No detectable differences in Nef-mediated downregulation of HLA-I and CD4 molecules among HIV-1 group M lineages circulating in Cameroon, where the pandemic originated.

HIV-1 group M (HIV-1M) lineages downregulate HLA-I and CD4 expression via their Nef proteins. We hypothesized that these Nef functions may be partially responsible for the differences in prevalence of viruses from different lineages that co-circulate within an epidemic. Here, we characterized these two Nef activities in HIV-1M isolates from Cameroon, where multiple variants have been circulating since the pandemic's origin. Single HIV-1 Nef clones from 234 HIV-1-ART naïve individuals living in remote villages and two cosmopolitan cities of Cameroon, sampled between 2000 and 2013, were isolated from plasma HIV RNA and analyzed for their capacity to downregulate HLA-I and CD4 molecules. We found that, despite a large degree of within- and inter- lineage variation, the ability of Nef to downregulate HLA-I was similar across these different viruses. Moreover, Nef-mediated CD4 downregulation activity was also well conserved across the different lineages found in Cameroon. In addition, we observed a trend towards higher HLA-I downregulation activity of viruses circulating in the cosmopolitan cities versus the remote villages, whereas the CD4 downregulation activities were similar across the two settings. Furthermore, we noted a significant decline of HLA-I downregulation activity from 2000 to 2013, providing additional evidence supporting the attenuation of the global HIV-1M population over time. Finally, we identified 18 amino acids associated with differential HLA-I downregulation and 13 amino acids associated with differential CD4 downregulation within the dominant CRF02_AG lineage. Our lack of observation of HIV lineage-related differences in Nef-mediated HLA-I and CD4 downregulation function suggests that these activities do not substantively influence the prevalence of different HIV-1M lineages in Cameroon.

Structural Features and Genetic Diversity in Gag Gene of Rare HIV-1 Subtypes from the Democratic Republic of Congo.

Type-1 HIV (HIV-1) group M (HIV-1M) genetic diversity is highest in the Congo Basin where the epidemic ignited a century ago. HIV-1M has diversified into multiple subtypes, sub-subtypes, and circulating and unique recombinant forms (CRFs/URFs). An unanswered question is why some rare subtypes never reached epidemic levels despite their age. Several studies identified the role of HIV-1M accessory genes nef and vpu in virus adaptation to human hosts and subsequent spread. Other reports also pointed out the pivotal role of gag in transmissibility, virulence, and replication capacity. In this study we characterized the HIV-1 gag gene of 148 samples collected in different localities of the Democratic Republic of the Congo (DRC) between 1997 and 2013. We used nested polymerase chain reaction (PCR) to amplify the whole gag gene. PCR products were sequenced either by Sanger method or by next generation sequencing on Illumina MiSeq or iSeq100 platforms. Generated sequences were used for subsequent analyses using different bioinformatic tools. Phylogenetic analysis of the generated sequences revealed a high genetic diversity with up to 22 different subtypes, sub-subtypes, CRFs. Up to 15% (22/148) URFs were identified, in addition to rare subtypes such as H, J, and K. At least two amino acid motifs present in the gag gene have been shown to modulate HIV-1 replication, budding, and fitness: the P(T/S)AP and the LYPXnL motifs. Structural analysis revealed the presence of P(T/S)AP in all the 148 sequences with the majority (136/148) bearing the PTAP. Three samples presented a duplication of this motif. The LYPXnL motif was identified in 38 of 148 sequences. There was no clear link between the frequency of these motifs and HIV-1M subtypes. In summary, we confirmed a high genetic diversity of HIV-1M in the DRC. We observed the presence of amino acid motifs important for viral replication and budding even in some rare HIV-1 subtypes. Their impact on viral fitness needs be further evaluated by in vitro studies.

Genomic surveillance of SARS-CoV-2 reveals highest severity and mortality of delta over other variants: evidence from Cameroon.

While the SARS-CoV-2 dynamic has been described globally, there is a lack of data from Sub-Saharan Africa. We herein report the dynamics of SARS-CoV-2 lineages from March 2020 to March 2022 in Cameroon. Of the 760 whole-genome sequences successfully generated by the national genomic surveillance network, 74% were viral sub-lineages of origin and non-variants of concern, 15% Delta, 6% Omicron, 3% Alpha and 2% Beta variants. The pandemic was driven by SARS-CoV-2 lineages of origin in wave 1 (16 weeks, 2.3% CFR), the Alpha and Beta variants in wave 2 (21 weeks, 1.6% CFR), Delta variants in wave 3 (11 weeks, 2.0% CFR), and omicron variants in wave 4 (8 weeks, 0.73% CFR), with a declining trend over time (p = 0.01208). Even though SARS-CoV-2 heterogeneity did not seemingly contribute to the breadth of transmission, the viral lineages of origin and especially the Delta variants appeared as drivers of COVID-19 severity in Cameroon.

An epidemiological synthesis of emerging and re-emerging zoonotic disease threats in Cameroon, 2000-2022: a systematic review.

Introduction: Population factors such as urbanization, socio-economic, and environmental factors are driving forces for emerging/re-emerging zoonotic diseases in Cameroon. To inform preparedness and prioritization efforts, this study mapped out epidemiological data (including prevalence) of zoonotic diseases occurring in Cameroon between 2000 and 2022 by demographic factors. Methods: Following the PRISMA guidelines, a protocol was registered in the PROSPERO database (CRD42022333059). Independent reviewers searched the PubMed, Embase, CINAHL, Cochrane, and Scopus databases on May 30, 2022 for relevant articles; duplicates were removed, and the titles, abstracts, and full texts were screened to identify eligible articles. Results: Out of 4142 articles identified, 64 eligible articles were retrieved in the database search and an additional 12 from the cited literature (N = 76). Thirty-five unique zoonoses (viral, bacterial, and parasitic) were indexed, including Cameroon priority zoonoses: anthrax, bovine tuberculosis, Ebola and Marburg virus disease, highly pathogenic avian influenza, and rabies. The number of studies varied by region, ranging from 12 in the Far North to 32 in the Centre Region. The most reported were as follows: brucellosis (random-effects pooled estimate proportion (effect size), ES 0.05%, 95% confidence interval (CI) 0.03-0.07; n = 6), dengue (ES 0.13%, 95% CI 0.06-0.22; n = 12), avian and swine influenza virus (ES 0.10%, 95% CI 0.04-0.20; n = 8), and toxoplasmosis (ES 0.49%, 95% CI 0.35-0.63; n = 11), although I 2 values were greater than 75%, thus there was high inter-study heterogeneity (P < 0.01). Conclusions: This understanding of the distribution of emerging and re-emerging zoonotic threats in Cameroon is vital to effective preventive and resource prioritization measures.

Comparative evaluation of SARS-CoV-2 serological tests shows significant variability in performance across different years of infection and between the tests.

Introduction: While the global COVID-19 pandemic is slowly coming under control, current efforts are focused on understanding the epidemiology of endemic SARS-CoV-2. The tool of choice for doing so remains serological tests that detect SARS-CoV-2 induced antibodies. However, the performance of these tests should be evaluated to ensure they comply with the specific performance criteria desired by each country that they are used in. Methods: Here, we use pre-COVID-19 plasma and plasma from SARS-CoV-2-infected individuals collected in 2020, 2021 and 2022 to evaluate the performance of two commercial Rapid Lateral Flow (RLF) tests (the PANBIO™ COVID-19 IgG/IgM rapid test and the LABNOVATION™ COVID-19 (SARS-CoV-2) IgG/IgM rapid test) and one commercial ELISA test (the PLATELIA™ SARS-CoV-2 total Ab). Results: We find that whereas the specificity of the two RLF tests is ≥ 95%, it was 91% for the ELISA tests. However, at 14 days post-COVID-19 date of diagnosis (DoD), only the ELISA test constantly achieved a sensitivity of ≥80% over all the three years. In addition, the rate of detection of the two RLF tests varied across the years with a sensitivity ranging from <80% in 2021 to >80% in 2022. More importantly the capacity of these two RLF tests to detect IgG antibodies decreased with time. On the contrary, the sensitivity of the ELISA test was still above 80% more than six months post DoD. Conclusion: We recommend that sero-epidemiological surveys focused on testing antibodies should not rely on performances reported by the assay manufacturers. They should include a formal evaluation of the selected assays to ensure its limitations and strengths conform with the data-accuracy requirements of the surveys.

Rapid Increase of Community SARS-CoV-2 Seroprevalence during Second Wave of COVID-19, Yaoundé, Cameroon.

We conducted 2 independent population-based SARS-CoV-2 serosurveys in Yaoundé, Cameroon, during January 27-February 6 and April 24-May 19, 2021. Overall age-standardized SARS-CoV-2 IgG seroprevalence increased from 18.6% in the first survey to 51.3% in the second (p<0.001). This finding illustrates high community transmission during the second wave of COVID-19.

Subtype-specific differences in Gag-protease replication capacity of HIV-1 isolates from East and West Africa.

BACKGROUND: The HIV-1 epidemic in sub-Saharan Africa is heterogeneous with diverse unevenly distributed subtypes and regional differences in prevalence. Subtype-specific differences in disease progression rate and transmission efficiency have been reported, but the underlying biological mechanisms have not been fully characterized. Here, we tested the hypothesis that the subtypes prevalent in the East Africa, where adult prevalence rate is higher, have lower viral replication capacity (VRC) than their West African counterparts where adult prevalence rates are lower. RESULTS: Gag-protease sequencing was performed on 213 and 160 antiretroviral-naïve chronically infected participants from West and East Africa respectively and bioinformatic tools were used to infer subtypes and recombination patterns. VRC of patient-derived gag-protease chimeric viruses from West (n = 178) and East (n = 114) Africa were determined using a green fluorescent protein reporter-based cell assay. Subtype and regional differences in VRC and amino acid variants impacting VRC were identified by statistical methods. CRF02_AG (65%, n = 139), other recombinants (14%, n = 30) and pure subtypes (21%, n = 44) were identified in West Africa. Subtypes A1 (64%, n = 103), D (22%, n = 35), or recombinants (14%, n = 22) were identified in East Africa. Viruses from West Africa had significantly higher VRC compared to those from East Africa (p < 0.0001), with subtype-specific differences found among strains within West and East Africa (p < 0.0001). Recombination patterns showed a preference for subtypes D, G or J rather than subtype A in the p6 region of gag, with evidence that subtype-specific differences in this region impact VRC. Furthermore, the Gag A83V polymorphism was associated with reduced VRC in CRF02_AG. HLA-A*23:01 (p = 0.0014) and HLA-C*07:01 (p = 0.002) were associated with lower VRC in subtype A infected individuals from East Africa. CONCLUSIONS: Although prevalent viruses from West Africa displayed higher VRC than those from East Africa consistent with the hypothesis that lower VRC is associated with higher population prevalence, the predominant CRF02_AG strain in West Africa displayed higher VRC than other prevalent strains suggesting that VRC alone does not explain population prevalence. The study identified viral and host genetic determinants of virus replication capacity for HIV-1 CRF02_AG and subtype A respectively, which may have relevance for vaccine strategies.

Elucidation of Early Evolution of HIV-1 Group M in the Congo Basin Using Computational Methods.

The Congo Basin region is believed to be the site of the cross-species transmission event that yielded HIV-1 group M (HIV-1M). It is thus likely that the virus has been present and evolving in the region since that cross-species transmission. As HIV-1M was only discovered in the early 1980s, our directly observed record of the epidemic is largely limited to the past four decades. Nevertheless, by exploiting the genetic relatedness of contemporary HIV-1M sequences, phylogenetic methods provide a powerful framework for investigating simultaneously the evolutionary and epidemiologic history of the virus. Such an approach has been taken to find that the currently classified HIV-1 M subtypes and Circulating Recombinant Forms (CRFs) do not give a complete view of HIV-1 diversity. In addition, the currently identified major HIV-1M subtypes were likely genetically predisposed to becoming a major component of the present epidemic, even before the events that resulted in the global epidemic. Further efforts have identified statistically significant hot- and cold-spots of HIV-1M subtypes sequence inheritance in genomic regions of recombinant forms. In this review we provide ours and others recent findings on the emergence and spread of HIV-1M variants in the region, which have provided insights into the early evolution of this virus.

Dolutegravir-based and low-dose efavirenz-based regimen for the initial treatment of HIV-1 infection (NAMSAL): week 96 results from a two-group, multicentre, randomised, open label, phase 3 non-inferiority trial in Cameroon.

BACKGROUND: Updated WHO guidelines recommend a dolutegravir-based regimen as the preferred first-line treatment for HIV infection and low-dose efavirenz (400 mg) as an alternative. We aimed to report the non-inferior efficacy of dolutegravir compared with efavirenz 400 mg at week 96. METHODS: We did a multicentre, randomised, open label, phase 3 trial in in three hospitals in Yaoundé, Cameroon, in HIV-1 infected antiretroviral-naive adults with an HIV RNA viral load of greater than 1000 copies per mL to compare dolutegravir 50 mg with efavirenz 400 mg (reference treatment), both combined with lamivudine and tenofovir disoproxil fumarate. The primary endpoint was the proportion with a viral load of less than 50 copies per mL at week 48 (10% non-inferiority margin). The study is registered with ClinicalTrials.gov, NCT02777229 and is ongoing. FINDINGS: Between July, 2016, and August, 2019, of 820 patients assessed, 613 were randomly assigned to receive at least one dose of study medication, with 310 in the dolutegravir group and 303 in the efavirenz 400 mg group. At week 96 in the intention-to-treat analysis, 229 (74%) of 310 patients receiving dolutegravir and 219 (72%) of 303 patients receiving efavirenz, achieved plasma HIV-1 RNA less than 50 copies per mL (difference 1·6%, 95% CI -5·4 to 8·6; p=0.66). Viral load suppression was reached significantly more rapidly in the dolutegravir group (p<0·001). Virological failure (>1000 copies per mL) was observed in 27 patients (eight in the dolutegravir group, among which, three women switched to efavirenz 600 mg because of the dolutegravir teratogeneicity signal, and 19 in the efavirenz 400 mg group). No acquired resistance mutations to dolutegravir were observed against 17 mutations to efavirenz with or without mutations to lamivudine and tenofovir disoproxil fumarate among the 19 efavirenz 400 mg participants with virological failure. Weight gain was greater in the dolutegravir group (median weight gain, 5·0 kg in the dolutegravir group and 3·0 kg in the efavirenz 400 mg group, p<0·001, and incidence of obesity, 22% in the dolutegravir group and 16% in the efavirenz 400 mg group, p=0·043). The incidence of new WHO HIV-related stage 3 and 4 events was similar in each group (12 [4%] in each group). The two groups had similar rates of serious adverse events (28 [9%] of 310 in the dolutegravir group and 21 [7%] of 303 in the efavirenz 400 mg group). 18 deaths were observed during the 96-week follow-up (eight in the dolutegravir group and ten in the efavirenz 400 mg group). INTERPRETATION: The non-inferior efficacy of the dolutegravir-based regimen and non-emergence of dolutegravir resistance at 96 weeks supports its use as a first-line regimen for antiretroviral-naive adults with HIV-1 infection. Viral load suppression was reached more quickly in the dolutegravir group and weight gain was significantly higher. FUNDING: UNITAID and the French National Agency for AIDS Research.

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.

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.

Patterns of genomic site inheritance in HIV-1M inter-subtype recombinants delineate the most likely genomic sites of subtype-specific adaptation.

Recombination between different HIV-1 group M (HIV-1M) subtypes is a major contributor to the ongoing genetic diversification of HIV-1M. However, it remains unclear whether the different genome regions of recombinants are randomly inherited from the different subtypes. To elucidate this, we analysed the distribution within 82 circulating and 201 unique recombinant forms (CRFs/URFs), of genome fragments derived from HIV-1M Subtypes A, B, C, D, F, and G and CRF01_AE. We found that viruses belonging to the analysed HIV-1M subtypes and CRF01_AE contributed certain genome fragments more frequently during recombination than other fragments. Furthermore, we identified statistically significant hot-spots of Subtype A sequence inheritance in genomic regions encoding portions of Gag and Nef, Subtype B in Pol, Tat and Env, Subtype C in Vif, Subtype D in Pol and Env, Subtype F in Gag, Subtype G in Vpu-Env and Nef, and CRF01_AE inheritance in Vpu and Env. The apparent non-randomness in the frequencies with which different subtypes have contributed specific genome regions to known HIV-1M recombinants is consistent with selection strongly impacting the survival of inter-subtype recombinants. We propose that hotspots of genomic region inheritance are likely to demarcate the locations of subtype-specific adaptive genetic variations.

Unravelling the complicated evolutionary and dissemination history of HIV-1M subtype A lineages.

Subtype A is one of the rare HIV-1 group M (HIV-1M) lineages that is both widely distributed throughout the world and persists at high frequencies in the Congo Basin (CB), the site where HIV-1M likely originated. This, together with its high degree of diversity suggests that subtype A is amongst the fittest HIV-1M lineages. Here we use a comprehensive set of published near full-length subtype A sequences and A-derived genome fragments from both circulating and unique recombinant forms (CRFs/URFs) to obtain some insights into how frequently these lineages have independently seeded HIV-1M sub-epidemics in different parts of the world. We do this by inferring when and where the major subtype A lineages and subtype A-derived CRFs originated. Following its origin in the CB during the 1940s, we track the diversification and recombination history of subtype A sequences before and during its dissemination throughout much of the world between the 1950s and 1970s. Collectively, the timings and numbers of detectable subtype A recombination and dissemination events, the present broad global distribution of the sub-epidemics that were seeded by these events, and the high prevalence of subtype A sequences within the regions where these sub-epidemics occurred, suggest that ancestral subtype A viruses (and particularly sub-subtype A1 ancestral viruses) may have been genetically predisposed to become major components of the present epidemic.

High Degree of HIV-1 Group M (HIV-1M) Genetic Diversity within Circulating Recombinant Forms: Insight into the Early Events of HIV-1M Evolution.

The existence of various highly divergent HIV-1 lineages and of recombination-derived sequence tracts of indeterminate origin within established circulating recombinant forms (CRFs) strongly suggests that HIV-1 group M (HIV-1M) diversity is not fully represented under the current classification system. Here we used a fully exploratory screen for recombination on a set of 480 near-full-length genomes representing the full known diversity of HIV-1M. We decomposed recombinant sequences into their constituent parts and then used maximum-likelihood phylogenetic analyses of this mostly recombination-free data set to identify rare divergent sequence lineages that fall outside the major named HIV-1M taxonomic groupings. We found that many of the sequence fragments occurring within CRFs (including CRF04_cpx, CRF06_cpx, CRF11_cpx, CRF18_cpx, CRF25_cpx, CRF27_cpx, and CRF49_cpx) are in fact likely derived from divergent unclassified parental lineages that may predate the current subtypes, even though they are presently identified as derived from currently defined HIV-1M subtypes. Our evidence suggests that some of these CRFs are descended predominantly from what were or are major previously unidentified HIV-1M lineages that were likely epidemiologically relevant during the early stages of the HIV-1M epidemic. The restriction of these divergent lineages to the Congo basin suggests that they were less infectious and/or simply not present at the time and place of the initial migratory wave that triggered the global epidemic.IMPORTANCE HIV-1 group M (HIV-1M) likely spread to the rest of the world from the Congo basin in the mid-1900s (N. R. Faria et al., Science 346:56-61, 2014, http://dx.doi.org/10.1126/science.1256739) and is today the principal cause of the AIDS pandemic. Here, we show that large sequence fragments from several HIV-1M circulating recombinant forms (CRFs) are derived from divergent parental lineages that cannot reasonably be classified within the nine established HIV-1M subtypes. These lineages are likely to have been epidemiologically relevant in the Congo basin at the onset of the epidemic. Nonetheless, they appear not to have undergone the same explosive global spread as other HIV-1M subtypes, perhaps because they were less transmissible. Concerted efforts to characterize more of these divergent lineages could allow the accurate inference and chemical synthesis of epidemiologically key ancestral HIV-1M variants so as to directly test competing hypotheses relating to the viral genetic factors that enabled the present pandemic.

Near full-length HIV type 1M genomic sequences from Cameroon : Evidence of early diverging under-sampled lineages in the country.

BACKGROUND: Cameroon is the country in which HIV-1 group M (HIV-1M) likely originated and is today a major hotspot of HIV-1M genetic diversity. It remains unclear, however, whether the highly divergent HIV-1M lineages found in this country arose during the earliest phases of the global HIV-1M epidemic, or whether they arose more recently as a result of recombination events between globally circulating HIV-1M lineages. METHODOLOGY: To differentiate between these two possibilities, we performed phylogenetic analyses of the near full genome sequences of nine newly sequenced divergent HIV-1M isolates and 15 previously identified, apparently unique recombinant forms (URFs) from Cameroon. RESULTS: Although two of the new genome sequences were clearly classifiable within subtype G, the remaining seven were highly divergent and phylogenetically branched either outside of, or very near the bases of clades containing the well characterised globally circulating viral lineages that they were most closely related to. Recombination analyses further revealed that these divergent viruses were likely complex URFs. We show, however that substantial portions (>1 Kb) of three of the new genome sequences and 15 of the previously characterised Cameroonian URFs have apparently been derived from divergent parental viruses that branch phylogenetically near the bases of the major HIV-1M clades. CONCLUSIONS AND IMPLICATIONS: Our analyses indicate the presence in Cameroon of contemporary descendants of numerous early-diverging HIV-1M lineages. Further efforts to sample and sequence viruses from such lineages could be crucial both for retracing the earliest evolutionary steps during the emergence of HIV-1M in humans, and accurately reconstructing the ancestral sequences of the major globally circulating HIV-1M lineages.

Phylogenetics of HIV-1 subtype G env: Greater complexity and older origins than previously reported.

HIV-1 subtype G has played an early and central role in the emergent complexity of the HIV-1 group M (HIV-1M) epidemic in central/west Africa. Here, we analysed new subtype G env sequences sampled from 8 individuals in Yaoundé, Cameroon during 2007-2010, together with all publically available subtype G-attributed full-length env sequences with known sampling dates and locations. We inferred that the most recent common ancestor (MRCA) of the analysed subtype G env sequences most likely occurred in ∼1953 (95% Highest Posterior Density interval [HPD] 1939-1963): about 15 years earlier than previous estimates. We found that the subtype G env phylogeny has a complex structure including seven distinct lineages, each likely dating back to the late 1960s or early 1970s. Sequences from Angola, Gabon and the Democratic Republic of Congo failed to group consistently in these lineages, possibly because they are related to more ancient sequences that are poorly sampled. The circulating recombinant form (CRF), CRF06_cpx env sequences but not CRF25_cpx env sequences are phylogenetically nested within the subtype G clade. This confirms that the CRF06_cpx env plausibly was derived through recombination from a subtype G parent, and suggests that the CRF25_cpx env was likely derived from an HIV-1M lineage related to the MRCA of subtype G that has remained undiscovered and may be extinct. Overall, this fills important gaps in our knowledge of the early events in the spread of HIV-1M.

Evaluating potential T-cell epitope peptides for detecting HIV-specific T cell responses in a highly diverse HIV-1 epidemic from Cameroon.

HIV genetic diversity is a major obstacle for vaccine development. To define whether potential T-cell epitope (PTE) peptide usage improves the detection of T cell responses in a highly diverse HIV-1 epidemic, we compared the magnitude, breadth and depth of group M PTE peptide responses to consensus M peptides in Gag and Nef proteins. Gag PTE responses were detected at a higher magnitude, more Nef PTE responses were detected at a cohort (but not individual) level and depth was detected in both Gag and Nef responses.

Challenges in the design of a T cell vaccine in the context of HIV-1 diversity.

The extraordinary variability of HIV-1 poses a major obstacle to vaccine development. The effectiveness of a vaccine is likely to vary dramatically in different populations infected with different HIV-1 subtypes, unless innovative vaccine immunogens are developed to protect against the range of HIV-1 diversity. Immunogen design for stimulating neutralizing antibody responses focuses on "breadth" - the targeting of a handful of highly conserved neutralizing determinants on the HIV-1 Envelope protein that can recognize the majority of viruses across all HIV-1 subtypes. An effective vaccine will likely require the generation of both broadly cross-neutralizing antibodies and non-neutralizing antibodies, as well as broadly cross-reactive T cells. Several approaches have been taken to design such broadly-reactive and cross-protective T cell immunogens. Artificial sequences have been designed that reduce the genetic distance between a vaccine strain and contemporary circulating viruses; "mosaic" immunogens extend this concept to contain multiple potential T cell epitope (PTE) variants; and further efforts attempt to focus T cell immunity on highly conserved regions of the HIV-1 genome. Thus far, a number of pre-clinical and early clinical studies have been performed assessing these new immunogens. In this review, the potential use of these new immunogens is explored.

Striking lack of T cell immunodominance in both a multiclade and monoclade HIV-1 epidemic: implications for vaccine development.

Understanding the impact of HIV diversity on immunological responses to candidate immunogens is critical for HIV vaccine development. We investigated the reactivity and immunodominance patterns of HIV-1 consensus group M Gag and Nef in (i) Cameroon, where individuals infected with the predominant CRF02_AG clade were compared with those infected with diverse non-CRF02_AG clades; and (ii) in a multiclade epidemic, namely Cameroon, compared with a monoclade C epidemic, South Africa. We analyzed 57 HIV-infected individuals from Cameroon and 44 HIV-infected individuals from South Africa for differences in detecting HIV-1 consensus M Gag and Nef T cell responses using the IFN-γ ELISpot assay. We found no difference in the predicted epitope coverage between CRF02_AG and non-CRF02_AG viruses for either Gag or Nef. There were no differences in the magnitude and breadth of responses for CRF02_AG and non-CRF02_AG-infected individuals. In contrast, the specificity of epitope targeting was markedly different between the two groups, with fewer than one third (11/38) of peptides commonly recognized in Gag. Furthermore, only one peptide was commonly recognized by at least three individuals from both AG and non-AG groups, indicating poor immunodominance. For Nef, more than half of all targeted peptides (14/27) were recognized by both groups, and four peptides were commonly targeted by at least three individuals. Three times more peptides were exclusively targeted in the diverse non-CRF02_AG group compared to the CRF02_AG group (10 vs. 3). Of note, similar results were obtained when South Africa, a monoclade C epidemic, and Cameroon, a multiclade epidemic, were compared. The central nature of HIV-1 consensus M sequences resulted in their broad recognition, but failed to identify highly immunodominant peptides between homogeneous and diverse HIV epidemics.