HIV-1 Antisense protein (ASP) is an antisense open reading frame encoding for a highly hydrophobic protein of 20 kDa encompassing Env at the junction of gp120-gp41. Although initially suggested in 1988, existence of ASP remains controversial and its function has yet to be defined. A previous study shows that sera from HIV+ individuals can recognize ASP, suggesting actual production of this protein during infection. In these experiments however, the same sera were used for both immunoprecipitations and immunoblots. This, together with the fact that defective ribosome products with immune response have been reported for HIV-1, may have allowed for cross-reactivity leading to false positives. In this study we report the detection of anti-ASP antibodies in sera of HIV patients by ELISA assay using a panel of overlapping synthetic peptides spanning the whole length of the protein.
ASP-specific antibodies in sera of 14 HIV+ individuals and 9 healthy donors were determined by ELISA using 7 overlapping peptides spanning the whole ASP. 96-well flat-bottom plates were coated overnight with the synthetic peptides (1 peptide/well). Sera were diluted 1:200 prior to screening. Goat anti-human AP-conjugated secondary antibodies were diluted 1:2000. ELISA cut-off was calculated by adding 3 SDs to the mean OD from antigen-negative wells (healthy donors).
Of the 14 patients analyzed, 5 (35.7%) had a positive ELISA result against each of the seven peptides tested (p<0.05) (Figure 1). Interestingly, the peptides characterized by the lowest OD values were P58, corresponding in the sense orientation to gp41, and P28 and P43, who corresponding to Env hypervariable regions V4 and V5. Two healthy donors were found to have OD values slightly above the cut-off, probably due to the low number of samples tested.
Our data show that anti-ASP antibodies are present in sera from HIV+ individuals. The use of overlapping fragments covering the entire length of the protein indicates that the antibody response is triggered by the whole protein and not by incomplete ribosome products. In addition, peptides derived from different regions of the protein appear to be more or less immunogenic depending on the Env region present in the opposite orientation. Unfortunately no clinical information is available for the patients tested. We are currently planning a large scale testing of sera from HIV-infected individuals before and after seroconversion and/or undergoing different therapeutic regimens.