Infecting HIV-1 Subtype Predicts Disease Progression in Women of Sub-Saharan Africa

Abstract

Introduction: Long-term natural history cohorts of HIV-1 in the absence of treatment provide the best measure of virulence by different viral subtypes.

Methods: Newly HIV infected Ugandan and Zimbabwean women (N=303) were recruited and monitored for clinical, social, behavioral, immunological and viral parameters for 3 to 9.5years.

Results: Ugandan and Zimbabwean women infected with HIV-1 subtype C had 2.5-fold slower rates of CD4 T-cell declines and higher frequencies of long-term non-progression than those infected with subtype A or D (GEE model, P<0.001), a difference not associated with any other clinical parameters. Relative replicative fitness and entry efficiency of HIV-1 variants directly correlated with virulence in the patients, subtype D>A>C (P<0.001, ANOVA).

Discussion: HIV-1 subtype C was less virulent than either A or D in humans; the latter being the most virulent. Longer periods of asymptomatic HIV-1 subtype C could explain the continued expansion and dominance of subtype C in the global epidemic.

Keywords: Africa; Disease progression; HIV-1 diversity; Pathogenesis; Subtypes.

Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Figures

Fig. 1

The number of people living with HIV-1 between 1980 and 2015 in the four UNAIDS Regions with the greatest number of infections as well as globally. The distribution of subtypes in 1990 and 2015 are shown in the inset pie graphs, scaled by the number of infections. Relative amounts of subtype C infections per UNAIDS Region are reported on the pie graphs and via red shading. Data compiled from the World Health Organization, UNAIDS and published literature.

Fig. 2

CD4 + cell declines in HIV-1 infected Ugandan (n = 112) and Zimbabwean women (n = 174) during the natural course of disease. (a) The mean loss of CD4 cells per mm3 per week of infection in Ugandan and Zimbabwean women and (c) in those women infected with subtypes A, C, and D. (b) The percentage of rapid progressors and slow progressors/controllers in Ugandan and Zimbabwean women and (d) in those women infected with subtypes A, C, and D. Rapid progressors are defined as HIV-1 infected women with a drop to 200 CD4 cell counts/mm3 within 2 years of infection and sustained viral RNA loads > 2000 copies/ml. Slow progressors/controllers had viral RNA loads stable CD4 cell counts above 350 and viral loads < 2000 copies/ml in plasma for > 3 years.

Fig. 3

Comparing disease progression in a subset of 56 women in Uganda and Zimbabwe infected by different subtypes. (a)(b)(c)(d)(e) CD4 cell counts/ml were plotted over time in subtype C infected women defined as slow progressors/controllers (a), as typical progressors (b), in subtype A infected women defined as slow progressors/controllers (c), as typical progressors (d), and in subtype D infected women defined as typical progressors (e). (f)(g)(h)(i)(j) Viral RNA loads in plasma of subtype C slow progressors, C typical progressors, A slow progressors, A typical progressors and D typical progressors, respectively.

Fig. 4

Relative replicative fitness of HIV-1 subtype A, B, C, and D in human peripheral blood mononuclear cells. (a) 12 subtype A, 8 subtype D, and 27 subtype C HIV-1 primary isolates were competed against 25 subtype B HIV-1 isolates in PHA-activated, IL2-treated PBMCs of HIV-negative donors in series of published and unpublished studies. All competitions in PBMCs involved the use of 0.001 infectious units of virus to 1 cell. The relative production of two viruses in each competition was measured by a radiolabeled heteroduplex tracking assays or using next generation sequencing (Abraha et al., 2009, Ball et al., 2003, Arien et al., 2005). Production of individual HIV-1 isolates in a dual infection (f0) divided by its initial proportion in the inoculum (i0) is referred to as the relative fitness (w = f0/i0). In this study, w is expressed as percent replication of the subtype A, C, or D HIV-1 isolates relative to subtype B HIV-1. Assay sensitivity is 100-fold differences in replicative fitness. (b) The relative replicative fitness of the subtype B HIV-1 isolates in competition with the subtype A, C, and D HIV-1 (the reciprocal of a). Symbols with thick outlines represent replicative fitness of the classified virus at > 100-fold or < 0.01-fold to competitor virus. The shaded symbols represent the replicative fitness values of primary HIV-1 isolates. The open/white symbols represent the replicative fitness values of replication-competent chimeric viruses containing the env gene of a subtype A, C, or D virus within the NL4-3 subtype B backbone. (c) and (d) Replicative fitness of chimeric viruses derived from env gene of 5 subtype A, 3 subtype D, and 5 subtype C AHI of this cohort. These 13 AHI chimeric viruses were competed against the reference subtype B HIV-1 isolates, B-QO (c) and B-K44 (triplicate).

Fig. 5

Function, cell-to-cell transmission efficiency and viral fusion of the Env glycoprotein derived from acute subtype A, C, and D HIV-1 infections. The HIV-1 env gene of acute/early infections in Ugandan and Zimbabwean women was cloned into the pREC-nfl HIV-1 genomic vector by yeast-based recombination/gap repair as described. The function and co-receptor usage of the Env glycoprotein from these 14 subtype A, 4 subtype D, and 16 subtype C acute/early infections was accessed using the Veritrop cell-to-cell fusion assay. None of the 34 Env produced in the context of virus-like particles (VLPs) could mediate cell fusion via the CXCR4 co-receptor within U87.CD4.CXCR4 cells (below negative control and 1000 RLUs) (data below range of graph in (a)). (a) Cell-to-cell fusion between the U87.CD4.CCR5 target cells expressing Firefly Luciferase upon Tat/Rev-mediate expression from pDM1.1 and the 293T effector cells expressing the 34 Env glycoproteins in context with HIV-1 VLPs. The NL4-3 nfl VLPs are morphologically identical to wild type HIV but is incapable of reverse transcription and cannot induce luciferase expression in the target cells. (b) Schematic of the time-of-drug-addition experiment using the Veritrop assay. 2 μM of Enfuvirtide was added at 30 min to 10 h post incubation of the effector and target cells. (c) Box plot of the time to 50% inhibition by Enfuvirtide of cell-to-cell fusion mediated by 34 Env glycoproteins expressed in context with the HIV-1 VLPs. (c) Schematic of the viral fusion assay which can be monitored by HIV-1 carrying a BlaM-Vpr fusion protein that cleaves the CCF2 dye in target cells and changes the fluorescent spectrum. (d) Relative entry over time (0–600 min) into U87.CD4.CCR5 cells by the chimeric HIV nfl carrying the 14 subtype A, 4 subtype D, and 16 subtype C Env glycoproteins from acute/early infections. (e) Spectral shift curves and box plot of the time required for maximal virus entry into U87.CD4.CCR5 cells carrying the CCF2 dye.