CTL epitope distribution patterns in the Gag and Nef proteins of HIV-1 from subtype A infected subjects in Kenya: use of multiple peptide sets increases the detectable breadth of the CTL response.

BACKGROUND: Subtype A is a major strain in the HIV-1 pandemic in eastern Europe, central Asia and in certain regions of east Africa, notably in rural Kenya. While considerable effort has been focused upon mapping and defining immunodominant CTL epitopes in HIV-1 subtype B and subtype C infections, few epitope mapping studies have focused upon subtype A. RESULTS: We have used the IFN-gamma ELIspot assay and overlapping peptide pools to show that the pattern of CTL recognition of the Gag and Nef proteins in subtype A infection is similar to that seen in subtypes B and C. The p17 and p24 proteins of Gag and the central conserved region of Nef were targeted by CTL from HIV-1-infected Kenyans. Several epitope/HLA associations commonly seen in subtype B and C infection were also observed in subtype A infections. Notably, an immunodominant HLA-C restricted epitope (Gag 296-304; YL9) was observed, with 8/9 HLA-CW0304 subjects responding to this epitope. Screening the cohort with peptide sets representing subtypes A, C and D (the three most prevalent HIV-1 subtypes in east Africa), revealed that peptide sets based upon an homologous subtype (either isolate or consensus) only marginally improved the capacity to detect CTL responses. While the different peptide sets detected a similar number of responses (particularly in the Gag protein), each set was capable of detecting unique responses not identified with the other peptide sets. CONCLUSION: Hence, screening with multiple peptide sets representing different sequences, and by extension different epitope variants, can increase the detectable breadth of the HIV-1-specific CTL response. Interpreting the true extent of cross-reactivity may be hampered by the use of 15-mer peptides at a single concentration and a lack of knowledge of the sequence that primed any given CTL response. Therefore, reagent choice and knowledge of the exact sequences that prime CTL responses will be important factors in experimentally defining cross-reactive CTL responses and their role in HIV-1 disease pathogenesis and validating vaccines aimed at generating broadly cross-reactive CTL responses.

Circulating and unique recombinant forms of HIV type 1 containing subsubtype A2.

HIV-1 strains containing subsubtype A2 are relatively rare in the pandemic but have been repeatedly identified in Kenya, where candidate vaccines based in part on subtype A, but not A2 strains, may be evaluated. Among the most recent is CRF16_A2D, a circulating recombinant form (CRF) whose prototypes are complete or partial HIV-1 sequences from Kenya, Korea, and Argentina. Using samples from blood bank discards in Kenya and complete genome sequencing, this report further documents CRF16_A2D and related recombinants and identifies a second CRF, CRF21_A2D. The two A2-containing CRFs, and two recombinants related to CRF16_A2D, share common structural elements but appear to have been independently derived. Concerted selection may have influenced the emergence and spread of certain A2-containing strains in Kenya. The second complete subtype C sequence from Kenya is also reported here. Monitoring of A2-containing recombinants and subtype C strains, both relatively rare in Kenya, may be informative in the course of cohort development and evaluation of candidate vaccines.

RhC Phenotyping, Adsorption/Elution Test, and SSP-PCR: The Combined Test for D-Elute Phenotype Screening in Thai RhD-Negative Blood Donors.

The Rhesus (Rh) blood group is the most polymorphic human blood group and it is clinically significant in transfusion medicine. Especially, D antigen is the most important and highly immunogenic antigen. Due to anti-D, it is the cause of the hemolytic disease of the newborn and transfusion reaction. About 0.1%-0.5% of Asian people are RhD-negative, whereas in the Thai population, the RhD-negative blood type only occurs in 0.3%. Approximately 10%-30% of RhD-negative in Eastern Asian people actually were D-elute (DEL) phenotype, the very weak D antigen that cannot be detected by indirect antiglobulin test (IAT). There are many reports about anti-D immunization in RhD-negative recipients through the transfusion of red blood cells from individuals with DEL phenotype. D-elute phenotype screening in Thai RhD-negative blood donors was studied to distinguish true RhD-negative from DEL phenotype. A total of 254 Thai serologically RhD-negative blood donors were tested for RhCE phenotypes and anti-D adsorption/elution test. In addition, RhC(+) samples were tested for RHD 1227A allele by SSP-PCR technique. The RhD-negative phenotype samples consisted of 131 ccee, 4 ccEe, 1 ccEE, 101 Ccee, 16 CCee, and 1 CcEe. The 42 Ccee and 8 CCee phenotype samples were typed as DEL phenotype and 96% of DEL samples were positive for RHD 1227A allele. The incidence of RhC(+) was 46.4%, and 48 of the 118 RhC(+) samples were positive for both anti-D adsorption/elution test and SSP-PCR technique for RHD 1227A allele. The sensitivity and specificity were 96% and 100%, respectively, for RHD 1227A detection as compared with the adsorption/elution test. In conclusion, RhC(+) phenotype can combine with anti-D adsorption/elution test and RHD 1227A allele SSP-PCR technique for distinguishing true RhD-negative from DEL phenotype.