Persistence of frequently transmitted drug-resistant HIV-1 variants can be explained by high viral replication capacity

Background: In approximately 10% of newly diagnosed individuals in Europe, HIV-1 variants harboring transmitted drug resistance mutations (TDRM) are detected. For some TDRM it has been shown that they revert to wild type while other mutations persist in the absence of therapy. To understand the mechanisms explaining persistence we investigated the in vivo evolution of frequently transmitted HIV-1 variants and their impact on in vitro replicative capacity. Results: We selected 31 individuals infected with HIV-1 harboring frequently observed TDRM such as M41L or K103N in reverse transcriptase (RT) or M46L in protease. In all these samples, polymorphisms at non-TDRM positions were present at baseline (median protease: 5, RT: 6). Extensive analysis of viral evolution of protease and RT demonstrated that the majority of TDRM (51/55) persisted for at least a year and even up to eight years in the plasma. D

Cross-genotype AR3-specific neutralizing antibodies confer long-term protection in injecting drug users after HCV clearance

Background & AimsUnderstanding immune protection against hepatitis C virus (HCV) infection is necessary for designing an effective vaccine. A number of broadly-reactive, neutralizing antibodies have been isolated from B cells of HCV-infected subjects. However, it remains unclear whether B cells producing such antibodies contribute to the clearance and long-term immune protection against HCV.MethodsWe analysed the B-cell repertoire of thirteen participants from the Amsterdam Cohort Study among injecting drug users with a median follow-up of 17.5 years. Five subjects ultimately became chronically infected either after primary infection or after reinfection. Eight subjects, at the end of study follow-up, were HCV RNA negative following spontaneous clearance of one or multiple infections. From each subject, 10,000 CD27+IgG+ B cells, collected 0.75 year after HCV infection, were cultured to characterize the antibody repertoire.ResultsUsing a multiplex flow cytometry-based assay to study the antibody binding to E1E2 from genotype 1 to 6, we found that a high frequency of cross-genotype antibodies was associated with spontaneous clearance of one or multiple infections (p-value=0.03). Epitope specificity of these cross-genotype antibodies was determined by alanine mutant scanning in four subjects, who were HCV RNA negative following spontaneous clearance of one or multiple infections. Interestingly, the cross-genotype antibodies were mainly AR3-specific and showed cross-neutralizing activity against HCV. In addition to AR3 antibodies, three subjects developed antibodies recognizing AR4 of which one monoclonal antibody showed cross-neutralizing capacity.ConclusionsTogether, these data suggest that a strong B-cell response producing cross-genotype and neutralizing antibodies, especially targeting AR3, contribute to HCV clearance and long-term immune protection against HCV

Persistence of frequently transmitted drug-resistant HIV-1 variants can be explained by high viral replication capacity

Background: In approximately 10% of newly diagnosed individuals in Europe, HIV-1 variants harboring transmitted drug resistance mutations (TDRM) are detected. For some TDRM it has been shown that they revert to wild type while other mutations persist in the absence of therapy. To understand the mechanisms explaining persistence we investigated the in vivo evolution of frequently transmitted HIV-1 variants and their impact on in vitro replicative capacity. Results: We selected 31 individuals infected with HIV-1 harboring frequently observed TDRM such as M41L or K103N in reverse transcriptase (RT) or M46L in protease. In all these samples, polymorphisms at non-TDRM positions were present at baseline (median protease: 5, RT: 6). Extensive analysis of viral evolution of protease and RT demonstrated that the majority of TDRM (51/55) persisted for at least a year and even up to eight years in the plasma. During follow-up only limited selection of additional polymorphisms was observed (median: 1). To investigate the impact of frequently observed TDRM on the replication capacity, mutant viruses were constructed with the most frequently encountered TDRM as site-directed mutants in the genetic background of the lab strain HXB2. In addition, viruses containing patient-derived protease or RT harboring similar TDRM were made. The replicative capacity of all viral variants was determined by infecting peripheral blood mononuclear cells and subsequently monitoring virus replication. The majority of site-directed mutations (M46I/M46L in protease and M41L, M41L + T215Y and K103N in RT) decreased viral replicative capacity; only protease mutation L90M did not hamper viral replication. Interestingly, most patient-derived viruses had a higher in vitro replicative capacity than the corresponding site-directed mutant viruses. Conclusions: We demonstrate limited in vivo evolution of protease and RT harbouring frequently observed TDRM in the plasma. This is in line with the high in vitro replication capacity of patient-derived viruses harbouring TDRM compared to site-directed mutant viruses harbouring TDRM. As site-directed mutant viruses have a lower replication capacity than the patient-derived viruses with similar mutational patterns, we propose that (baseline) polymorphisms function as compensatory mutations improving viral replication capacity

Differential in vitro kinetics of drug resistance mutation acquisition in HIV-1 RT of subtypes B and C.

BACKGROUND: HIV-1 subtype B is the most prevalent in developed countries and, consequently, it has been extensively studied. On the other hand, subtype C is the most prevalent worldwide and therefore is a reasonable target for future studies. Here we evaluate the acquisition of resistance and the viability of HIV-1 subtype B and C RT clones from different isolates that were subjected to in vitro selection pressure with zidovudine (ZDV) and lamivudine (3TC). METHODS/PRINCIPAL FINDINGS: MT4 cells were infected with chimeric virus pseudotyped with RT from subtype B and C clones, which were previously subjected to serial passage with increasing concentrations of ZDV and 3TC. The samples collected after each passage were analyzed for the presence of resistance mutations and VL. No differences were found between subtypes B and C in viral load and resistance mutations when these viruses were selected with 3TC. However, the route of mutations and the time to rebound of subtype B and C virus were different when subjected to ZDV treatment. In order to confirm the role of the mutations detected, other clones were generated and subjected to in vitro selection. RT subtype B virus isolates tended to acquire different ZDV resistance mutations (Q151M and D67N or T215Y, D67D/N and F214L) compared to subtype C (D67N, K70R, T215I or T215F). CONCLUSIONS/SIGNIFICANCE: This study suggests that different subtypes have a tendency to react differently to antiretroviral drug selection in vitro. Consequently, the acquisition of resistance in patients undergoing antiretroviral therapy can be dependent on the subtypes composing the viral population

Multiplex flow cytometry-based assay to study the breadth of antibody responses against E1E2 glycoproteins of hepatitis C virus

Hepatitis C virus (HCV) infection is a major global public health problem. Early induction of cross-reactive neutralizing antibodies during acute infection correlates with the spontaneous clearance of HCV. Understanding the antibody response in multiple subjects in large-scale studies would greatly benefit vaccine development. To determine the breadth of a polyclonal-serum antibody response, and or, the monoclonal antibodies against the different HCV E1E2 genotypes, we developed a quick and high throughput flow cytometry assay using fluorescent cell barcoding to distinguish cells transfected with different E1E2 sequences in a single measurement. HCV-specific antibodies recognizing conformational epitopes were tested for binding to cells transfected with E1E2 from six genotypes. In this assay, 1500 samples can be analyzed for specific binding to 6 different HCV E1E2 sequences within 8h. Plasma of HCV infected subjects were tested in our assay allowing us to determine the breadth of their antibody response. In summary, we developed a quick and high throughput assay to study the specificity of an antibody response against multiple HCV E1E2 sequences simultaneously. This assay can also be used to facilitate the discovery of novel antibodies, and because other flavi- and picornaviruses have similar intracellular assembly mechanisms, this approach can be used to study the antibody response against such viruses