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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.


Binding of antibodies to untriggered envelope trimers. The effect of fusion inhibitor resistance-conferring mutations on the binding of anti-CD4bs MAbs, VRC01, 49G2, and 42F9, and of anti-V3 MAbs, KD247, 16G6, and 0.5γ, was determined by performing flow cytometry on 293T cells expressing mutant envelopes. The geometric MFI was calculated and normalized by the MFI of 2G12. The changes in antibody binding levels are presented as the fold change of binding with respect to WT. The dash line represents the change in antibody binding (fold change = 1). The results are shown as the means ± SEs of three replicas
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Fig4: Binding of antibodies to untriggered envelope trimers. The effect of fusion inhibitor resistance-conferring mutations on the binding of anti-CD4bs MAbs, VRC01, 49G2, and 42F9, and of anti-V3 MAbs, KD247, 16G6, and 0.5γ, was determined by performing flow cytometry on 293T cells expressing mutant envelopes. The geometric MFI was calculated and normalized by the MFI of 2G12. The changes in antibody binding levels are presented as the fold change of binding with respect to WT. The dash line represents the change in antibody binding (fold change = 1). The results are shown as the means ± SEs of three replicas

Mentions: To investigate the mechanism of neutralization enhancement, we used flow cytometry to assess the binding of antibodies against CD4bs (VRC01, 42F9, and 49G2) and V3 (KD247, 16G6, and 0.5γ) to mutant envelopes expressed on the cell surface and evaluated the exposure level of epitopes on un-triggered Env trimers (Fig. 4). A striking enhancement was observed for anti-CD4bs antibodies, 42F9 (twofold) and 49G2 (1.6–1.7 fold), against C34r, I37K and I37K/L204I mutants. These mutants all had the I37K mutation, suggesting that the increased binding affinity of 42F9 and 49G2 to viruses with I37K enhances the neutralization by these antibodies (Fig. 2a). N126K, the other C34-resistant mutation that is critical for enhanced neutralization, did not affect the antibody binding, and, instead, it abrogated the enhancement effect of I37K in the I37K/N126K mutant. This finding suggests that the N126K mutation enhances neutralization by a mechanism other than an increase in the affinity between the envelope and the antibody. We observed some increase in VRC01 binding, but this occurred in the absence of any correlation with neutralizing sensitivity (Fig. 4). This lack of correlation between neutralization sensitivity and binding affinity indicates that the enhanced neutralization sensitivity of I37K and N126K to VRC01 is mediated by some other unknown mechanism (Fig. 2a).Fig. 4


Enhanced antibody-mediated neutralization of HIV-1 variants that are resistant to fusion inhibitors
Binding of antibodies to untriggered envelope trimers. The effect of fusion inhibitor resistance-conferring mutations on the binding of anti-CD4bs MAbs, VRC01, 49G2, and 42F9, and of anti-V3 MAbs, KD247, 16G6, and 0.5γ, was determined by performing flow cytometry on 293T cells expressing mutant envelopes. The geometric MFI was calculated and normalized by the MFI of 2G12. The changes in antibody binding levels are presented as the fold change of binding with respect to WT. The dash line represents the change in antibody binding (fold change = 1). The results are shown as the means ± SEs of three replicas
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5037607&req=5

Fig4: Binding of antibodies to untriggered envelope trimers. The effect of fusion inhibitor resistance-conferring mutations on the binding of anti-CD4bs MAbs, VRC01, 49G2, and 42F9, and of anti-V3 MAbs, KD247, 16G6, and 0.5γ, was determined by performing flow cytometry on 293T cells expressing mutant envelopes. The geometric MFI was calculated and normalized by the MFI of 2G12. The changes in antibody binding levels are presented as the fold change of binding with respect to WT. The dash line represents the change in antibody binding (fold change = 1). The results are shown as the means ± SEs of three replicas
Mentions: To investigate the mechanism of neutralization enhancement, we used flow cytometry to assess the binding of antibodies against CD4bs (VRC01, 42F9, and 49G2) and V3 (KD247, 16G6, and 0.5γ) to mutant envelopes expressed on the cell surface and evaluated the exposure level of epitopes on un-triggered Env trimers (Fig. 4). A striking enhancement was observed for anti-CD4bs antibodies, 42F9 (twofold) and 49G2 (1.6–1.7 fold), against C34r, I37K and I37K/L204I mutants. These mutants all had the I37K mutation, suggesting that the increased binding affinity of 42F9 and 49G2 to viruses with I37K enhances the neutralization by these antibodies (Fig. 2a). N126K, the other C34-resistant mutation that is critical for enhanced neutralization, did not affect the antibody binding, and, instead, it abrogated the enhancement effect of I37K in the I37K/N126K mutant. This finding suggests that the N126K mutation enhances neutralization by a mechanism other than an increase in the affinity between the envelope and the antibody. We observed some increase in VRC01 binding, but this occurred in the absence of any correlation with neutralizing sensitivity (Fig. 4). This lack of correlation between neutralization sensitivity and binding affinity indicates that the enhanced neutralization sensitivity of I37K and N126K to VRC01 is mediated by some other unknown mechanism (Fig. 2a).Fig. 4

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.