among the HLA alleles that are present.

Meta-analysis reveals association between most common class II haplotype in full-heritage Native Americans and rheumatoid arthritis.

The association of RA with the alleles at the HLA system was tested among Pima and Tohono O'odham Indians (Pimans) of the Gila River Indian Community of Arizona. Serologic class I (HLA-A, -B, and -C) alleles were typed in 51 individuals with RA and in 302 without RA. Serologic class II (HLA-DR, DQ; DR52 DR53) alleles were typed in a subset of 47 with RA and 147 without RA. Molecular subtypes of DR3X6, DRB1*1402, and *1406 were determined in 29 individuals, 16 with RA and 13 without RA. Among the cases with RA, 46 of 47 had the serologic antigen HLA-DR3X6, as did 140 of 147 of those without the disease. However, this association was not statistically significant because of the high prevalence of the antigen in the controls. Data from Pimans were analyzed with similar results from the Tlingit and Yakima Indians. A meta-analysis employing the Mantel-Haenszel procedure, stratified by tribe, revealed a statistically significant association between the most common haplotype, DRB1*1402 DQA1*0501 DQB1*0301 DRB3*0101, and RA (summary odds ratio = 2.63, 95% confidence interval = 1.08, 6.46). There was also a statistically significant difference in the genotype distributions of one class I locus, HLA-C, between those with and without RA (chi 2 = 12.4, 5 df; p = 0.03). It is concluded that the association with the most common class II haplotype in full-heritage Native Americans might help explain their high prevalence of RA.

Analysis of HLA haplotype segregation in the Schmiedeleut Hutterites of South Dakota.

HLA data from 1,085 South Dakotan Schmiedeleut Hutterites were examined for evidence of nonrandom transmission of haplotypes. The inheritance of haplotypes was viewed as a series of genetic contests between competing pairs of parental haplotypes using a maximum likelihood approach first put forward by Carlisle and Woodbury. Haplotype transmission probabilities were expressed in terms of weights, and the null hypothesis that the inheritance pattern was a random stochastic process, equivalent to the equality of the weights, was tested via the likelihood ratio. A total of 1,517 competitions was subjected to analysis, first globally using all data, and then for particular haplotypes of interest. Transmission of haplotype observed to compete with only a single other haplotype was also assessed using an exact procedure. No evidence of preferential transmission of HLA haplotypes was found. These results do not rule out transmission biases that may arise because of selection against homozygotes, mechanisms specifically affecting particular alleles or haplotypes not considered in the present study, or biases arising from maternal-fetal interactions.

Level of human TCRBV3S1 (V beta 3) expression correlates with allelic polymorphism in the spacer region of the recombination signal sequence.

One of the causes of variations in the expressed human T cell receptor (TCR) BV (V beta) repertoire is genetic variation in the germline DNA. Herein evidence is provided that allelic polymorphism may affect recombination frequency for a specific V gene. Two alleles of the TCR BV3 differ only at a single nucleotide position (C/T) within the 23-bp spacer region of the recombination signal sequence. These alleles are associated with variable percentages of BV3 cells in the peripheral blood, as shown in families and in unrelated normal donors. Individuals homozygous for allele 2 have a mean of 8.1% BV3 cells, heterozygous individuals have a mean of 4.7% BV3 cells, and homozygotes for allele 1 have a mean of 1.2% BV3 cells in CD3+ CD4+ peripheral blood T cells. Since the correlation is tight in unrelated individuals and other genetic differences were not found in the vicinity of BV3, we suggest that the spacer region sequence itself modifies recombination efficiency. This allelic system provides an example of a novel mechanism by which cis-acting genetic elements may affect recombination in a natural in vivo system.

HLA: fertile territory for developmental genes?

The HLA gene complex is well known for several reasons, for example, for immunologic studies of peptide presentation, the remarkable polymorphism and the ensuing problems in transplantation, and the association of particular alleles and haplotypes with susceptibility to autoimmune and inflammatory diseases. But for nearly 20 years, there have also been sporadic reports of HLA associations with neural tube defects, spontaneous abortion and infertility, and observations of transmission distortion and deficits of homozygotes. Part of the interest in these reports is because of the identification of developmental genes in or near the murine MHC that affect embryonic and germ cell differentiation. The first part of this review discusses current gene mapping of murine chromosome 17 and human chromosome 6, in light of the spectrum of new genes that have been identified and the colinearity of genes in the two MHCs. The second part scrutinizes published data on HLA associations.

A universally applicable diagnostic approach to filarial and other infections.

Antibody-based assays for the diagnosis of filarial and other infections cannot reliably distinguish between past and current infection, nor can they be used to assess the efficacy of chemotherapy. In this article, Thomas Nutmon, Peter Zimmerman, Joseph Kubofcik and Donna Kostyu discuss how the detection of parasite-specific PCR products using on ELISA-based assay may overcome these problems.

Rapid HLA-DR oligotyping by an enzyme-linked immunosorbent assay performed in microtiter trays.

A simple, sensitive ELISA that is performed in 96-well microtiter plates and that requires less than 90 minutes to complete was developed for HLA-DRB oligotyping. The second exon of HLA-DRB1 was amplified using an unlabeled forward primer and a biotinylated reverse primer and the PCR product was immobilized in avidin-coated wells. Subsequent treatment included exposure to 0.4 N NaOH to remove the nonbiotinylated sense strand, addition of a fluorescein-labeled oligonucleotide probe, one or more 5-minute stringency washes, addition of an alkaline-phosphatase-labeled anti-FL FAB, and then alkaline-phosphatase substrate and amplifier. An intense red-violet color developed within 15 minutes in positive wells and could be quantitated by OD readings at 490-495 nm. To control for stringency and to establish threshold OD values for positive reactions, biotin-labeled antisense oligos that were complementary to the probe or that differed by one or more bases were immobilized in wells in place of PCR products. The assay was sensitive to < 0.05 pmol (approximately 4 ng)/well and required only standard incubators and waterbaths and an optional microplate reader. All reagents were commercially available. The method should facilitate oligotyping of both class I and class II alleles and is adaptable for analysis of other polymorphic gene products.

Deficit of HLA homozygotes in a Caucasian isolate.

Deficits of HLA-A, -B homozygotes observed many years ago in two inbred populations suggested negative selection against HLA homozygotes. To determine whether a similar deficiency would be observed in the S-leut Hutterites, a well-characterized Caucasian isolate of European ancestry, and to determine whether selection operated at the allele, locus, or haplotype level, observed and expected numbers of homozygotes were compared in 852 adult Hutterites. Deficits ranging from 11% to 24% were observed for all five loci examined (HLA-A, -B, -C, -DR, and -DQ). However, these deficits were secondary to, and almost completely accounted for by, a 64% loss of individuals homozygous for the haplotype. There was no evidence of deficits affecting only a single allele or locus. The data indicate strong negative selection against HLA homozygotes. This could be due, at least in part, to decreased fecundability among couples sharing HLA-DR. However, these data suggest that additional selective factors acting at the level of the haplotype also operate in this population.

Decreased fecundability in Hutterite couples sharing HLA-DR.

To study the effects of parental HLA sharing on pregnancy outcome, we initiated population-based studies in the Hutterites. We previously reported longer intervals from marriage to each birth among couples sharing HLA, particularly HLA-DR. In the present report, we present the results of a prospective, 5-year study of fecundability and fetal loss rates in this population. Between April 1986 and April 1991, 154 pregnancies were observed in 104 couples. The median number of months of unprotected intercourse to a positive pregnancy test was significantly longer among couples sharing HLA-DR who stopped nursing prior to the first menses as compared with couples not sharing HLA-DR who stopped nursing prior to the first menses (5.1 vs. 2.0 mo, respectively; P = .016). Fetal loss rates were increased among couples sharing HLA-B as compared with couples not sharing HLA-B (.23 vs. .12, respectively; P = .041, adjusted for age, gravidity, and kinship). These data suggest that our earlier observations of increased birth interval lengths among Hutterite couples sharing HLA were predominantly due to longer intervals until a clinical pregnancy among couples sharing HLA-DR and, to a lesser degree, were due to increased fetal loss rates among couples sharing HLA-B.

Induction of HLA-specific CTL to nonimmunogenic, heat-inactivated lymphocytes by interleukin 2.

Human lymphocytes that have been heat-inactivated (1 hr, 45 degrees C) were used as stimulator cells in a model system to study the requirements of allogeneic T cell activation in vitro. Cytotoxic T lymphocytes were not generated in either primary or secondary mixed lymphocyte cultures after exposure to heated stimulator cells. Successful reconstitution of cytolytic activity in primary cultures was achieved by the addition of rIL-2. Further, cytotoxic T cell lines could be maintained in culture for several weeks by stimulation with heated allogeneic cells and periodic addition of exogenous IL-2. The cytotoxic T cells generated in primary cultures or in the T cell lines were specific for the HLA class I antigens of the stimulating cells. Thus, the combination of heated cells and IL-2 stimulated antigen-specific cytotoxic cells, and not merely lymphokine-activated killers. Although IL-2 production appeared to be a crucial missing component of MLCs with heated lymphocytes, the addition of IL-1, a factor known to act as a second signal for stimulating IL-2 production, did not reconstitute cytolytic activity. These results indicate that (1) heat treatment does not appreciably affect class I structure; (2) HLA class I/T cell receptor interactions are intact, resulting in responsiveness to IL-2 but not IL-1; and (3) heating creates a defect that has a minimal effect on CTL precursor activation but does disrupt a T helper cell/stimulator cell interaction critical for IL-2 production.

Epitopes within the HLA-B5,B35 cross-reacting group.

The HLA-B5,B35 cross-reacting group is a large and serologically complex antigen family which includes the World Health Organization-recognized specificities HLA-B5,B51,Bw52,B35,Bw53,B18,Bw70,Bw71, and Bw72. In addition, several variants of antigens in this cross-reacting group have been described in the past but have not yet gained official recognition. A genetic basis for the complexity and the protein and molecular bases of this highly cross-reactive and polymorphic cross-reacting group have yet to be established. The potential contributions of shared amino acid sequences, the occurrence of multiple epitopes on a single HLA-B molecule, and the presence of new HLA-C antigens have been difficult to resolve. To address this issue, we have carefully examined the serologic reactions of more than 900 allo- and monoclonal antibodies (Tenth International Workshop, Third Asia-Oceanic Workshop, and local reagents) versus lymphocytes from 92 individuals of diverse ethnic origin (North American Caucasians, North American blacks, Amerindians, Middle Eastern Caucasians), 84 of whom were informative for the HLA-B5,B35 cross-reacting group and related antigens. Our results demonstrate that the HLA-B5,B35 gene products share different combinations of distinct epitopes. We have constructed a model for the evolution of this cross-reacting group by assigning polarity to distinct diversification steps utilizing principles of maximum parsimony.