Caractérisation moléculaire et phylogénétique de l’enveloppe du VIH-1 transmis/fondateur

Overcoming HIV infection effectively requires further refinement of antiretroviral therapy as well as intensifying prevention strategies to stop the spread of the disease. It involves the capacity to develop a preventive or therapeutic broad-spectrum vaccine. Reaching this goal requires a better understanding of the acute and early events mediated by HIV founder viruses’ envelope glycoproteins during primary infection. While efforts of the scientific community are focused on finding a preventive vaccine, it is also necessary to limit the rate of new infections in the population. This thesis contributes to the efforts focusing on HIV prevention by studying the transmission dynamics of disease transmission and progression. The first part of my work is detailed in Chapter III and presented in a manuscript published in the journal “PLOS ONE” bears the title: « HIV-1 envelope sequence-based measures for identifying recent infections » (Article 1). The objective of this study was to evaluate and determine which molecular method, among envelope genetic diversity measures, can qualify the current status of HIV-1 infection. Four genetic diversity measures of short segments of HIV-1 envelope sequences were evaluated for their efficacy to characterize infection recency. They included i) the percent complexity, ii) the percent of diversity, iii) the number of haplotypes and iv), the Shannon entropy. We have identified Shannon entropy as the best diversity measure which can effectively predict the recency of HIV-1 infection when associated to three short segments (less than 100 base pairs) of the HIV-1 envelope gp120 C2 segments 1 and 3 and the V3 loop as envelopes sequenced based diversity measure (s) and segments. The performance index of Shannon entropy for these 3 segments: gp120 C2 segment 1; C2 segment 3; V3 loop was 0,806, 0, 0805, and 0,812 respectively. It could serve as a molecular biomarker for identifying new infections in newly diagnosed HIV-1-positive patients. The ability to differentiate recent infections from established (chronic) infections is important for public health. In fact, newly infected people are the most infectious and able to spread the disease quickly in the population. Since these people are usually unaware of their HIV status, they may engage in risky behaviours. This population generally has a high viral load with a relatively high-replicating capacity (fitness) that accelerates HIV-1 forward transmission. Being able to identify these individuals as soon as possible could limit and prevent new transmissions. Our work demonstrated that Shannon entropy measuring of these nucleic sequences can identify new infections.  The second part of my work is reported in Chapter III and published in the journal « AIDS Research and Human Retroviruses » entitled: « A short-term assessment of HIV-1 transmission dynamics among newly diagnosed individuals using envelope sequence-based phylogenetic clustering » (Article 2). This study consists of identifying the phylogenetic links or clusters that exist between the viral sequences of the HIV-1 envelope of newly diagnosed patients in Québec. The formation of clusters between the viral sequences of several individuals could reflect the relationship of transmitted viruses between individuals who might share characteristics or commons risk factors. The results of this study identified 15 minor transmission clusters (2-5 individuals per cluster) between study participants. We also assessed and compared the ability of a fragment of a larger nucleotide sequence of the HIV-1 envelope to that of the much shorter V3 loop to independently predict and identify individuals that might be included in a transmission cluster. They demonstrated a moderate agreement based on the kappa coefficient (k= 0.59) between the two fragments of the viral envelope used. The use of the V3 loop sequences indeed confirmed 66.70% (10/15) of the identified clusters with a longer fragment of the HIV-1 envelope sequence. The number of infected individuals accessing genotyping tests that use V3 sequence to determine viral tropism in routine clinical practice is globally limited. However, the sequences of the viral envelope and particularly those of the V3 loop could easily be integrated into a public health program where they could be available in addition to those of the pol genes (available in clinical laboratories for antiretroviral resistance testing). We also included the characteristics of the subjects (sociodemographic, clinical and risk factors) in a logistic regression analysis model with cluster formation as an outcome). The results of the analysis demonstrate that in the study population, the average age (<38.8 versus>38.8 years) and the viral subtype (subtype B versus non-B) are two factors significantly associated with HIV transmission clustering among participants. Underlying HIV-1 transmission chains among HIV-1+ individuals in Québec and identifying the common risk factors should help the efforts to implement better prevention strategies in the target groups.  The results of the third and last part of this work reported in this thesis (Chapter III, Article 3), were published in « VIRUSES » journal entitled: « HIV-1 Envelope Glycoprotein Amino Acids Signatures Associated with Clade B Transmitted/Founder and Recent Viruses ». It consists in identifying genetic signatures in the viral envelope sequences of the first HIV-1 variants called Transmitted/Founder (TF) viruses selected during the acute stage of infection. These variants can establish a productive infection at the time of infection in the new host. They are indeed selected among a multitude of viral quasi-species exposed during virus transmission. They may possess molecular and phenotypic properties that may govern their functions of the successful establishment of infection. Identifying such early HIV-1 genetic signatures may inform the design of novel immunologic or chemical inhibitors and serve as a benchmark for designing an effective HIV-1 vaccine. This study identified a few genetic signatures among HIV-1 TF viruses’ envelope amino acid sequences. One of these signatures consisting of a mutation/substitution of an arginine (R) by an isoleucine (I) associated with TF virus seems to be particularly important. This mutation is located at position 841 (R841I) specifically in the cytoplasmic domain of gp41 in segment 1 of lentivirus lytic peptides (LLP-1). The isoleucine is selected at more than 33% by TF viruses compared to chronic infection viruses (9%). The difference of selected amino acid between the two types of infectious viruses is statistically significant, OR = 0.2, 95% CI [0.09, 0.44], p = 0.00001. The cytoplasmic domain of GP41 and specifically the LLP-1 is strongly implicated in viral replication by mediating intracellular trafficking and the envelope incorporation into virions. A mutation in this area may suggest a molecular mechanism (polymorphism) contributing to HIV-1 transmission and/or viral escape from immune response.

Keyphrases: HIV, Recent infection, Envelope genetic signatures, Transmission clusters, Phylogenetic analysis