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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.


Related in: MedlinePlus

Effect of C34 resistance-conferring mutations on the sensitivities to anti-CD4bs MAb and sCD4. Three mutations that together confer C34 resistance, I37K, N126K, and L204I, were examined for their effect on the neutralization sensitivity to anti-CD4bs MAbs, VRC01, 49G2, and 42F9, (a) and to sCD4 (b) using single (left) and double (right) mutants. In each graph, the x axis represents the log concentration of MAbs in µg/mL and the y axis represents the percent inhibition compared with the corresponding no inhibitor control. The results are shown as the means ± SEs of three independent experiments
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Fig2: Effect of C34 resistance-conferring mutations on the sensitivities to anti-CD4bs MAb and sCD4. Three mutations that together confer C34 resistance, I37K, N126K, and L204I, were examined for their effect on the neutralization sensitivity to anti-CD4bs MAbs, VRC01, 49G2, and 42F9, (a) and to sCD4 (b) using single (left) and double (right) mutants. In each graph, the x axis represents the log concentration of MAbs in µg/mL and the y axis represents the percent inhibition compared with the corresponding no inhibitor control. The results are shown as the means ± SEs of three independent experiments

Mentions: To investigate the effect on the neutralization sensitivity of mutations conferring fusion inhibitor resistance, we focused on the C34r mutant that comprises three mutations (I37K/N126K/L204I). The impact of the individual mutations in the C34r mutant on its sensitivity to antibodies was analyzed by performing neutralization assays using antibodies targeting the CD4bs (VRC01, 49G2, and 42F9), V3 (16G6, KD247, and 0.5γ), and MPER (10E8). As shown in Fig. 2a, I37K (5.9-fold) and N126K (7.2-fold) mutants are more sensitive to VRC01 than WT, based on their corresponding IC50 values. However, the neutralization sensitivity of the combination of these two mutations (I37K/N126K) was unaltered with respect to WT. Additionally, neutralization by VRC01 was unaffected by the combinations of mutations I37K/L204I and N126K/L204I compared to WT. Anti-CD4bs antibodies, 49G2 and 42F9, failed to neutralize WT virus, but the inhibition by these antibodies reached over 50 % against I37K, N126K, and I37K/L204I mutants. These results indicate that I37K and N126K are the key mutations responsible for increased neutralization sensitivity against anti-CD4bs antibodies, and that the combination of these mutations with L204I caused the C34-resistant phenotype in C34r. Interestingly, the I37K and N126K mutations were unable to increase the sensitivity to all of these anti-CD4bs antibodies in combination, suggesting that the effects of these mutations on the sensitivity of the viruses to anti-CD4bs antibodies were antagonized by each other (Fig. 2a).Fig. 2


Enhanced antibody-mediated neutralization of HIV-1 variants that are resistant to fusion inhibitors
Effect of C34 resistance-conferring mutations on the sensitivities to anti-CD4bs MAb and sCD4. Three mutations that together confer C34 resistance, I37K, N126K, and L204I, were examined for their effect on the neutralization sensitivity to anti-CD4bs MAbs, VRC01, 49G2, and 42F9, (a) and to sCD4 (b) using single (left) and double (right) mutants. In each graph, the x axis represents the log concentration of MAbs in µg/mL and the y axis represents the percent inhibition compared with the corresponding no inhibitor control. The results are shown as the means ± SEs of three independent experiments
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig2: Effect of C34 resistance-conferring mutations on the sensitivities to anti-CD4bs MAb and sCD4. Three mutations that together confer C34 resistance, I37K, N126K, and L204I, were examined for their effect on the neutralization sensitivity to anti-CD4bs MAbs, VRC01, 49G2, and 42F9, (a) and to sCD4 (b) using single (left) and double (right) mutants. In each graph, the x axis represents the log concentration of MAbs in µg/mL and the y axis represents the percent inhibition compared with the corresponding no inhibitor control. The results are shown as the means ± SEs of three independent experiments
Mentions: To investigate the effect on the neutralization sensitivity of mutations conferring fusion inhibitor resistance, we focused on the C34r mutant that comprises three mutations (I37K/N126K/L204I). The impact of the individual mutations in the C34r mutant on its sensitivity to antibodies was analyzed by performing neutralization assays using antibodies targeting the CD4bs (VRC01, 49G2, and 42F9), V3 (16G6, KD247, and 0.5γ), and MPER (10E8). As shown in Fig. 2a, I37K (5.9-fold) and N126K (7.2-fold) mutants are more sensitive to VRC01 than WT, based on their corresponding IC50 values. However, the neutralization sensitivity of the combination of these two mutations (I37K/N126K) was unaltered with respect to WT. Additionally, neutralization by VRC01 was unaffected by the combinations of mutations I37K/L204I and N126K/L204I compared to WT. Anti-CD4bs antibodies, 49G2 and 42F9, failed to neutralize WT virus, but the inhibition by these antibodies reached over 50 % against I37K, N126K, and I37K/L204I mutants. These results indicate that I37K and N126K are the key mutations responsible for increased neutralization sensitivity against anti-CD4bs antibodies, and that the combination of these mutations with L204I caused the C34-resistant phenotype in C34r. Interestingly, the I37K and N126K mutations were unable to increase the sensitivity to all of these anti-CD4bs antibodies in combination, suggesting that the effects of these mutations on the sensitivity of the viruses to anti-CD4bs antibodies were antagonized by each other (Fig. 2a).Fig. 2

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.


Related in: MedlinePlus