X4 tropic multi-drug resistant quasi-species detected at the time of primary HIV-1 infection remain exclusive or at least dominant far from PHI

Abstract

Our objective was to analyze the evolution of resistance mutations (RM) and viral tropism of multi-drug-resistant (MDR) strains detected at primary HIV-1 infection (PHI). MDR HIV strain was defined as the presence of genotypic resistance to at least 1 antiretroviral of the 3 classes. Tropism determinations (CCR5 or CXCR4) were performed on baseline plasma HIV-RNA and/or PBMC-HIV-DNA samples, then during follow-up using population-based sequencing of V3 loop and phenotypic tests. Clonal analysis was performed at baseline for env, RT and protease genes, and for HIV-DNA env gene during follow-up. Five patients were eligible. At baseline, RT, protease and env clones from HIV-RNA and HIV-DNA were highly homogenous for each patient; genotypic tropism was R5 in 3 (A,B,C) and X4 in 2 patients (D,E). MDR strains persisted in HIV-DNA throughout follow-up in all patients. For patient A, tropism remained R5 with concordance between phenotypic and genotypic tests. Clonal analysis on Month (M) 78 HIV-DNA evidenced exclusively R5 (21/21) variants. In patient B, clonal analysis at M36 showed exclusively R5 variants (19/19) using both genotypic and phenotypic tests. In patient C, baseline tropism was R5 by genotypic test and R5/X4 by phenotypic test. An expansion of these X4 clones was evidenced by clonal analysis on M72 HIV-DNA (12/14 X4 and 2/14 R5 variants). In patient D, baseline tropism was X4 with concordance between both techniques and HIV-RNA and HIV-DNA remained X4-tropic up to M72, confirmed by the clonal analysis. Patient E harboured highly homogenous X4-using population at baseline; tropism was unchanged at M1 and M18. In all patients, the initial MDR population was highly homogenous initially, supporting the early expansion of a monoclonal population and its long-term persistence. X4-tropic variants present at baseline were still exclusive (patients D and E) or dominant (at least one time point, patient C) far from PHI.

Figure 1. Phylogenetic analysis, based on the neighbour-joining method, of clones of HIV-1 reverse transcriptase gene sequences (1A), protease gene sequences (1B) and envelope gene sequences (1C) from patients A–E at baseline, demonstrating highly homogenous circulating and archived variants in each patient.

Clones obtained from plasma HIV-1 RNA are in full squares and clones obtained from PBMC-HIV-1 DNA are in white circles. Resistance mutational pattern in reverse transcriptase gene and in protease gene is shown for each patient. Clones not harbouring all resistance mutations are indicated. HIV-1 tropism on Figure 1C is determined by genotypic test. Only bootstrap values ≥700 are shown. The reference sequence HXB2 was used as an outgroup. Genetic distance is indicated at the top of the figure, and represents the number of nucleotide substitutions per site. The numbers between brackets indicate the number of strictly identical sequences that segregate on the same branch.

Figure 2. Longitudinal phylogenetic analysis, based on the neighbour-joining method, of clones of HIV-1 envelope gene from patient A (2A), B (2B), C (2C) and E (2D).

Clones obtained from plasma HIV-1 RNA are in full squares and clones obtained from PBMC-HIV-1 DNA are in white circles. The most frequent amino-acid sequence of the V3-loop is indicated for each patient in a square. Amino-acid substitutions within the V3-loop are underlined and correspondent variants are indicated by an arrow for each patient. Only bootstrap values ≥700 are shown. The reference sequence HXB2 was used as an outgroup. Genetic distance is indicated at the top of the figure, and represents the number of nucleotide substitutions per site. The numbers between brackets indicate the number of strictly identical sequences that segregate on the same branch.