Unique Phenotypic Characteristics of Recently Transmitted HIV-1 Subtype C Envelope Glycoprotein gp120: Use of CXCR6 Coreceptor by Transmitted Founder Viruses

Abstract

Adequate information on the precise molecular and biological composition of the viral strains that establish HIV infection in the human host will provide effective means of immunization against HIV infection. In an attempt to identify the transmitted founder (TF) virus and differentiate the biological properties and infectious potential of the TF virus from those of the population of the early transmitted viruses, 250 patient-derived gp120 envelope glycoproteins were cloned in pMN-K7-Luc-IRESs-NefΔgp120 to obtain chimeric viruses. Samples were obtained from eight infants who had recently become infected with HIV through mother-to-child transmission (MTCT) and two adults who acquired infection through the heterosexual route and were in the chronic stage of infection. Among the 250 clones tested, 65 chimeric viruses were infectious, and all belonged to HIV-1 subtype C. The 65 clones were analyzed for molecular features of the envelope, per-infectious-particle infectivity, coreceptor tropism, drug sensitivity, and sensitivity to broadly neutralizing antibodies. Based on genotypic and phenotypic analysis of the viral clones, we identified 10 TF viruses from the eight infants. The TF viruses were characterized by shorter V1V2 regions, a reduced number of potential N-linked glycosylation sites, and a higher infectivity titer compared to the virus variants from the adults in the chronic stage of infection. CXCR6 coreceptor usage, in addition to that of the CCR5 coreceptor, which was used by all 65 chimeric viruses, was identified in 13 viruses. The sensitivity of the TF variants to maraviroc and a standard panel of neutralizing monoclonal antibodies (VRC01, PG09, PG16, and PGT121) was found to be much lower than that of the virus variants from the adults in the chronic stage of infection.IMPORTANCE Tremendous progress has been made during the last three and half decades of HIV research, but some significant gaps continue to exist. One of the frontier areas of HIV research which has not seen a breakthrough yet is vaccine research, which is because of the enormous genetic diversity of HIV-1 and the unique infectious fitness of the virus. Among the repertoire of viral variants, the virus that establishes successful infection (transmitted founder [TF] virus) has not been well characterized yet. An insight into the salient features of the TF virus would go a long way toward helping with the design of an effective vaccine against HIV. Here we studied the biological properties of recently transmitted viruses isolated from infants who acquired infection from the mother and have come up with unique characterizations for the TF virus that establishes infection in the human host.

Keywords: amino acid diversity; coreceptor tropism; human immunodeficiency virus; mother-to-child transmission; resistance to neutralization; transmitted founder virus.

FIG 1

Relative infectious potential of chimeric viruses on a per-infectious-particle basis. Infectivity values for chronic and recently transmitted viruses are expressed in terms of the number of relative light units (RLU) as log₁₀ values. Particle infectivity is defined by the infectious titer determined on TZM-bl cells. (a) Particle infectivity based on the number of RLU for each individual is presented. The box plot represents the infectivity of a single viral particle of multiple variants from each infected individual. Infections were performed in TZM-bl cells with virus at an MOI of 0.005 in the presence of DEAE-dextran. Pairwise comparisons were performed using the Mann-Whitney test, and a P value of <0.05 was considered statistically significant. (b) Particle infectivity of the chronic control, recently transmitted, and TF viruses. Pairwise comparisons were performed using the Mann-Whitney test, and a P value of <0.05 was considered statistically significant.

FIG 2

Coreceptor tropism and viral entry. The expression of luciferase upon infection with GHOST(3) cells (CXCR4⁺, CCR5⁺, CXCR4-CCR5⁺, and CXCR6⁺) by the recombinant viruses was measured and used for the identification of coreceptor usage. (a) The bar diagram represents the number of viral isolates that were identified as R5-, R5X4-, and R5X4X6-tropic based on the coreceptors used for viral entry. (b) Confocal microscopy images of green fluorescent protein expression by IN_09.09 viral isolates that use the R5, X4, and X6 coreceptors for viral entry. NC, no-drug control. (c) Quantification of p24 for detection of cellular entry by the two R5X4X6 chimeric viruses in CD8-depleted PBMCs. The experiments were performed in duplicate. The mean (SD) is presented.

FIG 3

Half-maximal effective concentration (EC₅₀) of a CCR5 antagonist for suppression of infection by chimeric viral variants. TZM-bl cells were infected with the chimeric virus at an MOI of 0.005 and cultured in the presence of the CCR5 antagonist maraviroc at concentrations ranging from 1 × 10⁴ nM to 1 × 10⁻³ nM with 10-fold serial dilutions. The box plots represent the EC₅₀ of maraviroc for each viral variant from the individuals (a), the susceptibilities of viral isolates derived from later and early stages of infection to maraviroc (b), and the susceptibility difference between R5- and R5X6-capable early-transmitted viruses (c). Inhibition data from replicates were plotted using GraphPad Prism software, and EC₅₀ were determined using variable-slope nonlinear regression analysis. Experiments were carried out in triplicate in two biological replicates. The statistical comparisons were performed using the Kruskal-Wallis test (among the individual patients) and the Mann-Whitney test (between groups), and a P value of <0.05 was considered statistically significant. ns, not significant.

FIG 4

Neutralizing potential of a panel of 4 monoclonal antibodies against the chimeric viruses. The figure represents the potency of neutralization of viral isolates by the VRC01, PG09, PG16, and PGT121 monoclonal antibodies. Twofold dilutions of all MAbs, except VRC01, starting from 1 μg/ml and going up to 8 serial dilutions, were tested for their potency against the 65 infectious viral variants; VRC01 was tested at a higher concentration of 5 μg/ml. Data from replicates were plotted using GraphPad Prism software, and IC₅₀ were determined using variable-slope nonlinear regression analysis. Experiments were carried out in duplicate. (a) The IC₅₀ of each dot represents the amount of MAbs required to neutralize half of the virus in each patient. (b) The difference in color intensity in the heatmap indicates the potency of neutralization. The darker and lighter colors indicate a stronger and a weaker neutralization potency of the virus, respectively. (c) The box plot represents the neutralization potency of MAbs against the TF and chronic viruses. The statistical comparisons were performed using the Mann-Whitney test (between groups), and a P value of <0.05 was considered statistically significant.

FIG 5

Representation of neutralization profile in association with coreceptor tropism. The graph depicts the percent inhibition of chronic control R5-capable and recently transmitted (R5 and R5X6) viruses against neutralizing monoclonal antibodies.