Received Date: April 25, 2017; Accepted Date: May 10, 2017; Published Date: May 15, 2017
Citation: Meseret Y, Mengistu Y, Howe RC, Messele T, Wolday D (2017) The HLA Profile of Ethiopian Discordant and Concordant Couples: In Comparison with AIDS Patients. J Mol Immunol 2:110.
Copyright: © 2017 Meseret Y, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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In order to analyze the effect of host HLA types on resistance or susceptibility to HIV infection and to study the HLA profile Of Ethiopian HIV/AIDS subjects DNA based HLA typing was carried out on 239 discordant, concordant couples and full blown HIV AIDS subjects. Our study showed that there was a clear difference between discordant positives and discordant negative couples, and discordant positives and concordant couples in their genetic profiles. Ethiopian AIDS patients were different from Ethiopian concordant couples in their very significant to significant association with HLA-A*29, *18, and *41; HLA-B*0705, *1517, *4101, *5001, *7301 and *18; HLA-C*0501, *0701, and *0740. AIDS patients were also very significantly different from discordant positives in their associations with HLA-A*68, HLA-B*39 and HLA- DR*11. AIDS patient were also different from discordant negatives in their very highly significant to highly significant association with HLA-*0801, *1817, *352001 and *4901; HLA-C*7 and HLADR* 40301. Discordant positive subjects were found to be more heterozygous at all loci (HLA-A, B, C and HLA-DR) when compared with concordant couples and HIV/AIDS subjects.
HLA Profile; HIV/AIDS; Ethiopia; Genotype; Discordant couples
Discordant couples (couples with different HIV-serostatus) and concordant couples (couples with similar HIV positive serostatus) are good models for comparison of HIV genetic studies. In this study, we compared HLA profile of these two groups with patients showing full blown or typical AIDS characteristic. Concordant couples and discordant positive subjects were HIV positive but did not show typical AIDS characteristics.
CD8+ cytotoxic T lymphocytes (CTL), which are the major antiviral adaptations, are also known against retroviral infections [1- 4]. If CTLs are important in controlling HIV infection, HLA class I type should play a major role in determining disease progression. HLA class I molecules have a direct and special connection to viruses. This is particularly true in that they play a central role in the task of alerting CTLs to cells that have been breached by virus . An individual’s HLA genotype is also predictive of whether HIV is likely to kill quickly or slowly. For example, there is a direct association between HLA class I types and rates of HIV disease progression , as was shown that heterozygosity for HLA-A, B, and C is known in delaying onset of AIDS. Studies with HIV infected long term non-progressors (LTNP) also showed increased frequency of specific HLA class I [6-12]. These and other studies indicated that there is a genetic background behind resistance and/or susceptibility to HIV infection. Even heterozygosity versus homozygosity of these HLA types are known in determining susceptibility and /or resistance to HIV [7,12].
Limited studies have been conducted to determine HLA class I allelic frequency in Ethiopia. One amongst this was a study carried out on 50 HIV- positive and HIV-negative subjects . Few studies, if at all were carried out, investigated HIV discordant and concordant couples separately and looked at their HIV profiles by comparing them with full-blown AIDS profile. Thus, the objective(s) of this study is to investigate the profile of HLA subtypes in discordant negatives (DSCN), discordant positive (DSCP), concordant couples (CONC) and full blown AIDS patients. This is particularly important because the study of HLA polymorphism facilitate diagnosis or prognosis of the potential AIDS patients and elucidates how susceptibility and resistance to HIV infection is related to genes.
The study was carried out on 239 HIV discordant, concordant and full blown AIDS subjects from January 2010-January 2012 in five Administrative Regions and Addis Ababa, the capital city of Ethiopia.
The study design was a single spot prospective cross sectional study involving comparisons of HLA subtypes in discordant and concordant couples and full blown AIDS patients.
Most of the subjects were counselled, tested and registered as HIV discordant or concordant couples and were on follow up by the respective health institutions (health centers and hospitals). That is, they were identified, counselled, tested and registered as discordant or concordant couples by the nurses and doctors of the respective health centers and/or hospitals.
The study was conducted in accordance with the ethical principles stipulated in the last revised version of the Helsinki declaration, the operational guideline for ethical committees of Ethiopia. The study was conducted after obtaining the national ethical clearance from the then Ethiopian Science and Technology Commission (ESTC) and the now Science and Technology Minister and the institutional clearance from Ethiopian Health and Nutrition Institute (EHNRI) now Ethiopian Public Health Institute and Addis Ababa University (AAU). An official letter of cooperation attached with ethical clearance copy from EHNRI, AAU was written to regional health Bureaus, and a similar letter was written from regional health Bureaus to the respective health centers and hospitals.
Participation in the study was voluntary. Detailed information about the study was made available for all patients in their language. Only patients who gave informed consent were included in the study. The consent form was completed only after the patient had understood the points enumerated in the information sheet. All study participants were able to withdraw from the study at any point without any consequence to his/her care and clinical management.
Sample collection, transportation and analysis
After the patients were identified and their willingness to participate in the research was approved, patients were asked to give samples (blood). Blood was collected by trained and experienced nurses. Twenty milliliter whole blood was collected from each study subject in vacationer tubes in EDTA and transported to the laboratory on the same day it was collected for analysis. Blood samples were always collected at the same time starting early in the mornings from 8:00 AM to 11:30 AM and was analyzed within 24 h.
The blood sample was rejected if it was haemolysed, turbid or had not been stored and transported properly, didn’t carry appropriate label, and the container had leaked. Two samples were rejected on these bases. Laboratory analysis was carried out at EHNRI and Center for clinical Immunology and Biostatistics (CCIB) research institute attached to Murdoch University, Perth, Australia.
The collected data was entered and analyzed using SPSS version 13 software. Mean, median, mode and standard deviation were collected for many parameters in the study. Results were compared in discordant and concordant couples. When the comparisons involved two groups, non-parametric (Mann-Whitney U-test) method was used. But when comparisons were made between three groups or more groups, the level of significance (α) was adjusted using Boferroni corrections (α=0.033). This association between several parameters was determined using a multivariate regression analysis. Correlation coefficients were calculated by the spearman’s test.
Peripheral blood mononuclear cell isolation
Venous blood was collected from the study subjects in EDTA vacutainer tubes and plasma and blood cells were separated by centrifugation. The plasma was separated and stored at -80°C until further analysis was carried out. Using Buffy coat isolation mechanism PBMC was also isolated from the plasma separated blood. The remaining blood cells were diluted with PBS and layered over Ficoll- Hypaque. After density gradient centrifugation on Ficoll-Hypaque, PBMC was collected and viable frozen in liquid nitrogen until further analysis was carried out.
Sequence based HLA typing
HLA-typing was determined using PCR amplification of exons 2 and 3 of the genes HLA-A, HLA-B, HLA-C and HLA-DR followed by DNA sequencing methods. Briefly, genomic DNA was extracted from Buffy coats, whole blood or plasma manually with the QIAamp DNA Blood Mini Kit (Qiagen) or the Agencourt Genfind DNA extraction kit (Beckman Coulter) with a method adapted for the Biomek FX robotic workstation. Exons two and three were amplified from each HLA gene and products were then purified using Agencourt Ampure (Beckman Coulter), sequenced with Big Dye terminator v3.1 methods (Applied Biosystems) and then cleaned up with Agencourt Cleanseq (Beckman Coulter). Finally, sample data was collected on an ABI PRISM 3730 Genetic Analyzer. Applied Biosystems 3730 Data Collection software v5.0 was used to collect electropherograms which are then analyzed with Applied Biosystems Software v5.2 and Assign v220.127.116.11 (Conexio Genomics Pty Ltd). The Assign program compared collected sample data against a database of known HLA sequences and assigned alleles accordingly.
HLA genotyping has been performed by DNA sequencing of the exon 2-3 region of HLA class I and exon 2 region of HLA-DRB1. To determine HLA class I and II frequencies and to investigate if the differences in HLA types and frequencies were associated with resistance or susceptibility to HIV, DNA based (molecular) HLA typing was carried out and the results of 10 discordant negatives, 52 discordant positives, 49 concordant couples and 138 HIV/AIDS subjects HLA frequencies were obtained. Five different types of HLA-A1, 6 different types of HLA-A2, 7 different kinds of HLA-B, 5 different kinds of HLA-B2, 3 different kinds of HLA-C1 and HLA-C2, 3 different kinds of HLA-DR1 and 2 different kinds of HLA-DR2 were found in discordant negatives. In discordant positives 16 different kinds of HLA-A1, HLA-A2, HLA-B1, 13 different kinds of HLA-B2, 6 different kinds of HLA-C1, 5 different kinds of HLA-C2 and HLA-DR1 and 4 different kinds of HLA-DR2 were found. In a similar way, 12 different kinds of HLA-A1, 14 different kinds of HLA-A2, 24 different kinds of HLA-B1 and 16 different kinds of HLA-B2, 4 different kinds of HLA-C1 and C2, 3 different kinds of HLA-DR1 and 1 type of HLADR2 was found in concordant couples. The results for HIV/AIDS were: 25 different kinds of HLA-A1 and A2, 31 different kinds of HLA-B1, 28 different kinds of HLA-B2, 18 different kinds of HLA-C1 and HLADR2 and 17 different kinds of HLA-DR1. In almost all cases more different kinds of HLA-B1 and B2 followed by HLA-C1 and C2 were found among HLA class I types. Similar results were obtained for HLA class II.
HLA genotyping has been performed by DNA sequencing of the exon 2-3 region of HLA class I and exon 2 region of HLA-DRB1. To determine HLA class I and II frequencies and to investigate if the differences in HLA types and frequencies were associated with resistance or susceptibility to HIV, DNA based (molecular) HLA typing was carried out and the results of 10 discordant negatives, 52 discordant positives, 49 concordant couples and 138 HIV/AIDS subjects HLA frequencies were obtained. Five different types of HLA-A1, 6 different types of HLA-A2, 7 different kinds of HLA-B, 5 different kinds of HLA-B2, 3 different kinds of HLA-C1 and HLA-C2, 3 different kinds of HLA-DR1 and 2 different kinds of HLA-DR2 were found in discordant negatives ( Tables 1 and 2). In discordant positives 16 different kinds of HLA-A1, HLA-A2, HLA-B1, 13 different kinds of HLA-B2, 6 different kinds of HLA-C1, 5 different kinds of HLA-C2 and HLA-DR1 and 4 different kinds of HLA-DR2 were found. In a similar way, 12 different kinds of HLA-A1, 14 different kinds of HLA-A2, 24 different kinds of HLA-B1 and 16 different kinds of HLA-B2, 4 different kinds of HLA-C1 and C2, 3 different kinds of HLA-DR1 and 1 type of HLA-DR2 was found in concordant couples (Table 1). The result for HIV/AIDS was: 25 different kinds of HLA-A1 and A2, 31 different kinds of HLA-B1, 28 different kinds of HLA-B2, 18 different kinds of HLA-C1 and HLA-DR2 and 17 different kinds of HLA-DR1. In almost all cases more different kinds of HLA-B1 and B2 followed by HLA-C1 and C2 were found among HLA class I types. Similar results were obtained for HLA class II (Table 1).
Table 1: The different kinds of HLA class I and II types in discordant negatives, discordant positives, concordant couples and HIV/AIDS subjects.
(HLA-A1=HLA-A allele1, HLA-A2=HLA-A allele 2, HLAB1= HLA-B allele1, HLA-B2=HLA-B allele2, HLA-C1=HLA-C allele1, HLA-C2=HLA-C allele2, HLA-DR1=HLA-DR allele1, HLADR2= HLA-DR allele 2).
DSCN=Discordant negative, DSCP=Discordant positive, CONC=Concordant couples, AIDS=AIDS patients.
The HLA alleles were then pooled out to determine the frequency, proportions and X2 –based p-value to see the associations between the different groups. Frequencies and proportions were compared between AIDS, concordant couples, discordant negatives and discordant positives. The results were as (Table 2).
Table 2: Comparison of HLA-A, HLA-B, HLA-C and HLA-DR aggregate subtypes in AIDS, DSCP (discordant positive), DSCN (discordant negative), and CONC (concordant couples) subjects. HST (HLA subtypes).
For the subjects in which 20% of the expected count was less than 5, p-value was calculated by Fishers Exact Test method and the result was as shown (Table 3).
|HLA/subtype||AIDS vs. CONC||AIDS vs. DSCP||AIDS vs. DCSN||CONC vs. DSCN||CONC vs. DSCP||DSCN vs. DSCP|
Table 3: Aggregate HLA subtypes Fishers Exact Test calculated p-value of those in which 20% of cells have expected count less than 5 (A=p<.001(very highly significant), B=p<.01 (very highly significant), C=p<.05 ( significant) AIDS (subjects at AIDS stage, CONC (concordant couples), DSCP (discordant positive subjects), DSCN(discordant negative Subjects).
HLA-B*49 (p<0.01), HLA-A*68 (p<0.01), and HLA-B41 (p<0.001) (Table 4) were found strongly associated with AIDS patients when compared with all others. The strongest association was observed for HLA-B*41(p<0.001) in AIDS patients. When AIDS patients were compared with concordant couples, HLA-A*41 (p<0.001), HLA-A*18 (p<0.01), HLA-A*29 (p<0.05) were found to be significantly associated with AIDS patients. HLA-B*41 (p<0.001) and HLA-B*18 (p<0.01) were also found to be strongly associated with AIDS patients (Table 3).
Table 4: Proportions and X2 values (likelihood ratios and Pearson’s p-values) of HLA-A subtypes in AIDS, concordant (CONC), discordant negative (DSCN) and discordant positive (DSCP) study subjects. HST (HLA subtypes).
When AIDS patients were compared with discordant positive subjects, three HLA subtypes: HLA-A*68 (p<0.01), HLA-B*39 (p<0.01) and HLA-DR*11 (p<0.01) were found to be very strongly associated with AIDS subjects, showing that discordant positive subjects were different from AIDS subjects. When AIDS subjects were compared with discordant negatives, the only HLA type found to be associated with AIDS subjects was HLA-C*7 (p<0.01) (Table 3).
Comparisons of associations of HLA subtypes between discordant negative and discordant positive showed that three HLA subtypes were strongly associated with discordant negative subjects. These were HLA-B*39 (p<0.01), HLA-B*41 and HLA-DR*11 (p<0.05) (Table 4).
The subtypes which were found associated with resistance to HIV in other studies, were not observed in our study. The frequency and the prevalence of the different kind of HLA subtypes associated with different clinical status are summarized in the following figure (Figure 1).
To investigate the frequency and proportions of the different forms within the subtypes and their relationships with the different groups, the different forms of the subtypes were analyzed and the result was as shown Table 4 (HLA-A), Table 5 (HLA-B), Table 6 (HLA-C), and Table 7 (HLA-DR).