No Difference in the Prevalence of HIV-1 gag Cytotoxic T-Lymphocyte-Associated Escape Mutations in Viral Sequences from Early and Late Parts of the HIV-1 Subtype C Pandemic in Botswana.

HIV is known to accumulate escape mutations in the gag gene in response to the immune response from cytotoxic T lymphocytes (CTLs). These mutations can occur within an individual as well as at a population level. The population of Botswana exhibits a high prevalence of HLA*B57 and HLA*B58, which are associated with effective immune control of HIV. In this retrospective cross-sectional investigation, HIV-1 gag gene sequences were analyzed from recently infected participants across two time periods which were 10 years apart: the early time point (ETP) and late time point (LTP). The prevalence of CTL escape mutations was relatively similar between the two time points-ETP (10.6%) and LTP (9.7%). The P17 protein had the most mutations (9.4%) out of the 36 mutations that were identified. Three mutations (A83T, K18R, Y79H) in P17 and T190A in P24 were unique to the ETP sequences at a prevalence of 2.4%, 4.9%, 7.3%, and 5%, respectively. Mutations unique to the LTP sequences were all in the P24 protein, including T190V (3%), E177D (6%), R264K (3%), G248D (1%), and M228L (11%). Mutation K331R was statistically higher in the ETP (10%) compared to the LTP (1%) sequences (p < 0.01), while H219Q was higher in the LTP (21%) compared to the ETP (5%) (p < 0.01). Phylogenetically, the gag sequences clustered dependently on the time points. We observed a slower adaptation of HIV-1C to CTL immune pressure at a population level in Botswana. These insights into the genetic diversity and sequence clustering of HIV-1C can aid in the design of future vaccine strategies.

Prediction of Coreceptor Tropism in HIV-1 Subtype C in Botswana.

It remains unknown whether the C-C motif chemokine receptor type 5 (CCR5) coreceptor is still the predominant coreceptor used by Human Immunodeficiency Virus-1 (HIV-1) in Botswana, where the HIV-1 subtype C predominates. We sought to determine HIV-1C tropism in Botswana using genotypic tools, taking into account the effect of antiretroviral treatment (ART) and virologic suppression. HIV-1 gp120 V3 loop sequences from 5602 participants were analyzed for viral tropism using three coreceptor use predicting algorithms/tools: Geno2pheno, HIV-1C Web Position-Specific Score Matrices (WebPSSM) and the 11/25 charge rule. We then compared the demographic and clinical characteristics of people living with HIV (PLWH) harboring R5- versus X4-tropic viruses using χ2 and Wilcoxon rank sum tests for categorical and continuous data analysis, respectively. The three tools congruently predicted 64% of viruses as either R5-tropic or X4-tropic. Geno2pheno and the 11/25 charge rule had the highest concordance at 89%. We observed a significant difference in ART status between participants harboring X4- versus R5-tropic viruses. X4-tropic viruses were more frequent among PLWH receiving ART (χ2 test, p = 0.03). CCR5 is the predominant coreceptor used by HIV-1C strains circulating in Botswana, underlining the strong potential for CCR5 inhibitor use, even in PLWH with drug resistance. We suggest that the tools for coreceptor prediction should be used in combination.

In Silico Prediction of Human Leukocytes Antigen (HLA) Class II Binding Hepatitis B Virus (HBV) Peptides in Botswana.

Hepatitis B virus (HBV) is the primary cause of liver-related malignancies worldwide, and there is no effective cure for chronic HBV infection (CHB) currently. Strong immunological responses induced by T cells are associated with HBV clearance during acute infection; however, the repertoire of epitopes (epi) presented by major histocompatibility complexes (MHCs) to elicit these responses in various African populations is not well understood. In silico approaches were used to map and investigate 15-mers HBV peptides restricted to 9 HLA class II alleles with high population coverage in Botswana. Sequences from 44 HBV genotype A and 48 genotype D surface genes (PreS/S) from Botswana were used. Of the 1819 epi bindings predicted, 20.2% were strong binders (SB), and none of the putative epi bind to all the 9 alleles suggesting that multi-epitope, genotype-based, population-based vaccines will be more effective against HBV infections as opposed to previously proposed broad potency epitope-vaccines which were assumed to work for all alleles. In total, there were 297 unique epi predicted from the 3 proteins and amongst, S regions had the highest number of epi (n = 186). Epitope-densities (Depi) between genotypes A and D were similar. A number of mutations that hindered HLA-peptide binding were observed. We also identified antigenic and genotype-specific peptides with characteristics that are well suited for the development of sensitive diagnostic kits. This study identified candidate peptides that can be used for developing multi-epitope vaccines and highly sensitive diagnostic kits against HBV infection in an African population. Our results suggest that viral variability may hinder HBV peptide-MHC binding, required to initiate a cascade of immunological responses against infection.

Incidence of hepatitis B virus infection among human immunodeficiency virus-infected treatment naïve adults in Botswana.

Hepatitis B virus (HBV) and human immunodeficiency virus (HIV) coinfection is highest in sub-Saharan Africa and results in accelerated clinical outcomes compared with HBV or HIV mono-infection. HBV clearance rates are higher in healthy adults; however, in sub-Saharan Africa, there are limited data on clearance of incident HBV in HIV-infected adults. Therefore, we sought to estimate HBV incidence and HBV surface antigen (HBsAg) clearance in HIV-infected adults in Botswana.This was a retrospective longitudinal study of 442 HIV-1C infected treatment naïve patients enrolled in a previous Botswana Harvard AIDS Institute Partnership study. Archived plasma samples from 435 HIV-infected treatment naïve participants were screened for HBsAg and HBV core antibody (anti-HBc). HBsAg was evaluated annually over a 4-year period, and HBV deoxyribonucleic acid (DNA) levels of HBsAg-positive chronic and incident patients were quantified.Baseline median CD4+ T-cell count was 458 cells/µL [Q1, Q3: 373, 593], and median HIV viral load was 4.15 copies/mL [Q1, Q3: 3.46, 4.64]. Twenty two HBV incident cases occurred, representing an incidence of 3.6/100 person-years [95% CI: 2.2-5.6]. All incident HBV cases with a follow-up sample available for screening (13/22) cleared HBsAg. Detectable HBV viral loads among chronic and incident cases ranged between 5.15 × 10 to 1.4 × 10 IU/L and 1.80 × 10 to 1.7 × 10 IU/mL, respectively.We report high HBV incidence associated with elevated HBV DNA levels despite high CD4+ T-cell counts in HIV-infected patients in Botswana. These incidence cases represent a potential source of HBV transmission in the population. Scaling-up of HIV treatment strategies utilizing antiretroviral therapy regimens with anti-HBV activity coupled with screening for HBV infections in households of the HBsAg-positive cases is recommended.

Molecular Characterization of Near Full-Length Genomes of Hepatitis B Virus Isolated from Predominantly HIV Infected Individuals in Botswana.

The World Health Organization plans to eliminate hepatitis B and C Infections by 2030. Therefore, there is a need to study and understand hepatitis B virus (HBV) epidemiology and viral evolution further, including evaluating occult (HBsAg-negative) HBV infection (OBI), given that such infections are frequently undiagnosed and rarely treated. We aimed to molecularly characterize HBV genomes from 108 individuals co-infected with human immunodeficiency virus (HIV) and chronic hepatitis B (CHB) or OBI identified from previous HIV studies conducted in Botswana from 2009 to 2012. Full-length (3.2 kb) and nearly full-length (~3 kb) genomes were amplified by nested polymerase chain reaction (PCR). Sequences from OBI participants were compared to sequences from CHB participants and GenBank references to identify OBI-unique mutations. HBV genomes from 50 (25 CHB and 25 OBI) individuals were successfully genotyped. Among OBI participants, subgenotype A1 was identified in 12 (48%), D3 in 12 (48%), and E in 1 (4%). A similar genotype distribution was observed in CHB participants. Whole HBV genome sequences from Botswana, representing OBI and CHB, were compared for the first time. There were 43 OBI-unique mutations, of which 26 were novel. Future studies using larger sample sizes and functional analysis of OBI-unique mutations are warranted.