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

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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.


Fusion kinetics of C34-resistant mutants. The effects of N126K, N126K/L204I, C34r, and I37K mutations on fusion kinetics were determined by dual split protein (DSP)-dependent cell–cell fusion assays. Renilla luciferase activity, which was detected in fused cells, was monitored periodically, and fusion is expressed as the percentage of fusion after 120 min. The results are shown as the means ± SEs of at least five replicas
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Fig6: Fusion kinetics of C34-resistant mutants. The effects of N126K, N126K/L204I, C34r, and I37K mutations on fusion kinetics were determined by dual split protein (DSP)-dependent cell–cell fusion assays. Renilla luciferase activity, which was detected in fused cells, was monitored periodically, and fusion is expressed as the percentage of fusion after 120 min. The results are shown as the means ± SEs of at least five replicas

Mentions: It has been suggested that enhancement of neutralization by anti-MPER antibodies is due to slower fusion kinetics, which lead to prolonged exposure of the epitope [25, 63–65]. To test this hypothesis, we performed fusion assays for I37K, N126K, N126K/L204I, and C34r mutants. As shown in Fig. 6, both I37K and C34r have reduced fusion kinetics with respect to WT. However, no such change was apparent for N126K or N126K/L204I mutants (Fig. 6). These data are consistent with the enhanced neutralization sensitivity of C34r and I37K mutants to 10E8 (Fig. 3b).Fig. 6


Enhanced antibody-mediated neutralization of HIV-1 variants that are resistant to fusion inhibitors
Fusion kinetics of C34-resistant mutants. The effects of N126K, N126K/L204I, C34r, and I37K mutations on fusion kinetics were determined by dual split protein (DSP)-dependent cell–cell fusion assays. Renilla luciferase activity, which was detected in fused cells, was monitored periodically, and fusion is expressed as the percentage of fusion after 120 min. The results are shown as the means ± SEs of at least five replicas
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5037607&req=5

Fig6: Fusion kinetics of C34-resistant mutants. The effects of N126K, N126K/L204I, C34r, and I37K mutations on fusion kinetics were determined by dual split protein (DSP)-dependent cell–cell fusion assays. Renilla luciferase activity, which was detected in fused cells, was monitored periodically, and fusion is expressed as the percentage of fusion after 120 min. The results are shown as the means ± SEs of at least five replicas
Mentions: It has been suggested that enhancement of neutralization by anti-MPER antibodies is due to slower fusion kinetics, which lead to prolonged exposure of the epitope [25, 63–65]. To test this hypothesis, we performed fusion assays for I37K, N126K, N126K/L204I, and C34r mutants. As shown in Fig. 6, both I37K and C34r have reduced fusion kinetics with respect to WT. However, no such change was apparent for N126K or N126K/L204I mutants (Fig. 6). These data are consistent with the enhanced neutralization sensitivity of C34r and I37K mutants to 10E8 (Fig. 3b).Fig. 6

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.