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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.


Neutralization sensitivities of mutants resistant to ENF, C34, SC34, or SC34EK. The neutralization sensitivities of fusion inhibitor-resistant mutants are shown as the average IC50 (µg/mL) of MAbs from several independent experiments. The fold change in neutralization sensitivity with respect to WT is presented in brackets below each IC50 values and highlighted by the following color code: yellow; 3–10-fold, red; >10 fold, and green; an emergence of neutralization sensitivity for MAbs that are not neutralizing to WT virus. Mutants for which calculation of fold change was not possible are presented with “–” in brackets
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Fig1: Neutralization sensitivities of mutants resistant to ENF, C34, SC34, or SC34EK. The neutralization sensitivities of fusion inhibitor-resistant mutants are shown as the average IC50 (µg/mL) of MAbs from several independent experiments. The fold change in neutralization sensitivity with respect to WT is presented in brackets below each IC50 values and highlighted by the following color code: yellow; 3–10-fold, red; >10 fold, and green; an emergence of neutralization sensitivity for MAbs that are not neutralizing to WT virus. Mutants for which calculation of fold change was not possible are presented with “–” in brackets

Mentions: To investigate the effect on antibody-mediated HIV-1 neutralization of mutations that confer fusion inhibitor resistance, we examined the sensitivity of fusion inhibitor-resistant pseudoviruses to a panel of MAbs targeting CD4bs, V3 loop, CD4i (CD4 induced epitope), or MPER epitopes. We observed enhanced neutralization sensitivity of C34-, SC34-, and SC34EK-resistant viruses by the selected antibodies with respect to the neutralization sensitivity of the WT virus (Fig. 1). In contrast, two of the three mutations conferring ENF resistance, V38A and Q40H, were unable to affect the neutralization sensitivity by these antibodies. The N43D mutation conferring ENF resistance enhanced the neutralization by anti-MPER antibodies 4E10 (>threefold) and 10E8 (>tenfold). These data are comparable to those of a previous report that showed HR1 mutations conferring ENF resistance enhance the neutralization by anti-MPER antibodies 4E10 and 2F5 [25]. In addition to the enhanced neutralization sensitivity to anti-MPER antibodies of the N43D mutant, we also observed an over threefold enhancement of neutralization by anti-V3 antibodies 0.5γ and KD247 for this mutant (Fig. 1).Fig. 1


Enhanced antibody-mediated neutralization of HIV-1 variants that are resistant to fusion inhibitors
Neutralization sensitivities of mutants resistant to ENF, C34, SC34, or SC34EK. The neutralization sensitivities of fusion inhibitor-resistant mutants are shown as the average IC50 (µg/mL) of MAbs from several independent experiments. The fold change in neutralization sensitivity with respect to WT is presented in brackets below each IC50 values and highlighted by the following color code: yellow; 3–10-fold, red; >10 fold, and green; an emergence of neutralization sensitivity for MAbs that are not neutralizing to WT virus. Mutants for which calculation of fold change was not possible are presented with “–” in brackets
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig1: Neutralization sensitivities of mutants resistant to ENF, C34, SC34, or SC34EK. The neutralization sensitivities of fusion inhibitor-resistant mutants are shown as the average IC50 (µg/mL) of MAbs from several independent experiments. The fold change in neutralization sensitivity with respect to WT is presented in brackets below each IC50 values and highlighted by the following color code: yellow; 3–10-fold, red; >10 fold, and green; an emergence of neutralization sensitivity for MAbs that are not neutralizing to WT virus. Mutants for which calculation of fold change was not possible are presented with “–” in brackets
Mentions: To investigate the effect on antibody-mediated HIV-1 neutralization of mutations that confer fusion inhibitor resistance, we examined the sensitivity of fusion inhibitor-resistant pseudoviruses to a panel of MAbs targeting CD4bs, V3 loop, CD4i (CD4 induced epitope), or MPER epitopes. We observed enhanced neutralization sensitivity of C34-, SC34-, and SC34EK-resistant viruses by the selected antibodies with respect to the neutralization sensitivity of the WT virus (Fig. 1). In contrast, two of the three mutations conferring ENF resistance, V38A and Q40H, were unable to affect the neutralization sensitivity by these antibodies. The N43D mutation conferring ENF resistance enhanced the neutralization by anti-MPER antibodies 4E10 (>threefold) and 10E8 (>tenfold). These data are comparable to those of a previous report that showed HR1 mutations conferring ENF resistance enhance the neutralization by anti-MPER antibodies 4E10 and 2F5 [25]. In addition to the enhanced neutralization sensitivity to anti-MPER antibodies of the N43D mutant, we also observed an over threefold enhancement of neutralization by anti-V3 antibodies 0.5γ and KD247 for this mutant (Fig. 1).Fig. 1

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.