The overall goal of this thesis was to identify HIV-1 Gag gene mutations associated to protease inhibitors (PI) resistance among non-B subtypes and the impact of viral diversity in the natural selection of these mutations. In particular, this thesis aimed to: i) Determine C-terminal Gag gene mutations selected under PI/ritonavir (PI/r) exposure and their covariations with protease major resistance mutations among HIV-1 non-B clades. ii) Evaluate HIV-1 Gag gene variability and drug resistance-associated mutations according to viral subtypes among drug-naïve individuals. iii) Determine HIV-1 Gag C-terminal genotypic profile among vertically infected adolescents failing PI/r regimen without major resistance mutations in protease gene. First, we characterized Gag mutations present in isolates from HIV-1 infected individuals treated with a PI/r-regimen (n = 143) and compared them with those obtained from individuals not treated with PI/r (ART-naïve [n = 101] or reverse transcriptase inhibitors (RTI) treated [n = 118]). The most frequent HIV-1 subtypes were CRF02_AG (54.69%), A (13.53%), D (6.35%) and G (4.69%). Eighteen Gag mutations showed a significantly higher prevalence in PI/r-treated isolates compared to ART-naïve (p < 0.05): Group 1 (prevalence < 1% in drugnaïve): L449F, D480N, L483Q, Y484P, T487V; group 2 (prevalence 1–5% in drug-naïve): S462L, I479G, I479K, D480E; group 3 (prevalence ≥ 5% in drug-naïve): P453L, E460A, R464G, S465F, V467E, Q474P, I479R, E482G, T487A. Five Gag mutations (L449F, P453L, D480E, S465F, Y484P) positively correlated (Phi ≥ 0.2, p < 0.05) with protease-resistance mutations. At PI/r-failure, no significant difference was observed between patients with and without these associated Gag mutations in term of viremia or CD4 count. This finding suggests that some Gag mutations show an increased frequency in patients failing PIs among HIV-1 nonB clades. Secondly, concerning the evaluation of HIV-1 Gag gene variability and drug resistanceassociated mutations according to viral subtypes among drug-naïve individuals, 2031 HIV-1 Gag sequences were retrieved from the Los Alamos HIV database and analyzed based on documented Gag mutations assoc ated to PI and Lenacapavir (LEN) resistance. Overall conservation analysis of the Gag gene revealed 255/500 (51.0%) amino acid positions were conserved (<5% variability) in the entire study population and p24 (capsid) protein presented the highest conservation rate (156/231; 67.5%). The prevalence of Gag mutations associated to PI/r resistance in vivo was as follows: Y79F (46.28%), R76K (39.73%), P453L (16.0%), K436E (13.24%), V128I (6.79%), T81A (5.76%), L449F (2.80%), I437T (1.23%), R452S (0.54%), A431V (0.44%) and K112E (0.09%). Importantly, Gag mutations associated to PI resistance were significantly higher in viral recombinant forms as compared to pure subtypes (72.3% [542/750] vs. 31.1% [398/1281], p<0.0001); the recombinant CRF01_AE had the highest drugresistance mutation prevalence (73.1% [326/446]). The overall prevalence of LEN resistance associated mutations was 0.6% (13/2031) and resistance mutations observed were: N74S (0.5%), M66I (0.05%), Q67H (0.05%). Interestingly, four Gag mutations associated to PI resistance (K436E, I437T, L449F and P453L) and Q67H associated to LEN resistance are significantly associated with a reduced binding affinity between T-lymphocyte cells’ epitopes and MHC molecules. Among antiretroviral-naïve patients, about half of amino acid positions within the Gag gene are conserved. Finally, higher rates of Gag mutations associated to PI resistance are found with recombinant viruses, especially the CRF01_AE. The low resistance to LEN (<1%) suggests a high effectiveness of LEN-based regimens. Some of these mutations are significantly associated with a reduced T-lymphocyte cells binding affinity to MHC, suggesting implications in HIV immune escape mechanism. Thirdly, we characterized HIV-1 Gag C-terminal genotypic profile among vertically infected adolescents (AVI) PI treatment without major resistance mutations in the protease gene in Cameroon. This was a cross sectional analysis among 23 HIV vertically infected adolescents in Cameroon failing PI/r-based therapy without PI major resistance. The mean duration of PI/rbased regimen was 27.6 months and ranged from 1 to 118 months. The mean viral load and CD4 count at time of measurement of drug resistance were 193246 copies/ml (range 2094– 733567) and 479 cells/mm3 (range 55–1168), respectively. Of the 23 AVI, fourteen adolescents (60.7%) had a CD4 count of <500 cells/mm3. Even in absence of major resistance mutations in the protease gene, protease mutations typical of non-B subtype were frequently found, as H69K (n=20, 89.9%), L89M (n=20, 86.9%), M36I (n=20, 86.9%), and I13V (n=18, 78.2%). In addition, PI mutations accessory drug resistance mutation (DRMS) polymorphisms (L10F, K20I, L33F) were also detected. The most prevalent Gag mutations were E460A (n=23, 100%), L449P (n=14, 94,4%), and R464G (n=15, 84.4%). Known HIV-1 drug resistance-associated Gag mutations K436R (n=8, 34,7%), L449P (n=14, 94,4%), P453L (n=2, 8,7%), and P453T (n=2, 8,7%) were found. Other Gag mutations known to be associated to PI/r exposure were also found Y441S (n= 11, 47.8%), P472S (n=11, 47.8%), Q474P (n=12, 52.2%), D480E (n=3, 13.0%) and E482G (n=9, 39.1%). In summary, several HIV-1 Gag mutations described as being associated to PI/r resistance and PI/r exposure have been found in these AVI on treatment failure in absence of protease major DRMS. Thus, this could support the involvement of HIV Gag gene mutations in PI/r treatment failure. In conclusion, the overall findings produced in this thesis demonstrated the implication of Gag mutations in protease inhibitors treatment failure among non-B subtypes and the hight natural selections of these mutations by viral recombinant forms. These results therefore support the implication of Gag gene mutations in PI/r treatment failure for a potential consideration in algorithms interpreting HIV drug resistance for clinical management.
L'obiettivo generale di questa tesi è stato di identificare le mutazioni del gene Gag dell'HIV-1 associate alla resistenza agli inibitori della proteasi tra i sottotipi non-B e l'impatto della diversità virale nella selezione naturale di queste mutazioni. In particolare, questa tesi si proponeva di: i) determinare le mutazioni C-terminali del gene Gag selezionate durante l'esposizione ai PI/r e le loro covariazioni con le mutazioni di resistenza maggiore alla proteasi tra i clades non-B dell'HIV-1. ii) Valutare la variabilità del gene Gag dell'HIV-1 e le mutazioni associate alla resistenza ai farmaci in base ai sottotipi virali tra gli individui drug-naïve. Determinare il profilo genotipico C-terminale del gene Gag dell'HIV-1 tra gli adolescenti infettati verticalmente che falliscono il regime PI/r senza mutazioni primarie di resistenza nel gene della proteasi. In primo luogo, abbiamo caratterizzato le mutazioni Gag presenti negli isolati di individui infetti da HIV-1 trattati con un regime PI/r (n = 143) e li abbiamo confrontati con quelli ottenuti da individui non trattati con PI/r (ART-naïve [n = 101] o trattati con inibitori della trascrittasi inversa (RTI) [n = 118]). I sottotipi di HIV-1 più frequenti sono stati CRF02_AG (54,69%), A (13,53%), D (6,35%) e G (4,69%). Diciotto mutazioni Gag hanno mostrato una prevalenza significativamente maggiore negli isolati trattati con PI/r rispetto a quelli ART-naïve (p < 0,05): Gruppo 1 (prevalenza < 1% nei drug-naïve): L449F, D480N, L483Q, Y484P, T487V; gruppo 2 (prevalenza 1-5% nei drug-naïve): S462L, I479G, I479K, D480E; gruppo 3 (prevalenza ≥ 5% nei drug-naïve): P453L, E460A, R464G, S465F, V467E, Q474P, I479R, E482G, T487A. Cinque mutazioni in Gag (L449F, P453L, D480E, S465F, Y484P) si sono correlate positivamente (Phi ≥ 0,2, p < 0,05) con le mutazioni di resistenza alla proteasi. Al momento del fallimento del PI/r, non è stata osservata alcuna differenza significativa tra i pazienti con e senza queste mutazioni Gag associate in termini di viremia o conta dei CD4. Questo dato suggerisce che alcune mutazioni Gag mostrano una maggiore frequenza nei pazienti che falliscono i PI tra i cladi di HIV-1 non-B. In secondo luogo, per quanto riguarda la valutazione della variabilità del gene Gag di HIV-1 e delle mutazioni associate alla resistenza ai farmaci in base ai sottotipi virali tra gli individui naïve ai farmaci, sono state recuperate 2031 sequenze di Gag dell'HIV-1 dal database dell'HIV di Los Alamos e analizzate in base alle mutazioni Gag documentate associate alla resistenza a PI e LEN. L'analisi complessiva della conservazione del gene Gag ha rivelato che 255/500 (51,0%) posizioni aminoacidiche erano conservate (<5% di variabilità) nell'intera popolazione di studio e la proteina p24 (capside) presentava il tasso di conservazione più elevato (156/231; 67,5%). La prevalenza delle mutazioni in Gag associate alla resistenza ai PI/r in vivo era la seguente: Y79F (46,28%), R76K (39,73%), P453L (16,0%), K436E (13,24%), V128I (6,79%), T81A (5,76%), L449F (2,80%), I437T (1,23%), R452S (0,54%), A431V (0,44%) e K112E (0,09%). È importante notare che le mutazioni di Gag associate alla resistenza ai PI erano significativamente più elevate nelle forme ricombinanti virali rispetto ai sottotipi puri (72,3% [542/750] vs. 31,1% [398/1281], p<0,0001); il ricombinante CRF01_AE aveva la più alta prevalenza di mutazioni di resistenza ai farmaci (73,1% [326/446]). La prevalenza complessiva di mutazioni associate alla resistenza a LEN è stata dello 0,6% (13/2031) e le mutazioni di resistenza osservate sono state: N74S (0,5%), M66I (0,05%), Q67H (0,05%). È interessante notare che quattro mutazioni Gag associate alla resistenza ai PI (K436E, I437T, L449F e P453L) e Q67H associata alla resistenza alle LEN sono significativamente associate a una ridotta affinità di legame tra gli epitopi dei linfociti T e le molecole MHC. Tra i pazienti naïve agli antiretrovirali, circa la metà delle posizioni aminoacidiche all'interno del gene Gag sono conservate. Infine, tassi più elevati di mutazioni di Gag associate alla resistenza ai PI si riscontrano nei virus ricombinanti, in particolare nel CRF01_AE. La bassa resistenza a LEN (<1%) suggerisce un'elevata efficacia dei regimi basati su LEN. Alcune di queste mutazioni sono significativamente associate a una ridotta affinità di legame dei linfociti T con l'MHC, suggerendo implicazioni nel meccanismo di fuga immunitaria dell'HIV. In terzo luogo, abbiamo caratterizzato il profilo genotipico del Gag C-terminale dell'HIV-1 tra gli adolescenti con infezione verticale (AVI) che hanno fallito il trattamento con inibitori della proteasi senza mutazioni importanti di resistenza nel gene della proteasi in Camerun. Si è trattato di un'analisi trasversale tra 23 adolescenti con infezione verticale da HIV in Camerun che hanno fallito la terapia a base di PI/r senza mutazioni maggiori di resistenza ai PI. La durata media del regime a base di PI/r era di 27,6 mesi e variava da 1 a 118 mesi. La carica virale media e la conta dei CD4 al momento della misurazione della resistenza ai farmaci erano rispettivamente di 193246 copie/ml (range 2094- 733567) e 479 cellule/mm3 (range 55-1168). Dei 23 AVI, quattordici adolescenti (60,7%) avevano una conta dei CD4 inferiore a 500 cellule/mm3. Le mutazioni della proteasi più diffuse erano H69K (n=20, 89,9%), L89M (n=20, 86,9%), M36I (n=20, 86,9%) e I13V (n=18, 78,2%). Inoltre, sono state rilevate mutazioni PI e polimorfismi di resistenza ai farmaci accessori (DRMS) (L10F, K20I, L33F). Le mutazioni Gag più diffuse sono state E460A (n=23, 100%), L449P (n=14, 94,4%) e R464G (n=15, 84,4%). Sono state riscontrate mutazioni Gag note associate alla resistenza ai farmaci dell'HIV-1: K436R (n=8, 34,7%), L449P (n=14, 94,4%), P453L (n=2, 8,7%) e P453T (n=2, 8,7%). Sono state riscontrate anche altre mutazioni Gag note per essere associate all'esposizione ai PI/r Y441S (n=11, 47,8%), P472S (n=11, 47,8%), Q474P (n=12, 52,2%), D480E (n=3, 13,0%) e E482G (n=9, 39,1%). In sintesi, diverse mutazioni Gag dell'HIV-1 descritte come associate alla resistenza e all'esposizione ai PI/r sono state riscontrate in questi AVVIH in fallimento di trattamento in assenza di DRMS proteasi maggiore. Pertanto, ciò potrebbe supportare il coinvolgimento delle mutazioni del gene Gag dell'HIV nel fallimento del trattamento con PI/r. In conclusione, i risultati complessivi prodotti in questa tesi hanno dimostrato l'implicazione delle mutazioni Gag nel fallimento del trattamento con inibitori della proteasi tra i sottotipi nonB e l'elevata selezione naturale di queste mutazioni da parte delle forme ricombinanti virali. Questi risultati supportano quindi l'implicazione delle mutazioni del gene Gag nel fallimento del trattamento con PI/r per una potenziale considerazione negli algoritmi di interpretazione della resistenza ai farmaci dell'HIV per la gestione clinica.
Identification and characterization of HIV-1 gag gene mutations associated to protease inhibitors resistance among Cameroonian patients
NKA, ALEX DURAND
2022
Abstract
The overall goal of this thesis was to identify HIV-1 Gag gene mutations associated to protease inhibitors (PI) resistance among non-B subtypes and the impact of viral diversity in the natural selection of these mutations. In particular, this thesis aimed to: i) Determine C-terminal Gag gene mutations selected under PI/ritonavir (PI/r) exposure and their covariations with protease major resistance mutations among HIV-1 non-B clades. ii) Evaluate HIV-1 Gag gene variability and drug resistance-associated mutations according to viral subtypes among drug-naïve individuals. iii) Determine HIV-1 Gag C-terminal genotypic profile among vertically infected adolescents failing PI/r regimen without major resistance mutations in protease gene. First, we characterized Gag mutations present in isolates from HIV-1 infected individuals treated with a PI/r-regimen (n = 143) and compared them with those obtained from individuals not treated with PI/r (ART-naïve [n = 101] or reverse transcriptase inhibitors (RTI) treated [n = 118]). The most frequent HIV-1 subtypes were CRF02_AG (54.69%), A (13.53%), D (6.35%) and G (4.69%). Eighteen Gag mutations showed a significantly higher prevalence in PI/r-treated isolates compared to ART-naïve (p < 0.05): Group 1 (prevalence < 1% in drugnaïve): L449F, D480N, L483Q, Y484P, T487V; group 2 (prevalence 1–5% in drug-naïve): S462L, I479G, I479K, D480E; group 3 (prevalence ≥ 5% in drug-naïve): P453L, E460A, R464G, S465F, V467E, Q474P, I479R, E482G, T487A. Five Gag mutations (L449F, P453L, D480E, S465F, Y484P) positively correlated (Phi ≥ 0.2, p < 0.05) with protease-resistance mutations. At PI/r-failure, no significant difference was observed between patients with and without these associated Gag mutations in term of viremia or CD4 count. This finding suggests that some Gag mutations show an increased frequency in patients failing PIs among HIV-1 nonB clades. Secondly, concerning the evaluation of HIV-1 Gag gene variability and drug resistanceassociated mutations according to viral subtypes among drug-naïve individuals, 2031 HIV-1 Gag sequences were retrieved from the Los Alamos HIV database and analyzed based on documented Gag mutations assoc ated to PI and Lenacapavir (LEN) resistance. Overall conservation analysis of the Gag gene revealed 255/500 (51.0%) amino acid positions were conserved (<5% variability) in the entire study population and p24 (capsid) protein presented the highest conservation rate (156/231; 67.5%). The prevalence of Gag mutations associated to PI/r resistance in vivo was as follows: Y79F (46.28%), R76K (39.73%), P453L (16.0%), K436E (13.24%), V128I (6.79%), T81A (5.76%), L449F (2.80%), I437T (1.23%), R452S (0.54%), A431V (0.44%) and K112E (0.09%). Importantly, Gag mutations associated to PI resistance were significantly higher in viral recombinant forms as compared to pure subtypes (72.3% [542/750] vs. 31.1% [398/1281], p<0.0001); the recombinant CRF01_AE had the highest drugresistance mutation prevalence (73.1% [326/446]). The overall prevalence of LEN resistance associated mutations was 0.6% (13/2031) and resistance mutations observed were: N74S (0.5%), M66I (0.05%), Q67H (0.05%). Interestingly, four Gag mutations associated to PI resistance (K436E, I437T, L449F and P453L) and Q67H associated to LEN resistance are significantly associated with a reduced binding affinity between T-lymphocyte cells’ epitopes and MHC molecules. Among antiretroviral-naïve patients, about half of amino acid positions within the Gag gene are conserved. Finally, higher rates of Gag mutations associated to PI resistance are found with recombinant viruses, especially the CRF01_AE. The low resistance to LEN (<1%) suggests a high effectiveness of LEN-based regimens. Some of these mutations are significantly associated with a reduced T-lymphocyte cells binding affinity to MHC, suggesting implications in HIV immune escape mechanism. Thirdly, we characterized HIV-1 Gag C-terminal genotypic profile among vertically infected adolescents (AVI) PI treatment without major resistance mutations in the protease gene in Cameroon. This was a cross sectional analysis among 23 HIV vertically infected adolescents in Cameroon failing PI/r-based therapy without PI major resistance. The mean duration of PI/rbased regimen was 27.6 months and ranged from 1 to 118 months. The mean viral load and CD4 count at time of measurement of drug resistance were 193246 copies/ml (range 2094– 733567) and 479 cells/mm3 (range 55–1168), respectively. Of the 23 AVI, fourteen adolescents (60.7%) had a CD4 count of <500 cells/mm3. Even in absence of major resistance mutations in the protease gene, protease mutations typical of non-B subtype were frequently found, as H69K (n=20, 89.9%), L89M (n=20, 86.9%), M36I (n=20, 86.9%), and I13V (n=18, 78.2%). In addition, PI mutations accessory drug resistance mutation (DRMS) polymorphisms (L10F, K20I, L33F) were also detected. The most prevalent Gag mutations were E460A (n=23, 100%), L449P (n=14, 94,4%), and R464G (n=15, 84.4%). Known HIV-1 drug resistance-associated Gag mutations K436R (n=8, 34,7%), L449P (n=14, 94,4%), P453L (n=2, 8,7%), and P453T (n=2, 8,7%) were found. Other Gag mutations known to be associated to PI/r exposure were also found Y441S (n= 11, 47.8%), P472S (n=11, 47.8%), Q474P (n=12, 52.2%), D480E (n=3, 13.0%) and E482G (n=9, 39.1%). In summary, several HIV-1 Gag mutations described as being associated to PI/r resistance and PI/r exposure have been found in these AVI on treatment failure in absence of protease major DRMS. Thus, this could support the involvement of HIV Gag gene mutations in PI/r treatment failure. In conclusion, the overall findings produced in this thesis demonstrated the implication of Gag mutations in protease inhibitors treatment failure among non-B subtypes and the hight natural selections of these mutations by viral recombinant forms. These results therefore support the implication of Gag gene mutations in PI/r treatment failure for a potential consideration in algorithms interpreting HIV drug resistance for clinical management.File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.14242/214557
URN:NBN:IT:UNIROMA2-214557