We have identified HLA-DRB1*11 as a candidate protective allele against trichiasis using DNA from buccal scrape samples obtained under harsh field conditions. Although this methodology has been used by others, to our knowledge it has not been adopted for trachoma field studies under the adverse field conditions found in Tanzanian villages.
This methodology was perfectly suitable for low-resolution HLA typing. However, we were unable to identify the entire HLA allele, likely because of the small amount of starting material in combination with some degree of sample degradation in the field. This resulted in our inability to generate enough DNA for the larger amplicons of HLA-B and -DQ for high-resolution allelic genotyping (4-digit alleles). The repeated attempts to use sequence-specific primer (SSP) and sequence-based typing (SBT) were not successful in amplifying the pertinent exons required for high-resolution genotyping of HLA-DRB*11. Although the reported statistical association in this study was based on low- to medium-resolution HLA typing, it could point to a trend for a legitimate genetic association. The evolution of typing methodologies from serologic to molecular methods confirms this trend. Indeed, many HLA-associated diseases were first reported at the low-resolution level and then confirmed at the allelic level.
We were unable to identify statistically significant HLA alleles that distinguished persistently infected children with severe disease from their control siblings who shared the same environmental exposure, perhaps in part because of the small sample size. More important, the data suggest that some of the sibling relationships might have been half-siblings or possibly non-siblings, which would invalidate using an analysis of sibling pairs discordant for the severe trachoma phenotype. The practice of polygamy and intervillage migration by the adults is prominent in this area of Tanzania. Affected sibling pair analysis has been used to determine the contribution of HLA class I and class II loci to the development of cervical cancer and human papillomavirus infection.27
Sibling pair analysis could be useful in HLA and linkage studies for other immune response genes in trachoma with carefully characterized families and with suitable sample size. Other considerations were that some participants in this study lived in villages that had been part of the initial azithromycin treatment trial, but this did not appear to influence the results.28
Acquisition of blood samples from the study population would allow us to go forward with identification of the entire candidate allele and to proceed with isolation of restricted CD4+
T cells. It is essential to perform high-resolution typing of HLA-DRB1*11 to validate the association of this allele with the lack of trichiasis. Preliminary data suggest that CD4+
T cells may protect against repeated bouts of infection leading to trichiasis. Additionally, we would be able to further our understanding of the role of linkage disequilibrium (LD) between particular DR-DQ alleles and innate immune markers in LD with HLA-B in ongoing studies. To our knowledge, there have been no studies aimed at identifying anti–C. trachomatis
T cells that are restricted by HLA-DRB*11 in trachoma. The few studies that have been conducted in trachoma have focused on HLA class I–restricted CD8+
effector cells for the outer membrane protein.14,16,18
Interestingly, one study demonstrated global inhibition of CD8+
T-cell activation in mice during primary and secondary chlamydia infection.29
In our study, we found that HLA-B*07 and HLA-B*08 were associated with trichiasis and that HLA-B*14 was associated with inflammatory follicular disease. As mentioned, it would be necessary to identify the complete allele before attempting to elucidate the contribution of the associated class I–restricted CD8+
cells to chlamydia pathogenesis.
The association of DRB1*11 with protection has not been found in other chlamydia infection association studies. Recurrent chlamydia genital and tubal factor infertility and blinding trachoma were associated with a variety of class II alleles.30,31
DRB1*11 was found to be associated with protective effects in other infections involving intracellular pathogens. For example, DRB1*11-DQB1*03 in LD was found more often in controls than in those with tuberculosis (TB), suggesting a link to TB resistance.32
DRB*11 has figured in an association with protection in the mild, restricted form of paracoccidiomycosis and mild liver damage in hepatitis C infection.33,34
Persons carrying HLA-DR11 express HLA-DR 52 and are in LD with HLA-DQ7 serotypes. The DQ7 serotype corresponds to the allotype DQB1*03, which includes more than 20 alleles. When DR11 is analyzed as part of its linked DQB1*03 allotype, the DRB1*1102-DQA1*0505-DQB1*0301 haplotype is associated with hepatitis B virus persistence.35
One of our goals was to compare HLA frequencies in several trachoma endemic regions with those obtained in the present Tanzanian study. There is a significant difference between cases/10,000 endemic population in West (1002) and East Africa (1031) compared with North (514) and South Africa (217) (WHO Global Health Atlas). This is because of the huge burden of severe disease in Ethiopia, Tanzania, and Burkina Faso. Tanzania is considered a mesoendemic region, with the TI/TF prevalence 5% to 40% in those younger than 6 years of age in 67 villages of 2500 to 10,000 inhabitants (according to the institutional review board of the Tanzania National Institute for Medical Research; Bobo L, personal communication, 2008). Gambia is considered hypoendemic because of a prevalence TI/TF of less than 10% in clusters of those younger than 10 years in 114 areas of 600 to 800 inhabitants (according to the institutional review board of the Joint Gambia Governor’s Medical Research Council Ethics; Bobo L, personal communication, 2008). On the other hand, Ethiopia is described as hyperendemic with a TI/TF greater than 40% in those younger than 10 years from 200 villages of 400 inhabitants, and trichiasis occurs at an earlier age (according to the institutional review board of the University of Gondor, Ethiopian Science and Technology; Bobo L, personal communication, 2008). It is unclear how the endemicity differences in these countries would contribute to the evolution of protective or pathogenic HLA alleles.
It is believed that evolving pathogens mainly evade presentation by the most common major histocompatibility complex (MHC) alleles in the host population by providing selective pressure for a large variety of rare MHC alleles.36-38
However, the more frequent allele in the present study, DRB1*11, was associated with lack of trichiasis and was significantly more represented in Tanzania (32.1%) than in the rest of East Africa (17.5%), West Africa (12%), and North Africa (17.2%) (dbMHC MHC database and Immunology/Histocompatibility Working group; www.ncbi.nlm.nih.gov/gv/mhc/ihwg.cgi
). Unfortunately, at present there are sparse data on HLA frequencies or linkage disequilibrium for infectious disease associations for African countries. This makes evaluation of the dynamics of chlamydia-MHC polymorphism in trachoma and how it would affect vaccine efficacy difficult across African countries.
In C. trachomatis
, recombination occurs within the immunodominant ompA
gene; there is little information on the mutability of other immunoaccessible chlamydia proteins.39,40
Pathogen-MHC coevolution is a dynamic process, but it is unknown whether HLA results from one geographic region can be generalized to another and how this process would affect vaccine efficacy. Additionally, it is unclear what the effects of coinfection with other pathogens would have on this process.
Another drawback of our study was that we did not identify who was coinfected with important pathogens, such as HIV-1, HIV-2, Mycobacterium tuberculosis, or Plasmodium species. In addition, the study participants were not assessed for malnutrition, lymphopenia, or vitamin A status, factors that are important in immune and ocular health. Therefore, it is possible that these variables might have biased the results for weaker HLA associations.
Recently, the coexistence of Chlamydiaceae
species in trachoma-endemic regions in Nepal has been identified, as has their association with clinical severity and their involvement in the immune response to Hsp60.41
Similarly, previous exposure to Chlamydia pneumoniae
, a common respiratory pathogen, was hypothesized to prime a Th1 T-cell response to certain C. trachomatis
antigens in the context of HLA-DRB1*0401.42
If this is true in other trachoma-endemic populations, such as in our Tanzanian study population, it will be important to determine which species should be taken into consideration for vaccine development using HLA-DRB1*11.