Viral parameters influencing clinical long-term non progression in HIV-1 infected subjects
Sammanfattning: A very small portion of the HIV-1 infected population is constituted by individuals, called long- term non progressors (LTNP), in whom after even a decade or more, no visible deterioration of the immune system is recognized and the infection remains therefore asymptomatic. The reasons for such "natural" prolonged well-being can be attributed to both the virus and the infected host and their understanding is likely to give important information for the development of an effective HIV- vaccine and for immune reconstitution therapy. The aim of the present thesis was to investigate and to evaluate parameters suspected to influence long-term non progression, and in specific the HIV-replication dynamic, the genetic "defects" in viral regulatory genes and the genetic susceptibility of the host. A multiple competitor PCR (mcPCR) assay was established, evaluated and finally used for quantifying HIV-1 DNA in blood cell samples from LTNP and from patients with advanced disease. A significantly higher FHV-1 DNA copy number was obtained in the latter subjects. Quantification of viral RNA and DNA loads was done in a longitudinal study, starting in 1993, in 20 HIV-infected subjects with 7 years of clinical non-progression. At study end in 1998, the 20 patients were classified as slow progressors (n=12) or LINP (n=8). Slow progressors displayed higher plasma HIV-1 RNA levels than LTNP already at inclusion, while HIV-1 DNA levels were comparable in the two groups. Slow progressors thereafter showed a continuously increasing RIV-1 RNA level, which was more pronounced than in LTNP. The majority of LTNP had a stable or a declining HIV- 1 DNA level. In a particular subject with almost undetectable plasma HIV-1 RNA and a remarkable progressive reduction of the HIV-1 DNA between 1994 and 1998, the evolution of HIV-1 env quasispecies heterogeneity in PBMC was analyzed. A very low heterogeneity was present in all the samples. The analysis of the quantification data, the phylogenetic distances and the amino acid conformation in the V3 indicated that the quasispecies present until 1996 had been substituted by a different one in 1998. Truncation or subtle mutations in HIV-1 long terminal repeat (LTR) and in the nef gene have been described in LTNP and suggested to be associated with an attenuated viral pathogenesis. We analyzed nef and LTR alleles in a large cohort of 21 LTNP and in a group of progressors. An untruncated intact nef open reading frame was observed as a major sequence in all LTNP. In the LTR, no specific mutations were discovered in LTNP, and none of the observed mutations was associated with in vivo HIV-1 RNA or DNA levels. To investigate if particular mutations in the nef are linked to LTNP, the amino acid sequences obtained from our cohort were compared with the previously reported sequences of 16 American LTNP and of 36 progressors. None of the substitutions in known functional sites of the Nef protein was linked to LTNP, but a valine/isoleucine at the variable position 1 1 was strongly associated with both European and American LTNP. A lower interpatient viral diversity was found in LTNP, but phylogenetic analysis did not showed any specific clustering of the LTNP sequences. The correlation between the presence of a deletion (A) in the human CCR5 gene, disease progression and HIV-1 specific immune responses was analyzed in 79 HIV-1 infected patients. The prevalence of the CCR5 allele was lower among patients with rapid progression as compared to patients with slow progression or to 25 HIV-1 uninfected blood donors. The antibody titers against gp41 and V3MN peptides in patients with a CCR5/CCR5 genotype were not significantly different from those in pair-matched CCR5/CCR5 controls. In conclusion, (1) LTNP express in general low and stable amounts of HIV-1 in plasma and stable, or even declining, cellular HIV-1 DNA levels; (2) the viral population in LTNP may exhibit a low heterogeneity; (3) defects in the nef and LTR are rare in LTNP and are not associated to a particular pattern of viral loads; (4) the heterozygous CCR5 mutation prolongs the AIDS-free interval, but is not associated to HIV-1 env antibody pattern nor is it a specific marker for LTNP.
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