Replicative fitness and pathogenicity of primate lentiviruses in lymphoid tissue, primary human and chimpanzee cells: relation to possible jumps to humans.

BACKGROUND: Simian immunodeficiency viruses (SIV) have been jumping between non-human primates in West/Central Africa for thousands of years and yet, the HIV-1 epidemic only originated from a primate lentivirus over 100 years ago. METHODS: This study examined the replicative fitness, transmission, restriction, and cytopathogenicity of 22 primate lentiviruses in primary human lymphoid tissue and both primary human and chimpanzee peripheral blood mononuclear cells. FINDINGS: Pairwise competitions revealed that SIV from chimpanzees (cpz) had the highest replicative fitness in human or chimpanzee peripheral blood mononuclear cells, even higher fitness than HIV-1 group M strains responsible for worldwide epidemic. The SIV strains belonging to the "HIV-2 lineage" (including SIVsmm, SIVmac, SIVagm) had the lowest replicative fitness. SIVcpz strains were less inhibited by human restriction factors than the "HIV-2 lineage" strains. SIVcpz efficiently replicated in human tonsillar tissue but did not deplete CD4+ T-cells, consistent with the slow or nonpathogenic disease observed in most chimpanzees. In contrast, HIV-1 isolates and SIV of the HIV-2 lineage were pathogenic to the human tonsillar tissue, almost independent of the level of virus replication. INTERPRETATION: Of all primate lentiviruses, SIV from chimpanzees appears most capable of infecting and replicating in humans, establishing HIV-1. SIV from other Old World monkeys, e.g. the progenitor of HIV-2, replicate slowly in humans due in part to restriction factors. Nonetheless, many of these SIV strains were more pathogenic than SIVcpz. Either SIVcpz evolved into a more pathogenic virus while in humans or a rare SIVcpz, possibly extinct in chimpanzees, was pathogenic immediately following the jump into human. FUNDING: Support for this study to E.J.A. was provided by the NIH/NIAID R01 AI49170 and CIHR project grant 385787. Infrastructure support was provided by the NIH CFAR AI36219 and Canadian CFI/Ontario ORF 36287. Efforts of J.A.B. and N.J.H. was provided by NIH AI099473 and for D.H.C., by VA and NIH AI AI080313.

Molecular epidemiology of SARS-CoV-2 in Northern South Africa: wastewater surveillance from January 2021 to May 2022.

Introduction: Wastewater-based genomic surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) provides a comprehensive approach to characterize evolutionary patterns and distribution of viral types in a population. This study documents the molecular epidemiology of SARS-CoV-2, in Northern South Africa, from January 2021 to May 2022. Methodology: A total of 487 wastewater samples were collected from the influent of eight wastewater treatment facilities and tested for SARS-CoV-2 RNA using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). SARS-CoV-2 positive samples with genome copies/mL ≥1,500 were subjected to allele-specific genotyping (ASG) targeting the Spike protein; 75 SARS-CoV-2 positive samples were subjected to whole genome sequencing (WGS) on the ATOPlex platform. Variants of concern (VoC) and lineages were assigned using the Nextclade and PangoLIN Software. Concordance for VoC between ASG and WGS analyses was determined. Sequence relationship was determined by phylogenetic analysis. Results: Seventy-five percent (365/487) of the influent samples were positive for SARS-CoV-2 RNA. Delta and Omicron VoC were more predominant at a prevalence of 45 and 32%, respectively, and they were detected as early as January and February 2021, while Beta VoC was least detected at a prevalence of 5%. A total of 11/60 (18%) sequences were assigned lineages and clades only, but not a specific VoC name. Phylogenetic analysis was used to investigate the relationship of these sequences to other study sequences, and further characterize them. Concordance in variant assignment between ASG and WGS was seen in 51.2% of the study sequences. There was more intra-variant diversity among Beta VoC sequences; mutation E484K was absent. Three previously undescribed mutations (A361S, V327I, D427Y) were seen in Delta VoC. Discussion and Conclusion: The detection of Delta and Omicron VoCs in study sites earlier in the outbreak than has been reported in other regions of South Africa highlights the importance of population-based approaches over individual sample-based approaches in genomic surveillance. Inclusion of non-Spike protein targets could improve the specificity of ASG, since all VoCs share similar Spike protein mutations. Finally, continuous molecular epidemiology with the application of sensitive technologies such as next generation sequencing (NGS) is necessary for the documentation of mutations whose implications when further investigated could enhance diagnostics, and vaccine development efforts.

COVID-19 vaccine acceptance and perceived risk among pregnant and non-pregnant adults in Cameroon, Africa.

BACKGROUND: The public health response to the global COVID-19 pandemic has varied widely by region. In Africa, uptake of effective COVID-19 vaccines has been limited by accessibility and vaccine hesitancy. The aim of this study was to compare perceptions of COVID-19 infection and vaccination between pregnant women and non-pregnant adults in four regions of Cameroon, located in Central Africa. METHODS: A cross-sectional survey study was conducted at urban and suburban hospital facilities in Cameroon. Participants were randomly selected from a convenience sample of adult pregnant and non-pregnant adults in outpatient clinical settings between June 1st and July 14th, 2021. A confidential survey was administered in person by trained research nurses after obtaining written informed consent. Participants were asked about self-reported sociodemographics, medical comorbidities, perceptions of COVID-19 infection, and vaccination. Descriptive statistics were used for survey responses and univariate and multivariable logistic regression models were created to explore factors associated with COVID-19 vaccine acceptability. RESULTS: Fewer than one-third of participants were interested in receiving the COVID-19 vaccine (31%, 257/835) and rates did not differ by pregnancy status. Overall, 43% of participants doubted vaccine efficacy, and 85% stated that the vaccine available in Africa was less effective than vaccine available in Europe. Factors independently associated with vaccine acceptability included having children (aOR = 1.5; p = 0.04) and higher education (aOR = 1.6 for secondary school vs primary/none; p = 0.03). Perceived risks of vaccination ranged from death (33%) to fetal harm (31%) to genetic changes (1%). Health care professionals were cited as the most trusted source for health information (82%, n = 681). CONCLUSION: COVID-19 vaccine hesitancy and misinformation in Cameroon was highly prevalent among pregnant and non-pregnant adults in 2021 while vaccine was available but not recommended for use in pregnancy. Based on study findings, consistent public health messaging from medical professionals about vaccine safety and efficacy and local production of vaccine are likely to improve acceptability.

Drug Resistance Mutations in a Population Before Antiretroviral Therapy Initiation in Northern South Africa.

South Africa introduced the "diagnose and treat" universal HIV treatment program in September 2016. This program enables all identified HIV-positive patients to immediately start first-line antiretroviral therapy (ART). However, the presence of drug-resistant (DR) viruses in the drug-naive population complicates the choice of ART. We used next-generation sequencing (NGS) to determine the prevalence and diversity of HIV DR mutations in patients entering HIV treatment programs in northern South Africa. RNA was isolated from plasma of drug-naive HIV-1-infected patients. Using reverse transcriptase polymerase chain reaction, the HIV-1-pol gene comprising the complete protease (PR) and the first 900 bp of reverse transcriptase (RT) was amplified and sequenced on an Illumina MiniSeq platform. Consensus sequences were derived at >20% threshold and at >5% threshold using Geneious PRIME® software version 2020.1.2. HIV-1 surveillance drug resistance mutations (SDRM) were inferred using Calibrated Population Resistance tool in HIV Drug Resistance Database. Viral subtypes were determined using REGA and RIP genotyping tools. The HIV PR/RT region was successfully sequenced from 241 patients. From these, 23 (9.5%) had at least one SDRM detected at >20% threshold, with a prevalence of 9.5% (n = 18), 3% (n = 7), and 0.4% (n = 1) for non-nucleoside reverse transcriptase inhibitors (NNRTI), nucleoside reverse transcriptase inhibitors (NRTI), and protease inhibitors (PI), respectively. The number of patients with SDRM increased to 31 (12.9%) when minority variants were accounted for at >5% threshold. The most frequent SDRMs based on drug class were; K103N (7.9%-NNRTI), K65R (2.5%-NRTI), and D30N (0.8%-PI). Four cases of dual NRTI/NNRTI mutations were identified. All consensus sequences were subtype C, except three, which were C/A1, C/F1, and C/G recombinants. NGS analysis confirms that individuals entering HIV treatment programs in northern South Africa, habor moderate levels of SDRM, including cases of dual-class drug resistance. Further SDRM studies may be required to better understand resistance in the drug-naive population in the era of "diagnose and treat" in Limpopo Province, South Africa.

Potential challenges to sustained viral load suppression in the HIV treatment programme in South Africa: a narrative overview.

BACKGROUND: South Africa, with one of the highest HIV prevalences in the world, introduced the universal test and treat (UTT) programme in September 2016. Barriers to sustained viral suppression may include drug resistance in the pre-treated population, non-adherence, acquired resistance; pharmacokinetics and pharmacodynamics, and concurrent use of alternative treatments. OBJECTIVE: The purpose of this review is to highlight potential challenges to achieving sustained viral load suppression in South Africa (SA), a major expectation of the UTT initiative. METHODOLOGY: Through the PRISMA approach, published articles from South Africa on transmitted drug resistance; adherence to ARV; host genetic factors in drug pharmacokinetics and pharmacodynamics, and interactions between ARV and herbal medicine were searched and reviewed. RESULTS: The level of drug resistance in the pre-treated population in South Africa has increased over the years, although it is heterogeneous across and within Provinces. At least one study has documented a pre-treated population with moderate (> 5%) or high (> 15%) levels of drug resistance in eight of the nine Provinces. The concurrent use of ARV and medicinal herbal preparation is fairly common in SA, and may be impacting negatively on adherence to ARV. Only few studies have investigated the association between the genetically diverse South African population and pharmacokinetics and pharmacodynamics of ARVs. CONCLUSION: The increasing levels of drug resistant viruses in the pre-treated population poses a threat to viral load suppression and the sustainability of first line regimens. Drug resistance surveillance systems to track the emergence of resistant viruses, study the burden of prior exposure to ARV and the parallel use of alternative medicines, with the goal of minimizing resistance development and virologic failure are proposed for all the Provinces of South Africa. Optimal management of the different drivers of drug resistance in the pre-treated population, non-adherence, and acquired drug resistance will be beneficial in ensuring sustained viral suppression in at least 90% of those on treatment, a key component of the 90-90-90 strategy.

HIV-1 subtype C predicted co-receptor tropism in Africa: an individual sequence level meta-analysis.

BACKGROUND: Entry inhibitors, such as Maraviroc, hold promise as components of HIV treatment and/or pre-exposure prophylaxis in Africa. Maraviroc inhibits the interaction between HIV Envelope gp120 V3-loop and CCR5 coreceptor. HIV-1 subtype C (HIV-1-C) is predominant in Southern Africa and preferably uses CCR5 co-receptor. Therefore, a significant proportion of HIV-1-C CXCR4 utilizing viruses (X4) may compromise the effectiveness of Maraviroc. This analysis examined coreceptor preferences in early and chronic HIV-1-C infections across Africa. METHODS: African HIV-1-C Envelope gp120 V3-loop sequences sampled from 1988 to 2014 were retrieved from Los Alamos HIV Sequence Database. Sequences from early infections (< 186 days post infection) and chronic infections (> 186 days post infection) were analysed for predicted co-receptor preferences using Geno2Pheno [Coreceptor] 10% FPR, Phenoseq-C, and PSSMsinsi web tools. V3-loop diversity was determined, and viral subtype was confirmed by phylogenetic analysis. National treatment guidelines across Africa were reviewed for Maraviroc recommendation. RESULTS: Sequences from early (n = 6316) and chronic (n = 7338) HIV-1-C infected individuals from 10 and 15 African countries respectively were available for analyses. Overall, 518/6316 (8.2%; 95% CI 0.7-9.3) of early sequences were X4, with Ethiopia and Malawi having more than 10% each. For chronic infections, 8.3% (95% CI 2.4-16.2) sequences were X4 viruses, with Ethiopia, Tanzania, and Zimbabwe having more than 10% each. For sequences from early chronic infections (< 1 year post infection), the prevalence of X4 viruses was 8.5% (95% CI 2.6-11.2). In late chronic infections (≥ 5 years post infection), X4 viruses were observed in 36% (95% CI - 16.3 to 49.9), with two countries having relatively high X4 viruses: South Africa (43%) and Malawi (24%). The V3-loop amino acid sequence were more variable in X4 viruses in chronic infections compared to acute infections, with South Africa, Ethiopia and Zimbabwe showing the highest levels of V3-loop diversity. All sequences were phylogenetically confirmed as HIV-1-C and clustered according to their co-receptor tropism. In Africa, Maraviroc is registered only in South Africa and Uganda. CONCLUSIONS: Our analyses illustrate that X4 viruses are present in significantly similar proportions in early and early chronic HIV-1 subtype C infected individuals across Africa. In contrast, in late chronic infections, X4 viruses increase 3-5 folds. We can draw two inferences from our observations: (1) to enhance the utility of Maraviroc in chronic HIV subtype C infections in Africa, prior virus co-receptor determination is needed; (2) on the flip side, research on the efficacy of CXCR4 antagonists for HIV-1-C infections is encouraged. Currently, the use of Maraviroc is very limited in Africa.

Characterization of APOBEC3 variation in a population of HIV-1 infected individuals in northern South Africa.

BACKGROUND: The apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3 (APOBEC3) genes A3D, A3F, A3G and A3H have all been implicated in the restriction of human immunodeficiency virus type 1 (HIV-1) replication. Polymorphisms in these genes are likely to impact viral replication and fitness, contributing to viral diversity. Currently, only a few studies indicate that polymorphisms in the A3 genes may be correlated with infection risk and disease progression. METHODS: To characterize polymorphisms in the coding regions of these APOBEC3 genes in an HIV-1 infected population from the Limpopo Province of South Africa, APOBEC3 gene fragments were amplified from genomic DNA of 192 HIV-1 infected subjects and sequenced on an Illumina MiSeq platform. SNPs were confirmed and compared to SNPs in other populations reported in the 1000 Genome Phase III and HapMap databases, as well as in the ExAC exome database. Hardy-Weinberg Equilibrium was calculated and haplotypes were inferred using the LDlink 3.0 web tool. Linkage Disequilibrium (LD) for these SNPS were calculated in the total 1000 genome and AFR populations using the same tool. RESULTS: Known variants compared to the GRCh37 consensus genome sequence were detected at relatively high frequencies (> 5%) in all of the APOBEC3 genes. A3H showed the most variation, with several of the variants present in both alleles in almost all of the patients. Several minor allele variants (< 5%) were also detected in A3D, A3F and A3G. In addition, novel R6K, L221R and T238I variants in A3D and I117I in A3F were observed. Four, five, four, and three haplotypes were identified for A3D, A3F, A3G, and A3H respectively. CONCLUSIONS: The study showed significant polymorphisms in the APOBEC3D, 3F, 3G and 3H genes in our South African HIV1-infected cohort. In the case of all of these genes, the polymorphisms were generally present at higher frequencies than reported in other 1000 genome populations and in the ExAC exome consortium database .

Next generation sequencing reveals a high frequency of CXCR4 utilizing viruses in HIV-1 chronically infected drug experienced individuals in South Africa.

BACKGROUND: Entry inhibitors, such as Maraviroc, bind to CCR5 inhibiting entry of CCR5 utilizing viruses (R5 viruses). In the course of HIV infection, CXCR4 utilizing viruses (X4 viruses) may emerge and outgrow R5 viruses, and potentially limit the effectiveness of Maraviroc. The use of Maraviroc is reserved for salvage therapy in South Africa. OBJECTIVE: In this study, we examined the frequency of R5 and X4 viruses, using next generation sequencing, in patients under treatment to draw inferences on the utility of Maraviroc in a South African population. STUDY DESIGN: Proviral DNA was isolated from peripheral blood mononuclear cells (PBMC) of 72 chronically HIV infected patients on antiretroviral treatment. HIV V3 loop gene was amplified and sequenced on an Illumina MiniSeq platform. Viral subtypes were determined by the jumping profile Hidden Markov Model (jpHMM) and REGA genotyping tools. De Novo consensus sequences were derived for the majority and minority populations for each patient using Geneious® software version 8.1.5. HIV-1 tropism was inferred using PSSMsinsi, Geno2pheno and Phenoseq-C web-based tools. RESULTS: Quality V3 loop sequences were obtained from 72 patients, with 5 years (range: 0-16) median duration on treatment. Subtypes A1, B and C viruses were identified at frequencies of 4% (3/72), 4% (3/72) and 92% (66/72) respectively. Fifty four percent (39/72) of patients exclusively harboured R5 viral quasispecies; and 21% (15/72) exclusively harbored X4 viral quasispecies. Twenty five percent of patients (18/72) harbored dual/mixture of R5X4 quasispecies. Of these 18 patients, about 28% (5/18) harbored the R5+X4, a mixture with a majority R5 and minority X4 viruses, while about 72% (13/18) harbored the R5X4+ mixture with a majority X4 and minority R5 viruses. The proportion of all patients who harbored X4 viruses either exclusively or dual/mixture was 46% (33/72). Thirty-five percent (23/66) of the patients who were of HIV-1 subtype C harboured X4 viruses (χ2 = 3.58; p = .058), and 57% of these (13/23) harbored X4 viruses exclusively. CD4+ cell count less than 350 cell/µl was associated with the presence of X4 viruses (χ2 = 4.99; p = .008). CONCLUSION: The effectiveness of Maraviroc as a component in salvage therapy may be compromised for a significant number of chronically infected patients harboring CXCR4 utilizing viruses.

Dendritic cell targeted HIV-1 gag protein vaccine provides help to a recombinant Newcastle disease virus vectored vaccine including mobilization of protective CD8+ T cells.

INTRODUCTION: Recombinant Newcastle Disease virus (rNDV) vectored vaccines are safe mucosal applicable vaccines with intrinsic immune-modulatory properties for the induction of efficient immunity. Like all viral vectored vaccines repeated inoculation via mucosal routes invariably results to immunity against viral vaccine vectors. To obviate immunity against viral vaccine vectors and improve the ability of rNDV vectored vaccines in inducing T cell immunity in murine air way we have directed dendritic cell targeted HIV-1 gag protein (DEC-Gag) vaccine; for the induction of helper CD4+ T cells to a Recombinant Newcastle disease virus expressing codon optimized HIV-1 Gag P55 (rNDV-L-Gag) vaccine. METHODS: We do so through successive administration of anti-DEC205-gagP24 protein plus polyICLC (DEC-Gag) vaccine and rNDV-L-Gag. First strong gag specific helper CD4+ T cells are induced in mice by selected targeting of anti-DEC205-gagP24 protein vaccine to dendritic cells (DC) in situ together with polyICLC as adjuvant. This targeting helped T cell immunity develop to a subsequent rNDV-L-Gag vaccine and improved both systemic and mucosal gag specific immunity. RESULTS: This sequential DEC-Gag vaccine prime followed by an rNDV-L-gag boost results to improved viral vectored immunization in murine airway, including mobilization of protective CD8+ T cells to a pathogenic virus infection site. CONCLUSION: Thus, complementary prime boost vaccination, in which prime and boost favor distinct types of T cell immunity, improves viral vectored immunization, including mobilization of protective CD8+ T cells to a pathogenic virus infection site such as the murine airway.

High level of HIV-1 drug resistance mutations in patients with unsuppressed viral loads in rural northern South Africa.

BACKGROUND: Combination antiretroviral therapy (cART) has significantly reduced HIV morbidity and mortality in both developed and developing countries. However, the sustainability of cART may be compromised by the emergence of viral drug resistance mutations (DRM) and the cellular persistence of proviruses carrying these DRM. This is potentially a more serious problem in resource limited settings. METHODS: DRM were evaluated in individuals with unsuppressed viral loads after first or multiple lines of cART at two sites in rural Limpopo, South Africa. Seventy-two patients with viral loads of >1000 copies/ml were recruited between March 2014 and December 2015. Complete protease (PR) and partial Reverse Transcriptase (RT) sequences were amplified from both plasma RNA and paired proviral DNA from 35 of these subjects. Amplicons were directly sequenced to determine subtype and DRM using the Stanford HIV Drug Resistance Interpretation algorithm. RESULTS: Among the 72 samples, 69 could be PCR amplified from RNA and 35 from both RNA and DNA. Sixty-five (94.2%) viruses were subtype C, while one was subtype B (1.4%), one recombinant K/C, one recombinant C/B and one unclassified. Fifty-eight (84%) sequences carried at least one DRM, while 11 (15.9%) displayed no DRM. DRM prevalence according to drug class was: NRTI 60.8% NNRTI 65.2%, and PI 5.8%. The most common DRMs were; M184V (51.7%), K103N (50%), V106M (20.6%), D67N (13.3%), K65R (12%). The frequency of the DRM tracked well with the frequency of use of medications to which the mutations were predicted to confer resistance. Interestingly, a significant number of subjects showed predicted resistance to the newer NNRTIs, etravirine (33%) and rilpivirine (42%), both of which are not yet available in this setting. The proportion of DRM in RNA and DNA were mostly similar with the exception of the thymidine analogue mutations (TAMs) D67N, K70R, K219QE; and K103N which were slightly more prevalent in DNA than RNA. Subjects who had received cART for at least 5 years were more likely to harbour >2 DRM (p < 0.05) compared to those treated for a shorter period. DRM were more prevalent in this rural setting compared to a neighbouring urban setting. CONCLUSION: We found a very high prevalence of NRTI and NNRTI DRM in patients from rural Limpopo settings with different durations of treatment. The prevalence was significantly higher than those reported in urban settings in South Africa. The dominance of NNRTI based mutations late in treatment supports the use of PI based regimens for second line treatment in this setting. The slight dominance of TAMs in DNA from infected PBMCs compared to plasma virus requires further studies that should include cART subjects with suppressed virus. Such studies will improve our understanding of the pattern of drug resistance and dynamics of viral persistence in these rural settings.

Effect of intercalator and Lewis acid-base branched peptide complex formation: boosting affinity towards HIV-1 RRE RNA.

High throughput screening of a 4096 compound library of boronic acid and acridine containing branched peptides revealed compounds that have dissociation constants in the low nanomolar regime for HIV-1 RRE IIB RNA. We demonstrate that branched peptide boronic acids A5, A6, and A7 inhibit the production of p24, an HIV-1 capsid protein, in a dose-dependent manner.

Rev-RRE Functional Activity Differs Substantially Among Primary HIV-1 Isolates.

The HIV-1 replication cycle requires the nucleocytoplasmic export of intron-containing viral RNAs, a process that is ordinarily restricted. HIV overcomes this by means of the viral Rev protein, which binds to an RNA secondary structure called the Rev response element (RRE) present in all unspliced or incompletely spliced viral RNA transcripts. The resulting mRNP complex is exported through interaction with cellular factors. The Rev-RRE binding interaction is increasingly understood to display remarkable structural plasticity, but little is known about how Rev-RRE sequence differences affect functional activity. To study this issue, we utilized a lentiviral vector assay in which vector titer is dependent on the activity of selected Rev-RRE pairs. We found that Rev-RRE functional activity varies significantly (up to 24-fold) between naturally occurring viral isolates. The activity differences of the Rev-RRE cognate pairs track closely with Rev, but not with RRE activity. This variation in Rev activity is not correlated with differences in Rev steady state protein levels. These data suggest that Rev sequence differences drive substantial variation in Rev-RRE functional activity between patients. Such variation may play a role in viral adaptation to different immune milieus within and between patients and may be significant in the establishment of latency. The identification of differences in Rev-RRE functional activity in naturally occurring isolates may also permit more efficient production of lentiviral vectors.

Characterization and in vitro activity of a branched peptide boronic acid that interacts with HIV-1 RRE RNA.

A branched peptide containing multiple boronic acids was found to bind RRE IIB selectively and inhibit HIV-1 p24 capsid production in a dose-dependent manner. Structure-activity relationship studies revealed that branching in the peptide is crucial for the low micromolar binding towards RRE IIB, and the peptide demonstrates selectivity towards RRE IIB in the presence of tRNA. Footprinting studies suggest a binding site on the upper stem and internal loop regions of the RNA, which induces enzymatic cleavage of the internal loops of RRE IIB upon binding.

HIV-1 Group O Genotypes and Phenotypes: Relationship to Fitness and Susceptibility to Antiretroviral Drugs.

Despite only 30,000 group O HIV-1 infections, a similar genetic diversity is observed among the O subgroups H (head) and T (tail) (previously described as subtypes A, B) as in the 9 group M subtypes (A-K). Group O isolates bearing a cysteine at reverse transcriptase (RT) position 181, predominantly the H strains are intrinsically resistant to non-nucleoside reverse transcriptase inhibitors (NNRTIs). However, their susceptibility to newer antiretroviral drugs such as etravirine, maraviroc, raltegravir (RAL), and elvitegravir (EVG) remains relatively unknown. We tested a large collection of HIV-1 group O strains for their susceptibility to four classes of antiretroviral drugs namely nucleoside RT, non-nucleoside RT, integrase, and entry inhibitors knowing in advance the intrinsic resistance to NNRTIs. Drug target regions were sequenced to determine various polymorphisms and were phylogenetically analyzed. Replication kinetics and fitness assays were performed in U87-CD4(+)CCR5 and CXCR4 cells and peripheral blood mononuclear cells. With all antiretroviral drugs, group O HIV-1 showed higher variability in IC50 values than group M HIV-1. The mean IC50 values for entry and nucleoside reverse transcriptase inhibitor (NRTI) were similar for group O and M HIV-1 isolates. Despite similar susceptibility to maraviroc, the various phenotypic algorithms failed to predict CXCR4 usage based on the V3 Env sequences of group O HIV-1 isolates. Decreased sensitivity of group O HIV-1 to integrase or NNRTIs had no relation to replicative fitness. Group O HIV-1 isolates were 10-fold less sensitive to EVG inhibition than group M HIV-1. These findings suggest that in regions where HIV-1 group O is endemic, first line treatment regimens combining two NRTIs with RAL may provide more sustained virologic responses than the standard regimens involving an NNRTI or protease inhibitors.

HIV-1 Group O Origin, Evolution, Pathogenesis, and Treatment: Unraveling the Complexity of an Outlier 25 Years Later.

Twenty-five years ago, an aberrant HIV-1 (now classified as HIV-1 group O) was described from a Cameroonian HIV patient living in Belgium. The epicenter of group O was later found to be in Central Africa, overlapping with the geographical location of the central chimpanzees (Pan troglodytes troglodytes) and western gorillas (Gorilla gorilla), the likely original hosts of group O. Although the prevalence of group O has remained low at 1-2% in Cameroon, some European countries (France, Spain, Belgium) with strong colonial ties to Central Africa have reported the highest prevalence out of Africa. The sequence diversity between HIV-1 group O and M strains is huge, reaching 50 and 30% in the envelope and pol, respectively. This diversity has hindered diagnosis, monitoring, and treatment of group O-infected patients. Due to the intrinsic presence of the C181 mutation in group O, more than 60% of the approximately 30,000 individuals that live with this virus are faced with the challenge of drug resistance to some currently used antiretroviral therapies, notably the non-nucleoside reverse transcriptase inhibitors. Despite its susceptibility to most antiretroviral therapies, some group Os show a high variable baseline susceptibility to enfuvirtide (T20) and maraviroc. Group O strains are the least fit among all HIV-1 and -2 and restrict tetherin using their Nef but not Vpu as reported for group M. Although limited follow-up studies indicate that the natural course of group O is similar to that of M, these viruses are dominantly CCR5 tropic even late in infection, suggesting slow disease progression. This review summarizes important findings that marked the discovery, origin, spread, evolution, pathogenesis, and treatment of group O within the last 25 years.

Defining the fitness of HIV-1 isolates with dual/mixed co-receptor usage.

BACKGROUND: CCR5-using (r5) HIV-1 predominates during asymptomatic disease followed by occasional emergence of CXCR4-using (x4) or dual tropic (r5x4) virus. We examined the contribution of the x4 and r5 components to replicative fitness of HIV-1 isolates. METHODS: Dual tropic r5x4 viruses were predicted from average HIV-1 env sequences of two primary subtype C HIV-1 isolates (C19 and C27) and from two patient plasma samples (B12 and B19). Chimeric Env viruses with an NL4-3 backbone were constructed from the B12 and B19 env sequences. To determine replicative fitness, these primary and chimeric dual tropic HIV-1 were then competed against HIV-1 reference isolates in U87.CD4 cells expressing CXCR4 or CCR5 or in PBMCs ± entry inhibitors. Contribution of the x4 and r5 clones within the quasispecies of these chimeric or primary HIV-1 isolates were then compared to the frequency of x4, r5, and dual tropic clones within the quasispecies as predicted by phenotypic assays, clonal sequencing, and 454 deep sequencing. RESULTS: In the primary HIV-1 isolates (C19 and C27), subtype C dual tropic clones dominated over x4 clones while pure r5 clones were absent. In two subtype B chimeric viruses (B12 and B19), r5 clones were >100-fold more abundant than x4 or r5/x4 clones. The dual tropic C19 and C27 HIV-1 isolates outcompeted r5 primary HIV-1 isolates, B2 and C3 in PBMCs. When AMD3100 was added or when only U87.CD4.CCR5 cells were used, the B2 and C3 reference viruses now out-competed the r5 component of the dual tropic C19 and C27. In contrast, the same replicative fitness was observed with dualtropic B12 and B19 HIV-1 isolates relative to x4 HIV-1 A8 and E6 or the r5 B2 and C3 viruses, even when the r5 or x4 component was inhibited by maraviroc (or AMD3100) or in U87.CD4.CXCR4 (or CCR5) cells. CONCLUSIONS: In the dual tropic HIV-1 isolates, the x4 replicative fitness is higher than r5 clones but the x4 or x4/r5 clones are typically at low frequency in the intrapatient virus population. Ex vivo HIV propagation promotes outgrowth of the x4 clones and provides an over-estimate of x4 dominance in replicative fitness within dual tropic viruses.

Similar replicative fitness is shared by the subtype B and unique BF recombinant HIV-1 isolates that dominate the epidemic in Argentina.

The HIV-1 epidemic in South America is dominated by pure subtypes (mostly B and C) and more than 7 BF and BC recombinant forms. In Argentina, circulating recombinant forms (CRFs) comprised of subtypes B and F make up more than 50% of HIV infections. For this study, 28 HIV-1 primary isolates were obtained from patients in Buenos Aires, Argentina and initially classified into subtype B (n = 9, 32.1%), C (n = 1, 3.6%), and CRFs (n = 18, 64.3%) using partial pol and vpu-env sequences, which proved to be inconsistent and inaccurate for these phylogenetic analyses. Near full length genome sequences of these primary HIV-1 isolates revealed that nearly all intersubtype BF recombination sites were unique and countered previous "CRF" B/F classifications. The majority of these Argentinean HIV-1 isolates were CCR5-using but 4 had a dual/mixed tropism as predicted by both phenotypic and genotypic assays. Comparison of the replicative fitness of these BF primary HIV-1 isolates to circulating B, F, and C HIV-1 using pairwise competitions in peripheral blood mononuclear cells (PBMCs) indicated a similarity in fitness of these BF recombinants to subtypes B and F HIV-1 (of the same co-receptor usage) whereas subtype C HIV-1 was significantly less fit than all as previously reported. These results suggest that the multitude of BF HIV-1 strains present within the Argentinean population do not appear to have gained replicative fitness following recent B and F recombination events.

Impact of mutations in highly conserved amino acids of the HIV-1 Gag-p24 and Env-gp120 proteins on viral replication in different genetic backgrounds.

It has been hypothesized that a single mutation at a highly conserved amino acid site (HCS) can be severely deleterious to HIV in most if not all isolate-specific genetic backgrounds. Consequently, potentially universal HIV-1 vaccines exclusively targeting highly conserved regions of the viral proteome have been proposed. To test this hypothesis, we examined the impact of 10 Gag-p24 and 9 Env-gp120 HCS single mutations on viral fitness. In the original founder sequence of the subject in whom these mutations were identified, all Gag-p24 HCS mutations significantly reduced viral replication fitness, including 7 that were lethal. Similar results were obtained at 9/10 sites when the same mutations were introduced into the founder sequences of two epidemiologically unlinked subjects. In contrast, none of the 9 Env-gp120 HCS mutations were lethal in the original founder sequence, and four had no fitness cost. Hence, HCS mutations in Gag-p24 are likely to be severely deleterious in different HIV-1 subtype B backgrounds; however, some HCS mutations in both Gag-p24 and Env-gp120 fragments can be well tolerated. Therefore, when designing HIV-1 immunogens that are intended to force the virus to nonviable escape pathways, the fitness constraints on the HIV segments included should be considered beyond their conservation level.

Treatment failure and drug resistance is more frequent in HIV-1 subtype D versus subtype A-infected Ugandans over a 10-year study period.

OBJECTIVES: To determine the impact of HIV-1 subtype on treatment outcomes and the emergence of drug resistance in the resource limited setting of Kampala, Uganda. DESIGN: The Joint Clinical Research Centre (JCRC) in Kampala, Uganda has provided over 2000 drug-resistant genotypes (DRGs) over the past 10 years as standard of care for patients failing therapy and 1403 from treatment-naive and experienced patients over the past 10 years have been analyzed for this study. METHOD: Viral loads, CD4 cell count, treatment histories and other relevant clinical data was compared with the infecting HIV-1 subtype and DRGs of Ugandan patients failing treatment. RESULTS: Patients failing HAART with DRGs (n = 937) were more frequently infected with subtype D than expected on the basis of the subtype distribution in the treatment-naive population (n = 655) in Kampala (P < 0.001). Higher proportions of treatment failures among subtype D-infected patients were driven by resistance to nucleoside reverse transcriptase inhibitors (NRTI) (P < 0.0002) more than to non-NRTIs (P > 0.04) or protease inhibitors. CONCLUSION: Higher rates of treatment failure among subtype D as compared with subtype A-infected Ugandans was analogous to the faster disease progression in subtype D-infected patients. The mechanism(s) by which drug resistance may emerge faster in subtype D HIV-1 may relate to higher replicative fitness and increased propensity for a CXCR4 tropism.

Effect of natural polymorphisms in the HIV-1 CRF02_AG protease on protease inhibitor hypersusceptibility.

Hypersusceptibility (HS) to inhibition by different antiretroviral drugs (ARVs) among diverse HIV-infected individuals may be a misnomer because clinical response to treatment is evaluated in relation to subtype B infections while drug susceptibility of the infecting virus, regardless of subtype, is compared to a subtype B HIV-1 laboratory strain (NL4-3 or IIIB). Mounting evidence suggests that HS to different ARVs may result in better treatment outcome just as drug resistance leads to treatment failure. We have identified key amino acid polymorphisms in the protease coding region of a non-B HIV-1 subtype linked to protease inhibitor HS, namely, 17E and 64M in CRF02_AG. These HS-linked polymorphisms were introduced in the BD6-15 CRF02_AG molecular clone and tested for inhibition using a panel of protease inhibitors. In general, suspected HS-linked polymorphisms did increase susceptibility to specific protease inhibitors such as amprenavir and atazanavir, but the combination of the 17E/64M polymorphisms showed greater HS. These two mutations were found at low frequencies but linked in a sequence database of over 700 protease sequences of CRF02_AG. In direct head-to-head virus competitions, CRF02_AG harboring the 17E/64M polymorphisms also had higher replicative fitness than did the 17E or the 64M polymorphism in the CFR02_AG clone. These findings suggest that subtype-specific, linked polymorphisms can result in hypersusceptibility to ARVs. Considering the potential benefit of HS to treatment outcome, screening for potential HS-linked polymorphisms as well as preexisting drug resistance mutations in treatment-naïve patients may guide the choice of ARVs for the best treatment outcome.