8th International AIDS Conference Amsterdam, Netherlands — July 19-24, 1992

Int Conf AIDS 1992 Jul 19-24; 8:We60 (abstract no. WeA 1083)
Cichutek K, Merget H, Norley S, Linde R, Kreutz W, Gahr M, Kurth R; Paul-Ehrlich-Institute, Langen, Germany.

OBJECTIVES: To analyse the quasispecies of human immunodeficiency virus type 1 (HIV-1) present in peripheral blood mononuclear cells (PBMC) of hemophiliacs probably infected from the same source.

METHODS: HIV-1 was isolated from PBMC by cocultivation with negative donor chord blood lymphocytes (CBL). Characterization of the env gene regions extending from variable region V3 to V4 was performed by nested PCR (polymerase chain reaction) amplification starting from PBMC or CBL DNA, molecular cloning and subsequent DNA sequencing of up to 20 subgenomic clones.

RESULTS: During treatment with one specific batch of blood clotting factor IX, a number of hemophilia B patients were infected in Germany with a clonal HIV-1 substrain belonging to the North-American/European group. The nucleotide sequences of cloned env gene regions between the variable V3-loop and variable V4 region analysed from cultured virus and from virus in the peripheral blood cell DNA of two different patients 5 months post infection and one of the patients 11 months post infection were shown to be highly homologous. Based on the assumption that the consensus sequence (termed HIV-1MBK) was identical with the genotype of the initially infecting virus we were able to construct phylogenetic trees of the quasispecies of both patients. True intermediate genotypes between input and multiply mutated genotypes were detected by 5 and 11 months post infection and genetic variation was shown to develop at a faster rate in one of the patients. Except for the HIV-1MBK genotype, the vast majority of all variants detected 5 months post infection in the blood of the other patient were replaced with new variants 11 months post infection. Variability was shown to reside in two small regions located 3' of the V3-loop and within the V4-region. Overlapping synthetic peptides corresponding to the entire sequenced region, including mutants, were produced and the antibody reactivity in patients sera tested by ELISA. Linear epitopes were found only within the V3 loop and fine mapping showed the sequence SINIGP to be the immunodominant epitope. The affinity of antibody to the only mutant found in this epitope (SINTGP) was reduced in comparison to the consensus.

CONCLUSION: This is the first report of the evolution of an HIV-1 quasispecies starting from a single and still detectable genotype.

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