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. 2011 Sep;85(17):8514-27.
doi: 10.1128/JVI.00736-11. Epub 2011 Jun 29.

Phenotypic and immunologic comparison of clade B transmitted/founder and chronic HIV-1 envelope glycoproteins

Affiliations

Phenotypic and immunologic comparison of clade B transmitted/founder and chronic HIV-1 envelope glycoproteins

Craig B Wilen et al. J Virol. 2011 Sep.

Abstract

Sexual transmission of human immunodeficiency virus type 1 (HIV-1) across mucosal barriers is responsible for the vast majority of new infections. This relatively inefficient process results in the transmission of a single transmitted/founder (T/F) virus, from a diverse viral swarm in the donor, in approximately 80% of cases. Here we compared the biological activities of 24 clade B T/F envelopes (Envs) with those from 17 chronic controls to determine whether the genetic bottleneck that occurs during transmission is linked to a particular Env phenotype. To maximize the likelihood of an intact mucosal barrier in the recipients and to enhance the sensitivity of detecting phenotypic differences, only T/F Envs from individuals infected with a single T/F variant were selected. Using pseudotyping to assess Env function in single-round infectivity assays, we compared coreceptor tropism, CCR5 utilization efficiencies, primary CD4(+) T cell subset tropism, dendritic cell trans-infections, fusion kinetics, and neutralization sensitivities. T/F and chronic Envs were phenotypically equivalent in most assays; however, T/F Envs were modestly more sensitive to CD4 binding site antibodies b12 and VRC01, as well as pooled human HIV Ig. This finding was independently validated with a panel of 14 additional chronic HIV-1 Env controls. Moreover, the enhanced neutralization sensitivity was associated with more efficient binding of b12 and VRC01 to T/F Env trimers. These data suggest that there are subtle but significant structural differences between T/F and chronic clade B Envs that may have implications for HIV-1 transmission and the design of effective vaccines.

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Figures

Fig. 1.
Fig. 1.
Phylogenetic relationships of T/F and chronic Envs selected for phenotypic analyses. The tree was constructed from Env amino acid sequences of T/F (red), Chronic 1 control (blue), and Chronic 2 (green) viruses (subtype B reference sequences from the database are shown in black). All sequences were derived by SGA methods; Env sequences from the same individuals form discrete subclusters. A bracket indicates epidemiologically linked infections from Trinidad and Tobago (14). The tree was inferred using maximum-likelihood methods (29). Numbers on nodes indicate posterior probabilities (only values above 0.95 are shown). The scale bar represents 0.05 amino acid substitution per site.
Fig. 2.
Fig. 2.
CCR5 utilization efficiency. (A) Viral pseudotypes were used to infect NP2/CD4/CCR5 cells in the presence of serial dilutions of the CCR5 antagonist maraviroc (MVC). Higher IC50s correspond to Envs that can utilize CCR5 more efficiently, and vice versa. T/F and chronic clade B Envs have similar MVC IC50s (P = 0.79), suggesting that they engage CCR5 comparably. (B) Since some Envs can utilize the MVC-bound conformation of CCR5 and since MVC is a candidate microbicide, we assessed the maximal percent inhibition (MPI) of MVC for each Env. All Envs were sensitive to MVC, and there was no difference in MPI between the T/F and chronic Envs (P = 0.17). All infections were done in at least triplicate in each of at least three independent experiments. Data were analyzed by a Mann-Whitney test.
Fig. 3.
Fig. 3.
CD4+ T cell subset tropism. To assess human CD4+ T subset tropism of the T/F and chronic Envs, cells were infected with Env pseudotypes expressing GFP and then stained and analyzed by flow cytometry. (A) Cells were gated as shown. Infected cells (GFP+) were then back gated on the memory markers CCR7 and CD45RO to evaluate differential subset infection. Naïve, CCR7+ CD45RO; central memory (TCM), CCR7+ CD45RO+; effector memory (TEM), CCR7 CD45RO+; effector memory RA (TEMRA), CCR7 CD45RO. (B) T/F and chronic Envs infected all four CD4+ T cell subsets comparably. TEM and TCM cells were infected most readily, followed by naïve and TEMRA cells. As expected, Envs that could utilize CXCR4 preferentially infected naïve cells compared to Envs that used exclusively CCR5. (C) T/F and chronic Env pseudotypes have comparable overall CD4+ T cell infection frequencies in each of the three donors examined. R5X4-tropic Envs are shown in red, and the one X4-tropic Env is shown in cyan. Tropism was assessed in cells obtained from three different, uninfected normal donors as indicated (ND218, ND335, and ND337). The horizontal lines indicate the mean value for each group of Envs.
Fig. 4.
Fig. 4.
Dendritic cell (DC) trans-infection. To assess differential DC binding and CD4+ T cell trans-infection of T/F and chronic Envs, we pulsed DCs with luciferase-expressing Env pseudotypes and then washed off unbound virus and added CD4+ T cells. Relative light units (RLUs) were then measured as a surrogate for infection. DC trans-infection efficiency was comparable between the T/F and chronic Envs (P = 0.44). Viral input was normalized based upon infectivity on NP2 cell lines. The data shown are from one of at least three independent experiments with cells from different donors, each done in at least triplicate. Data were analyzed by a Mann-Whitney test.
Fig. 5.
Fig. 5.
Entry kinetics and enfuvirtide sensitivity. (A) To examine differences in T/F and chronic Env endocytosis/fusion kinetics, we employed an indirect assay in which viral pseudotypes were bound to NP2 cells in the cold prior to the addition of prewarmed medium. A saturating concentration of enfuvirtide was added at various times postwarming. The time to half-maximal resistance to enfuvirtide (t1/2 max) was then calculated. The T/F and chronic Envs became resistant to enfuvirtide at equal rates (P = 0.55), with all of the Envs acquiring resistance to enfuvirtide more slowly than a prototypic R5-tropic HIV-1 control, JRFL. (B) Enfuvirtide potency, a compound measure of fusion kinetics and affinity, was assessed for all T/F and chronic Envs. There was no difference in enfuvirtide IC50 between the T/F and chronic Envs (P = 0.53), further suggesting that there is no difference in endocytosis/fusion rates between T/F and chronic Envs. Each infection condition was done in triplicate (A) or duplicate (B) for each Env in each of at least three independent experiments. Data were analyzed by a Mann-Whitney test.
Fig. 6.
Fig. 6.
Neutralization sensitivity. (A to H) The sensitivity to monoclonal antibodies b12, VRC01, PG9, and PG16 was assessed on both NP2 cells (A to D) and TZMbl cells (E to H). Neutralization sensitivity on NP2 cells was assessed by determining the maximal percent inhibition (MPI) to 10 μg/ml of the indicated MAb. IC50s were determined in the TZMbl assay. Clade B T/F Envs were more sensitive to b12 and VRC01 than the geographically matched panel of chronic Envs (Chronic 1). To confirm this finding, we assessed an independent panel of clade B chronic Envs from Washington state (Chronic 2). “All chronic” includes clade B chronic panels 1 and 2. (I) Clade B T/F Envs are also more sensitive to clade B HIV Ig on NP2 cells as measured by IC50. P values shown are from Mann-Whitney tests with the corresponding T/F Envs. NP2 and TZMbl experiments were performed in at least three and two independent experiments, respectively.
Fig. 7.
Fig. 7.
Correlation between MAb binding and neutralization. (A and B) Env was expressed on the surface of cells, and then binding to b12 (A) and VRC01 (B) relative to a CD4 control was assessed by ELISA. There is a strong positive correlation between binding and Env pseudotype neutralization sensitivity for both b12 and VRC01 for the T/F Envs and both panels of chronic Envs serving to validate the assay. (C and D) To assess the mechanism of enhanced neutralization sensitivity, we compared b12 (C) and VRC01 (D) binding between T/F and chronic Envs. This suggests that differences in MAb binding explain neutralization differences between T/F and chronic Envs. The data shown are the means from two independent experiments.

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References

    1. Abrahams M. R., et al. 2009. Quantitating the multiplicity of infection with human immunodeficiency virus type 1 subtype C reveals a non-Poisson distribution of transmitted variants. J. Virol. 83:3556–3567 - PMC - PubMed
    1. Alexander M., et al. 2010. Donor and recipient envs from heterosexual human immunodeficiency virus subtype C transmission pairs require high receptor levels for entry. J. Virol. 84:4100–4104 - PMC - PubMed
    1. Balotta C., et al. 1997. Homozygous delta 32 deletion of the CCR-5 chemokine receptor gene in an HIV-1-infected patient. AIDS 11:F67–F71 - PubMed
    1. Bar K. J., et al. 2010. Wide variation in the multiplicity of HIV-1 infection among injection drug users. J. Virol. 84:6241–6247 - PMC - PubMed
    1. Barbas C. F., III, et al. 1992. Recombinant human Fab fragments neutralize human type 1 immunodeficiency virus in vitro. Proc. Natl. Acad. Sci. U. S. A. 89:9339–9343 - PMC - PubMed

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