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Enhanced antibody-mediated neutralization of HIV-1 variants that are resistant to fusion inhibitors

View Article: PubMed Central - PubMed

ABSTRACT

Background: HIV-1 typically develops resistance to any single antiretroviral agent. Combined anti-retroviral therapy to reduce drug-resistance development is necessary to control HIV-1 infection. Here, to assess the utility of a combination of antibody and fusion inhibitor treatments, we investigated the potency of monoclonal antibodies at neutralizing HIV-1 variants that are resistant to fusion inhibitors.

Results: Mutations that confer resistance to four fusion inhibitors, enfuvirtide, C34, SC34, and SC34EK, were introduced into the envelope of HIV-1JR-FL, a CCR5-tropic tier 2 strain. Pseudoviruses with these mutations were prepared and used for the assessment of neutralization sensitivity to an array of antibodies. The resulting neutralization data indicate that the potencies of some antibodies, especially of those against the CD4 binding site, V3 loop, and membrane-proximal external region epitopes, were increased by the mutations in gp41 that conferred resistance to the fusion inhibitors. C34-, SC34-, and SC34EK-resistant mutants showed more sensitivity to monoclonal antibodies than enfuvirtide-resistant mutants. An analysis of C34-resistant mutations revealed that the I37K mutation in gp41 HR1 is a key mutation for C34 resistance, low infectivity, neutralization sensitivity, epitope exposure, and slow fusion kinetics. The N126K mutation in the gp41 HR2 domain contributed to C34 resistance and neutralization sensitivity to anti-CD4 binding site antibodies. In the absence of L204I, the effect of N126K was antagonistic to that of I37K. The results of a molecular dynamic simulation of the envelope trimer confirmation suggest that an I37K mutation induces the augmentation of structural fluctuations prominently in the interface between gp41 and gp120. Our observations indicate that the “conformational unmasking” of envelope glycoprotein by an I37K mutation is one of the mechanisms of neutralization sensitivity enhancement. Furthermore, the enhanced neutralization of C34-resistant mutants in vivo was shown by its high rate of neutralization by IgG from HIV patient samples.

Conclusions: Mutations in gp41 that confer fusion inhibitor resistance exert enhanced sensitivity to broad neutralizing antibodies (e.g., VRC01 and 10E8) and other conventional antibodies developed in HIV-1 infected patients. Therefore, next-generation fusion inhibitors and monoclonal antibodies could be a potential combination for future regimens of combined antiretroviral therapy.

Electronic supplementary material: The online version of this article (doi:10.1186/s12977-016-0304-7) contains supplementary material, which is available to authorized users.

No MeSH data available.


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Comparison of infectivity and envelope content among fusion inhibitor-resistant mutants. a The infectivity levels of ENF-resistant mutants, V38A, N43D, and Q40H, and of C34-resistant mutants, I37K, N126K, L204I, I37K/N126K, I37K/L204I, N126K/L204I, and C34r, are shown. The TCID50 and p24 concentrations were determined by single round infection assays using TZM-bl cells and ELISAs, respectively. Infectivity is shown as the TCID50/p24 amount ratio. b The envelope content per virion was compared among C34 resistance mutants. The concentrations of envelope and p24 in pseudovirus stocks were determined by ELISAs. As negative control pseudovirus expressing no envelope was used. The envelope content is shown as the envelope/p24 amount ratio. The results are expressed as the means ± SEs of three independent experiments. Asterisks correspond to values that are statistically different from those of the WT (p < 0.05 as calculated using the Mann–Whitney U test)
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Fig5: Comparison of infectivity and envelope content among fusion inhibitor-resistant mutants. a The infectivity levels of ENF-resistant mutants, V38A, N43D, and Q40H, and of C34-resistant mutants, I37K, N126K, L204I, I37K/N126K, I37K/L204I, N126K/L204I, and C34r, are shown. The TCID50 and p24 concentrations were determined by single round infection assays using TZM-bl cells and ELISAs, respectively. Infectivity is shown as the TCID50/p24 amount ratio. b The envelope content per virion was compared among C34 resistance mutants. The concentrations of envelope and p24 in pseudovirus stocks were determined by ELISAs. As negative control pseudovirus expressing no envelope was used. The envelope content is shown as the envelope/p24 amount ratio. The results are expressed as the means ± SEs of three independent experiments. Asterisks correspond to values that are statistically different from those of the WT (p < 0.05 as calculated using the Mann–Whitney U test)

Mentions: As shown in Fig. 5a, the infectivity of the C34r mutant was 1000-fold lower than of the WT, which can be compared with the mild effect on infectivity of mutations conferring ENF resistance. The I37K mutant, as well as combination mutants that included I37K, also showed a significant decrease in infectivity compared with the WT, similar to the C34r mutant, suggesting that I37K is the major mutation responsible for the reduced infectivity. Two ENF-resistant mutants, N43D and Q40H, showed low infectivity compared with WT (approximately tenfold), but the infectivity of the V38A mutant was at the same level as the WT. These data demonstrate that I37K is critical for C34 resistance in JR-FL, but its presence imposes a significant fitness cost (Fig. 5a). The effect of N126K on C34 resistance and its fitness cost were lower than that of I37K. We are unable to comment on the role of L204I in fitness because it was evaluated by single round infection assay in this study (14).Fig. 5


Enhanced antibody-mediated neutralization of HIV-1 variants that are resistant to fusion inhibitors
Comparison of infectivity and envelope content among fusion inhibitor-resistant mutants. a The infectivity levels of ENF-resistant mutants, V38A, N43D, and Q40H, and of C34-resistant mutants, I37K, N126K, L204I, I37K/N126K, I37K/L204I, N126K/L204I, and C34r, are shown. The TCID50 and p24 concentrations were determined by single round infection assays using TZM-bl cells and ELISAs, respectively. Infectivity is shown as the TCID50/p24 amount ratio. b The envelope content per virion was compared among C34 resistance mutants. The concentrations of envelope and p24 in pseudovirus stocks were determined by ELISAs. As negative control pseudovirus expressing no envelope was used. The envelope content is shown as the envelope/p24 amount ratio. The results are expressed as the means ± SEs of three independent experiments. Asterisks correspond to values that are statistically different from those of the WT (p < 0.05 as calculated using the Mann–Whitney U test)
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Fig5: Comparison of infectivity and envelope content among fusion inhibitor-resistant mutants. a The infectivity levels of ENF-resistant mutants, V38A, N43D, and Q40H, and of C34-resistant mutants, I37K, N126K, L204I, I37K/N126K, I37K/L204I, N126K/L204I, and C34r, are shown. The TCID50 and p24 concentrations were determined by single round infection assays using TZM-bl cells and ELISAs, respectively. Infectivity is shown as the TCID50/p24 amount ratio. b The envelope content per virion was compared among C34 resistance mutants. The concentrations of envelope and p24 in pseudovirus stocks were determined by ELISAs. As negative control pseudovirus expressing no envelope was used. The envelope content is shown as the envelope/p24 amount ratio. The results are expressed as the means ± SEs of three independent experiments. Asterisks correspond to values that are statistically different from those of the WT (p < 0.05 as calculated using the Mann–Whitney U test)
Mentions: As shown in Fig. 5a, the infectivity of the C34r mutant was 1000-fold lower than of the WT, which can be compared with the mild effect on infectivity of mutations conferring ENF resistance. The I37K mutant, as well as combination mutants that included I37K, also showed a significant decrease in infectivity compared with the WT, similar to the C34r mutant, suggesting that I37K is the major mutation responsible for the reduced infectivity. Two ENF-resistant mutants, N43D and Q40H, showed low infectivity compared with WT (approximately tenfold), but the infectivity of the V38A mutant was at the same level as the WT. These data demonstrate that I37K is critical for C34 resistance in JR-FL, but its presence imposes a significant fitness cost (Fig. 5a). The effect of N126K on C34 resistance and its fitness cost were lower than that of I37K. We are unable to comment on the role of L204I in fitness because it was evaluated by single round infection assay in this study (14).Fig. 5

View Article: PubMed Central - PubMed

ABSTRACT

Background: HIV-1 typically develops resistance to any single antiretroviral agent. Combined anti-retroviral therapy to reduce drug-resistance development is necessary to control HIV-1 infection. Here, to assess the utility of a combination of antibody and fusion inhibitor treatments, we investigated the potency of monoclonal antibodies at neutralizing HIV-1 variants that are resistant to fusion inhibitors.

Results: Mutations that confer resistance to four fusion inhibitors, enfuvirtide, C34, SC34, and SC34EK, were introduced into the envelope of HIV-1JR-FL, a CCR5-tropic tier 2 strain. Pseudoviruses with these mutations were prepared and used for the assessment of neutralization sensitivity to an array of antibodies. The resulting neutralization data indicate that the potencies of some antibodies, especially of those against the CD4 binding site, V3 loop, and membrane-proximal external region epitopes, were increased by the mutations in gp41 that conferred resistance to the fusion inhibitors. C34-, SC34-, and SC34EK-resistant mutants showed more sensitivity to monoclonal antibodies than enfuvirtide-resistant mutants. An analysis of C34-resistant mutations revealed that the I37K mutation in gp41 HR1 is a key mutation for C34 resistance, low infectivity, neutralization sensitivity, epitope exposure, and slow fusion kinetics. The N126K mutation in the gp41 HR2 domain contributed to C34 resistance and neutralization sensitivity to anti-CD4 binding site antibodies. In the absence of L204I, the effect of N126K was antagonistic to that of I37K. The results of a molecular dynamic simulation of the envelope trimer confirmation suggest that an I37K mutation induces the augmentation of structural fluctuations prominently in the interface between gp41 and gp120. Our observations indicate that the &ldquo;conformational unmasking&rdquo; of envelope glycoprotein by an I37K mutation is one of the mechanisms of neutralization sensitivity enhancement. Furthermore, the enhanced neutralization of C34-resistant mutants in vivo was shown by its high rate of neutralization by IgG from HIV patient samples.

Conclusions: Mutations in gp41 that confer fusion inhibitor resistance exert enhanced sensitivity to broad neutralizing antibodies (e.g., VRC01 and 10E8) and other conventional antibodies developed in HIV-1 infected patients. Therefore, next-generation fusion inhibitors and monoclonal antibodies could be a potential combination for future regimens of combined antiretroviral therapy.

Electronic supplementary material: The online version of this article (doi:10.1186/s12977-016-0304-7) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus