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Volume 6 Supplement 3

AIDS Vaccine 2009

P12-12. Analysis of antibody and B cell responses following inoculation with computationally designed HIV-1 2F5 epitope scaffold proteins

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Background

Two of the existing broadly neutralizing antibodies (NAbs) to HIV-1 Env, 2F5 and 4E10, recognize conserved continuous epitopes in the gp41 membrane-proximal external region. Re-elicitation of NAbs to the 2F5 epitope by immunization with peptides or chimeric proteins has so far proven difficult. To improve the efficiency of eliciting 2F5 epitope-directed NAbs, computationally designed scaffold proteins were used for more optimal presentation of the crystallographically defined 2F5 epitope (2F5E) to B cells. Here, we characterize 2F5E-specific B cell subsets and serum responses elicited by homologous and heterologous scaffold immunizations, with the overall goal to understand the rules of immuno-focusing in this novel system.

Methods

Computationally designed proteins, consisting of non-HIV proteins engrafted with the structurally defined 2F5E, were used in sequential immunizations to immuno-focus the humoral response on the 2F5E graft. A 2F5E-specific B cell ELISpot assay was developed and used for quantification of 2F5E-specific B cells in immunized mice. 2F5E-specific Ab levels in sera were analyzed by selected ELISA formats.

Results

A 2F5E-specific B cell ELISpot assay was optimized using a mouse hybridoma cell line specific for this epitope. Using this assay, we enumerated the number of 2F5E-specific antibody-secreting cells and memory B cells induced by homologous and heterologous scaffold immunization. Increases in the number of B cells specific for the 2F5 epitope were observed in groups of mice inoculated with heterologous scaffolds suggesting that the immuno-focusing strategy was successful. Increased Ab responses against the 2F5E were confirmed by ELISA.

Conclusion

By establishing a highly sensitive 2F5E-specific B cell ELISpot assay, we could quantify 2F5E specific B cell responses in mice immunized with different scaffold proteins presenting the 2F5E. Further analysis of the quality of the antibodies induced by homologous and heterologous scaffold immunizations is ongoing.

Author information

Correspondence to P Dosenovic.

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Open Access This article is published under license to BioMed Central Ltd. This is an Open Access article is distributed under the terms of the Creative Commons Attribution 2.0 International License (https://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Dosenovic, P., Guenaga, J., Ofek, G. et al. P12-12. Analysis of antibody and B cell responses following inoculation with computationally designed HIV-1 2F5 epitope scaffold proteins. Retrovirology 6, P178 (2009). https://doi.org/10.1186/1742-4690-6-S3-P178

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Keywords

  • Chimeric Protein
  • Scaffold Protein
  • Immunize Mouse
  • External Region
  • Optimal Presentation