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Abstract <p><b>Objective:</b> The development of a vaccine candidate for specific active immunoprophylaxis against HIV-1 infection, based on the structure-guided design of HIV-1 Env proteins, is considered one of the most promising approaches to creating an immunogen capable of inducing broadly neutralizing antibodies (bnAbs). However, the high diversity of HIV-1 implies that immunogens based on a single specific strain may be ineffective against other circulating variants. Consequently, there is a need to develop an antigen capable of neutralizing a broad spectrum of HIV-1 genetic variants while accounting for regional strain characteristics. <b>Methods:</b> In this study, based on the sequences of Russian strains, a HIV-1 Env surface antigen and its variant with additional mutations—optimized for engagement with germline precursors of VRC01-class bnAbs—were developed and characterized. <b>Results and Discussion:</b> The CON-RUS SOSIP-v.9.3 antigen was designed based on a consensus amino acid sequence derived from the most prevalent HIV-1 variants in the Russian Federation (A6, B, CRF02_AG, and CRF63_02A6), with stabilizing SOSIP.v9.3 and TD8 mutations. The CON-RUS SOSIP-v.9.3_GT01 variant was further engineered with additional mutations to enhance binding affinity for the germline precursor of the VRC01 antibody. Biochemical characterization by BN-PAGE and SEC-MALS confirmed that both constructs predominantly form trimers with a molecular weight consistent with the expected size (~220 kDa excluding glycans). Negative-stain electron microscopy revealed that the majority of CON-RUS SOSIP-v.9.3 molecules adopt a closed conformation, as demonstrated by 2D class averaging and 3D reconstruction fitting to the closed-state BG505 SOSIP.664 structure (PDB ID: 3J5M). ELISA showed that mature VRC01 (mVRC01) binds both CON-RUS SOSIP-v.9.3 and CON-RUS SOSIP-v.9.3_GT01 with affinity comparable to reference BG505 SOSIP proteins. However, while no binding of CON-RUS SOSIP-v.9.3 to germline VRC01 (gVRC01) was detected, the GT01 modification enabled weak binding to gVRC01, although this was significantly lower than that of the BG505 SOSIP variant carrying analogous mutations. <b>Conclusions:</b> Based on the consensus sequence of HIV-1 strains prevalent in the Russian Federation, two recombinant Env constructs (CON-RUS SOSIP-v.9.3 and CON-RUS SOSIP-v.9.3_GT01) were developed, which predominantly adopt a trimeric organization and effectively engage the mature VRC01 antibody.</p>

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Production and Structural-Functional Characterization of a Recombinant HIV-1 Env Trimer Based on the Consensus Sequence of the Most Prevalent Strains in the Russian Federation

  • 期刊论文

  • 发表日期：

    2026年5月6日

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  • I. A. Favorskaya,
  • I. O. Ilyasov,
  • I. A. Alekseeva,
  • E. I. Ryabova,
  • A. A. Antonova,
  • D. A. Kleymenov,
  • V. A. Gushchin,
  • A. D. Burtseva,
  • A. V. Moiseenko,
  • T. S. Trifonova,
  • V. O. Popov,
  • N. N. Sluchanko,
  • K. M. Boyko,
  • D. V. Shcheblyakov,
  • D. Yu. Logunov

摘要

Abstract

Objective: The development of a vaccine candidate for specific active immunoprophylaxis against HIV-1 infection, based on the structure-guided design of HIV-1 Env proteins, is considered one of the most promising approaches to creating an immunogen capable of inducing broadly neutralizing antibodies (bnAbs). However, the high diversity of HIV-1 implies that immunogens based on a single specific strain may be ineffective against other circulating variants. Consequently, there is a need to develop an antigen capable of neutralizing a broad spectrum of HIV-1 genetic variants while accounting for regional strain characteristics. Methods: In this study, based on the sequences of Russian strains, a HIV-1 Env surface antigen and its variant with additional mutations—optimized for engagement with germline precursors of VRC01-class bnAbs—were developed and characterized. Results and Discussion: The CON-RUS SOSIP-v.9.3 antigen was designed based on a consensus amino acid sequence derived from the most prevalent HIV-1 variants in the Russian Federation (A6, B, CRF02_AG, and CRF63_02A6), with stabilizing SOSIP.v9.3 and TD8 mutations. The CON-RUS SOSIP-v.9.3_GT01 variant was further engineered with additional mutations to enhance binding affinity for the germline precursor of the VRC01 antibody. Biochemical characterization by BN-PAGE and SEC-MALS confirmed that both constructs predominantly form trimers with a molecular weight consistent with the expected size (~220 kDa excluding glycans). Negative-stain electron microscopy revealed that the majority of CON-RUS SOSIP-v.9.3 molecules adopt a closed conformation, as demonstrated by 2D class averaging and 3D reconstruction fitting to the closed-state BG505 SOSIP.664 structure (PDB ID: 3J5M). ELISA showed that mature VRC01 (mVRC01) binds both CON-RUS SOSIP-v.9.3 and CON-RUS SOSIP-v.9.3_GT01 with affinity comparable to reference BG505 SOSIP proteins. However, while no binding of CON-RUS SOSIP-v.9.3 to germline VRC01 (gVRC01) was detected, the GT01 modification enabled weak binding to gVRC01, although this was significantly lower than that of the BG505 SOSIP variant carrying analogous mutations. Conclusions: Based on the consensus sequence of HIV-1 strains prevalent in the Russian Federation, two recombinant Env constructs (CON-RUS SOSIP-v.9.3 and CON-RUS SOSIP-v.9.3_GT01) were developed, which predominantly adopt a trimeric organization and effectively engage the mature VRC01 antibody.