Detailed characterization of transmitted HIV-1 variants in Uganda is fundamentally important to inform vaccine design, yet studies on the transmitted full-length strains of subtype D viruses are limited. Here, we amplified single genomes and characterized viruses, some of which were previously classified as subtype D by sub-genomic pol sequencing that were transmitted in Uganda between December 2006 to June 2011. Analysis of 5′ and 3′ half genome sequences showed 73% (19/26) of infections involved single virus transmissions, whereas 27% (7/26) of infections involved multiple variant transmissions based on predictions of a model of random virus evolution. Subtype analysis of inferred transmitted/founder viruses showed a high transmission rate of inter-subtype recombinants (69%, 20/29) involving mainly A1/D, while pure subtype D variants accounted for one-third of infections (31%, 9/29). Recombination patterns included a predominance of subtype D in the gag/pol region and a highly recombinogenic envelope gene. The signal peptide-C1 region and gp41 transmembrane domain (Tat2/Rev2 flanking region) were hotspots for A1/D recombination events. Analysis of a panel of 14 transmitted/founder molecular clones showed no difference in replication capacity between subtype D viruses (n = 3) and inter-subtype mosaic recombinants (n = 11). However, individuals infected with high replication capacity viruses had a faster CD4 T cell loss. The high transmission rate of unique inter-subtype recombinants is striking and emphasizes the extraordinary challenge for vaccine design and, in particular, for the highly variable and recombinogenic envelope gene, which is targeted by rational designs aimed to elicit broadly neutralizing antibodies.

HIV care programs in resource-limited settings have hitherto concentrated on antiretroviral therapy (ART) access, but HIV drug resistance is emerging. In a cross-sectional study of HIV-positive adults on ART for ≥6 months enrolled into a prospective cohort in Uganda, plasma HIV RNA was measured and genotyped if ≥1000 copies/ml. Identified Drug resistance mutations (DRMs) were interpreted using the Stanford database, 2009 WHO list of DRMs and the IAS 2014 update on DRMs, and examined and tabulated by ART drug classes. Between July 2013 and August 2014, 953 individuals were enrolled, 119 (12.5%) had HIV-RNA ≥1000 copies/ml and 110 were successfully genotyped; 74 (67.3%) were on first-line and 36 (32.7%) on second-line ART regimens. The predominant HIV-1 subtypes were D (34.5%), A (33.6%) and Recombinant forms (21.8%). The commonest clinically significant major resistance mutations associated with the highest levels of reduced susceptibility or virological response to the relevant Nucleoside Reverse Transcriptase Inhibitor (NRTI) were; the Non-thymidine analogue mutations (Non-TAMS) M184V—20.7% and K65R—8.0%; and the TAMs M41L and K70R (both 8.0%). The major Non-NRTI (NNRTI) mutations were K103N—19.0%, G190A—7.0% and Y181C—6.0%. A relatively nonpolymorphic accessory mutation A98G—12.0% was also common. Seven of the 36 patients on second line ART had major Protease Inhibitor (PI) associated DRMS including; V82A—7.0%, I54V, M46I and L33I (all 5.0%). Also common were the accessory PI mutations L10I—27%, L10V—12.0% and L10F—5.0% that either reduce PI susceptibility or increase the replication of viruses containing PI-resistance mutations. Of the 7 patients with major PI DRMs, five had high level resistance to ritonavir boosted Lopinavir and Atazanavir, with Darunavir as the only susceptible PI tested. In resource-limited settings, HIV care programs that have previously concentrated on ART access, should now consider availing access to routine HIV viral load monitoring, targeted HIV drug resistance testing and availability of third-line ART regimens.

The General Population Cohort (GPC) in south-western Uganda has a low HIV-1 incidence rate (25 years (aOR = 1.52; 95% CI, 1.16–2.0) and being a resident in the GPC (aOR = 6.90; 95% CI, 5.22–9.21). Phylogeographic analysis showed significant viral dissemination (Bayes Factor test, BF > 3) from the GPC without significant viral introductions (BF < 3) into the GPC. The findings suggest localized HIV-1 transmission in the GPC. Intensifying geographically focused combination interventions in the GPC would contribute towards controlling HIV-1 infections.

The observation that HIV-1 subtype D progresses faster to disease than subtype A prompted us to examine cytokine levels early after infection within the predominant viral subtypes that circulate in Uganda and address the following research questions: (1) Do cytokine levels vary between subtypes A1 and D? (2) Do cytokine profiles correlate with disease outcomes? To address these questions, HIV-1 subtypes were determined by population sequencing of the HIV-1 pol gene and 37 plasma cytokine concentrations were evaluated using V-Plex kits on Meso Scale Discovery platform in 65 recent sero-converters. HIV-1 subtype D (pol) infections exhibited significantly higher median plasma concentrations of IL-5, IL-16, IL-1α, IL-7, IL-17A, CCL11 (Eotaxin-1), CXCL10 (IP-10), CCL13 (MCP-4) and VEGF-D compared to subtype A1 (pol) infections. We also found that IL-12/23p40 and IL-1α were associated with faster CD4⁺T cell count decline, while bFGF was associated with maintenance of CD4+ counts above 350 cells/microliter. Our results suggest that increased production of cytokines in early HIV infection may trigger a disruption of the immune environment and contribute to pathogenic mechanisms underlying the accelerated disease progression seen in individuals infected with HIV-1 subtype D in Uganda.

The ability to efficiently establish a new infection is a critical property for human immunodeficiency virus type 1 (HIV-1). Although the envelope protein of the virus plays an essential role in receptor binding and internalization of the infecting virus, the structural proteins, the polymerase and the assembly of new virions may also play a role in establishing and spreading viral infection in a new host. We examined Ugandan viruses from newly infected patients and focused on the contribution of the Gag-Pol genes to replication capacity. A panel of Gag-Pol sequences generated using single genome amplification from incident HIV-1 infections were cloned into a common HIV-1 NL4.3 pol/env backbone and the influence of Gag-Pol changes on replication capacity was monitored. Using a novel protein domain approach, we then documented diversity in the functional protein domains across the Gag-Pol region and identified differences in the Gag-p6 domain that were frequently associated with higher in vitro replication.

Initial interactions between HIV-1 and the immune system at mucosal exposure sites play a critical role in determining whether the virus is eliminated or progresses to establish systemic infection. The virus that successfully crosses the mucosal barrier to establish infection in the new host is referred to as the transmitted/founder (TF) virus. Following mucosal HIV-1 transmission, type 1 interferons (IFN-I) are rapidly induced at sites of initial virus replication. The resistance of TF variants to these antiviral effects of the IFN-I has been studied among HIV-1 subtypes B and C. However, their role in restricting HIV-1 replication among subtypes D and AD recombinant remains unexplored. This study assessed the sensitivity of HIV-1 subtype D and AD recombinant TF viruses to IFN-I by infecting peripheral blood mononuclear cells in vitro with infectious molecular clones of these viruses. Cells were exposed to varying concentrations of interferon-α and interferon-β, and viral replicative capacity was measured using HIV-1 p24 antigen ELISA from culture supernatants. Sensitivity to IFN-I was quantified based on viral replication levels. The results showed that interferon-α was more effective in inhibiting viral replication than interferon-β, regardless of the varying amounts of IFN-I used. However, recombinant AD viruses were found to be more resistant to the antiviral effects of IFN-I compared to subtype D viruses. These findings highlight the differential sensitivity of HIV-1 subtypes AD recombinant and D TF viruses to IFN-I and underscore the potential of IFN-I as a therapeutic strategy to target TF viruses and reduce HIV-1 transmission, particularly in populations where subtype D is prevalent.