Multimethod Longitudinal HIV Drug Resistance Analysis in Antiretroviral-Therapy-Naive Patients.

The global intensification of antiretroviral therapy (ART) can lead to increased rates of HIV drug resistance (HIVDR) mutations in treated and also in ART-naive patients. ART-naive HIV-1-infected patients from Cameroon were subjected to a multimethod HIVDR analysis using amplification-refractory mutation system (ARMS)-PCR, Sanger sequencing, and longitudinal next-generation sequencing (NGS) to determine their profiles for the mutations K103N, Y181C, K65R, M184V, and T215F/Y. We processed 66 ART-naive HIV-1-positive patients with highly diverse subtypes that underlined the predominance of CRF02_AG and the increasing rate of F2 and other recombinant forms in Cameroon. We compared three resistance testing methods for 5 major mutation sites. Using Sanger sequencing, the overall prevalence of HIVDR mutations was 7.6% (5/66) and included all studied mutations except K65R. Comparing ARMS-PCR with Sanger sequencing as a reference, we obtained a sensitivity of 100% (5/5) and a specificity of 95% (58/61), caused by three false-positive calls with ARMS-PCR. For 32/66 samples, we obtained NGS data and we observed two additional mismatches made up of minority variants (7% and 18%) that might not be clinically relevant. Longitudinal NGS analyses revealed changes in HIVDR mutations in all five positive subjects that could not be attributed to treatment. In one of these cases, superinfection led to the temporary masking of a resistant virus. HIVDR mutations can be sensitively detected by ARMS-PCR and sequencing methods with comparable performances. Longitudinal changes in HIVDR mutations have to be considered even in the absence of treatment.

Vaccination with immune complexes modulates the elicitation of functional antibodies against HIV-1.

Introduction: Neutralizing antibodies (Abs) are one of the immune components required to protect against viral infections. However, developing vaccines capable of eliciting neutralizing Abs effective against a broad array of HIV-1 isolates has been an arduous challenge. Objective: This study sought to test vaccines aimed to induce Abs against neutralizing epitopes at the V1V2 apex of HIV-1 envelope (Env). Methods: Four groups of rabbits received a DNA vaccine expressing the V1V2 domain of the CRF01_AE A244 strain on a trimeric 2J9C scaffold (V1V2-2J9C) along with a protein vaccine consisting of an uncleaved prefusion-optimized A244 Env trimer with V3 truncation (UFO-BG.ΔV3) or a V1V2-2J9C protein and their respective immune complexes (ICs). These IC vaccines were made using 2158, a V1V2-specific monoclonal Ab (mAb), which binds the V2i epitope in the underbelly region of V1V2 while allosterically promoting the binding of broadly neutralizing mAb PG9 to its V2 apex epitope in vitro. Results: Rabbit groups immunized with the DNA vaccine and uncomplexed or complexed UFO-BG.ΔV3 proteins (DNA/UFO-UC or IC) displayed similar profiles of Env- and V1V2-binding Abs but differed from the rabbits receiving the DNA vaccine and uncomplexed or complexed V1V2-2J9C proteins (DNA/V1V2-UC or IC), which generated more cross-reactive V1V2 Abs without detectable binding to gp120 or gp140 Env. Notably, the DNA/UFO-UC vaccine elicited neutralizing Abs against some heterologous tier 1 and tier 2 viruses from different clades, albeit at low titers and only in a fraction of animals, whereas the DNA/V1V2-UC or IC vaccines did not. In comparison with the DNA/UFO-UC group, the DNA/UFO-IC group showed a trend of higher neutralization against TH023.6 and a greater potency of V1V2-specific Ab-dependent cellular phagocytosis (ADCP) but failed to neutralize heterologous viruses. Conclusion: These data demonstrate the capacity of V1V2-2J9C-encoding DNA vaccine in combination with UFO-BG.ΔV3, but not V1V2-2J9C, protein vaccines, to elicit homologous and heterologous neutralizing activities in rabbits. The elicitation of neutralizing and ADCP activities was modulated by delivery of UFO-BG.ΔV3 complexed with V2i mAb 2158.

Corrigendum: Vaccination with immune complexes modulates the elicitation of functional antibodies against HIV-1.

[This corrects the article DOI: 10.3389/fimmu.2023.1271686.].

Development of a Versatile, Near Full Genome Amplification and Sequencing Approach for a Broad Variety of HIV-1 Group M Variants.

Near full genome sequencing (NFGS) of HIV-1 is required to assess the genetic composition of HIV-1 strains comprehensively. Population-wide, it enables a determination of the heterogeneity of HIV-1 and the emergence of novel/recombinant strains, while for each individual it constitutes a diagnostic instrument to assist targeted therapeutic measures against viral components. There is still a lack of robust and adaptable techniques for efficient NFGS from miscellaneous HIV-1 subtypes. Using rational primer design, a broad primer set was developed for the amplification and sequencing of diverse HIV-1 group M variants from plasma. Using pure subtypes as well as diverse, unique recombinant forms (URF), variable amplicon approaches were developed for NFGS comprising all functional genes. Twenty-three different genomes composed of subtypes A (A1), B, F (F2), G, CRF01_AE, CRF02_AG, and CRF22_01A1 were successfully determined. The NFGS approach was robust irrespective of viral loads (≥306 copies/mL) and amplification method. Third-generation sequencing (TGS), single genome amplification (SGA), cloning, and bulk sequencing yielded similar outcomes concerning subtype composition and recombinant breakpoint patterns. The introduction of a simple and versatile near full genome amplification, sequencing, and cloning method enables broad application in phylogenetic studies of diverse HIV-1 subtypes and can contribute to personalized HIV therapy and diagnosis.

Near full genome characterization of HIV-1 unique recombinant forms in Cameroon reveals dominant CRF02_AG and F2 recombination patterns.

INTRODUCTION: In Cameroon, a manifold diversity of HIV strains exists with CRF02_AG and unique recombinant forms (URFs) being the predominant strains. In recent years, a steady increase in URFs and clade F2 viruses has been monitored through partial genome sequencing. There is an information gap in the characterization of emerging URFs along the full genome, which is needed to address the challenges URFs pose towards diagnosis, treatment and HIV-1 vaccine design. METHOD: Eighteen Cameroonian URFs from samples collected between the years 2000 and 2015 were studied using a newly developed near full genome sequencing (NFGS) protocol based on variable nested RT-PCRs with a versatile primer set. Near full genomes were characterized for recombination patterns and sequence signatures with possible impact on antiretroviral treatment or Env-directed immune responses. Third-generation sequencing (3GS) of near full or half genomes (HGs) gave insight into intra-patient URF diversity. RESULTS: The characterized URFs were composed of a broad variety of subtypes and recombinants including A, F, G, CRF01_AE, CRF02_AG and CRF22_01A1. Phylogenetic analysis unveiled dominant CRF02_AG and F2 recombination patterns. 3GS indicated a high intra-patient URF diversity with up to four distinct viral sub-populations present in plasma at the same time. URF pol genomic analysis revealed a number of accessory drug resistance mutations (DRMs) in the ART-naïve participants. Genotypic env analysis suggests CCR5 usage in 14/18 samples and identified deviations at residues, critical for gp120/gp41 interphase and CD4 binding site broadly neutralizing antibodies in more than half of the studied URFs. V1V2 sites of immune pressure in the human RV144 vaccine study varied in more than a third of URFs. CONCLUSIONS: This study identified novel mosaic patterns in URFs in Cameroon. In line with the regional predominance of CRF_02AG and the increased prevalence of clade F2, prominent CRF_02AG and F2 background patterns were observed underlying the URFs. In the context of the novel mosaic genomes, the impact of the identified accessory DRMs and Env epitope variations on treatment and immune control remains elusive. The evolving diversity of HIV-1 URFs in Cameroon requires continuous monitoring to respond to the increasing challenges for diagnosis, antiretroviral treatment and prevention.

Immune Correlates of Disease Progression in Linked HIV-1 Infection.

Genetic and immunologic analyses of epidemiologically-linked HIV transmission enable insights into the impact of immune responses on clinical outcomes. Human vaccine trials and animal studies of HIV-1 infection have suggested immune correlates of protection; however, their role in natural infection in terms of protection from disease progression is mostly unknown. Four HIV-1+ Cameroonian individuals, three of them epidemiologically-linked in a polygamous heterosexual relationship and one incidence-matched case, were studied over 15 years for heterologous and cross-neutralizing antibody responses, antibody binding, IgA/IgG levels, antibody-dependent cellular cytotoxicity (ADCC) against cells expressing wild-type or CD4-bound Env, viral evolution, Env epitopes, and host factors including HLA-I alleles. Despite viral infection with related strains, the members of the transmission cluster experienced contrasting clinical outcomes including cases of rapid progression and long-term non-progression in the absence of strongly protective HLA-I or CCR5Δ32 alleles. Slower progression and higher CD4/CD8 ratios were associated with enhanced IgG antibody binding to native Env and stronger V1V2 antibody binding responses in the presence of viruses with residue K169 in V2. ADCC against cells expressing Env in the CD4-bound conformation in combination with low Env-specific IgA/IgG ratios correlated with better clinical outcome. This data set highlights for the first time that V1V2-directed antibody responses and ADCC against cells expressing open, CD4-exposed Env, in the presence of low plasma IgA/IgG ratios, can correlate with clinical outcome in natural infection. These parameters are comparable to the major correlates of protection, identified post-hoc in the RV144 vaccine trial; thus, they may also modulate the rate of clinical progression once infected. The findings illustrate the potential of immune correlate analysis in natural infection to guide vaccine development.

Contrasting antibody responses to intrasubtype superinfection with CRF02_AG.

HIV superinfection describes the sequential infection of an individual with two or more unrelated HIV strains. Intersubtype superinfection has been shown to cause a broader and more potent heterologous neutralizing antibody response when compared to singly infected controls, yet the effects of intrasubtype superinfection remain controversial. Longitudinal samples were analyzed phylogenetically for pol and env regions using Next-Generation Sequencing and envelope cloning. The impact of CRF02_AG intrasubtype superinfection was assessed for heterologous neutralization and antibody binding responses. We compared two cases of CRF02_AG intrasubtype superinfection that revealed complete replacement of the initial virus by superinfecting CRF02_AG variants with signs of recombination. NYU6564, who became superinfected at an early time point, exhibited greater changes in antibody binding profiles and generated a more potent neutralizing antibody response post-superinfection compared to NYU6501. In contrast, superinfection occurred at a later time point in NYU6501 with strains harboring significantly longer V1V2 regions with no observable changes in neutralization patterns. Here we show that CRF02_AG intrasubtype superinfection can induce a cross-subtype neutralizing antibody response, and our data suggest timing and/or superinfecting viral envelope characteristics as contributing factors. These results highlight differential outcomes in intrasubtype superinfection and provide the first insight into cases with CRF02_AG, the fourth most prevalent HIV-1 strain worldwide.