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. 2011 Jan 26;6(1):e16074.
doi: 10.1371/journal.pone.0016074.

Heterologous epitope-scaffold prime:boosting immuno-focuses B cell responses to the HIV-1 gp41 2F5 neutralization determinant

Javier Guenaga  1 , Pia Dosenovic, Gilad Ofek, David Baker, William R Schief, Peter D Kwong, Gunilla B Karlsson Hedestam, Richard T Wyatt
Affiliations

Heterologous epitope-scaffold prime:boosting immuno-focuses B cell responses to the HIV-1 gp41 2F5 neutralization determinant

Javier Guenaga et al. PLoS One. .

Abstract

The HIV-1 envelope glycoproteins (Env) gp120 and gp41 mediate entry and are the targets for neutralizing antibodies. Within gp41, a continuous epitope defined by the broadly neutralizing antibody 2F5, is one of the few conserved sites accessible to antibodies on the functional HIV Env spike. Recently, as an initial attempt at structure-guided design, we transplanted the 2F5 epitope onto several non-HIV acceptor scaffold proteins that we termed epitope scaffolds (ES). As immunogens, these ES proteins elicited antibodies with exquisite binding specificity matching that of the 2F5 antibody. These novel 2F5 epitope scaffolds presented us with the opportunity to test heterologous prime:boost immunization strategies to selectively boost antibody responses against the engrafted gp41 2F5 epitope. Such strategies might be employed to target conserved but poorly immunogenic sites on the HIV-1 Env, and, more generally, other structurally defined pathogen targets. Here, we assessed ES prime:boosting by measuring epitope specific serum antibody titers by ELISA and B cell responses by ELISpot analysis using both free 2F5 peptide and an unrelated ES protein as probes. We found that the heterologous ES prime:boosting immunization regimen elicits cross-reactive humoral responses to the structurally constrained 2F5 epitope target, and that incorporating a promiscuous T cell helper epitope in the immunogens resulted in higher antibody titers against the 2F5 graft, but did not result in virus neutralization. Interestingly, two epitope scaffolds (ES1 and ES2), which did not elicit a detectable 2F5 epitope-specific response on their own, boosted such responses when primed with the ES5. Together, these results indicate that heterologous ES prime:boost immunization regimens effectively focus the humoral immune response on the structurally defined and immunogen-conserved HIV-1 2F5 epitope.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. The 2F5 epitope scaffold (ES) fusion proteins.
(A) Left, in red the 2F5 gp41 epitope region is shown both in the post-fusogenic helical form (PDB 3K9A) and in the 2F5-bound conformation (PDB 1TJI). The gp41 membrane proximal external region (MPER) including the 2F5 epitope adopts most frequently an alpha-helical conformation, however, it forms an extended ß-turn loop conformation when bound to the 2F5 antibody, as described in . Structural models (pymol) of the ES proteins used as immunogens ES5 (blue), ES1 (green) and ES2 (red), respectively. Their molecular surfaces are rendered translucent to display the underlying secondary structure. Superimposed (in red) is the 2F5 antibody-bound peptide conformation. The conserved 2F5 epitope graft molecular surface is shown in yellow. (B) Partial structure of the 2F5 antibody Fab (gray) docked to the model of ES4 (orange). ES4 was used as an antigenic probe to measure epitope-specific responses to the conformationally constrained 2F5 epitope and was not used as an immunogen. (C) Alignment of the gp41 2F5 epitope and the ES graft sequences; the 2F5 antibody contact residues defined in the 2F5 antibody-peptide structure are emboldened and underlined.
Figure 2
Figure 2. Biophysical characterization of the ES proteins.
(A) SDS-PAGE gel of ES proteins used as immunogens after affinity purification. The ES proteins possessing the C-terminal heterologous T cell helper residues are denoted “+TH”. (B) The recognition of the ES proteins by the 2F5 monoclonal antibody was assessed by surface plasmon resonance (SPR) in a Biacore 3000 instrument. In red, the observed data obtained by flowing the ES proteins as analytes over a CM5 chip to which the 2F5 IgG antibody was immobilized. In black, fit curves when a 1∶1 Langmuir model is applied to the observed data. Affinity constant values are indicated above the curves and the rate constants are denoted below the curves.
Figure 3
Figure 3. ELISA binding results of ES-elicited sera following a homologous inoculation regimen.
(A) Anti-ES titers in serum of inoculated mice (5 mice per group); upper panels depict titers obtained after immunizations with constructs not possessing the T cell helper epitope TH, and are denoted as “−TH”; bottom panels corresponds to immunizations of constructs possessing the T cell helper epitope and are denoted as “+TH”. (B) ES-elicited serum binding titers measured against the 2F5 peptide adsorbed to the ELISA plate. (C) Competition between ES5-elicited serum (at a 1∶2000 dilution) and the 2F5 mAb (serial concentrations) for binding to peptide (left) and ES4 (right) absorbed to the ELISA plate. Open circles represent binding of ES5 sera in the absence of the 2F5 mAb competitor and closed circles represent binding of the ES5 sera in the presence of increasing amounts of the competitor 2F5 mAb.
Figure 4
Figure 4. Heterologous prime∶boost regimen ELISA titers.
(A) Schematic representation of the heterologous prime∶boosting regimen. (B) 2F5 peptide (top) and not inoculated ES4 (bottom) binding serum titers of pooled sera from 5 mice following a heterologous prime∶boost immunization regimen (ES5-ES1-ES2) with or without T cell helper epitope (+/−TH). (C) To confirm that the epitope specific responses obtained in the heterologous regimen were not a result of the first (priming) inoculation with the immunogenic ES5 protein, which then increase over the time of the experiment, we inoculated 5 mice once with ES5 and measured sera binding titers after the same time interval of the complete heterologous prime-boosting regimen (34 days).
Figure 5
Figure 5. Validation of the B ELISpot using the biotinylated 2F5 peptide as a probe to measure epitope specific antigen secreting cells (ASC).
(A) Schematic depiction of the modified B cell ELISpot assay where all secreted antibodies are captured by rabbit anti-mouse IgG, then a biotinylated anti-mouse IgG probe is used to determine total IgG responses or a biotinylated 2F5 peptide probe is used to determine the 2F5 peptide-specific ASC. (B) ELISpot plate showing immuno spots generated by the hybridoma cells expressing the murine monoclonal antibody 1D9 which binds the 2F5 epitope. (C) As shown by the 1∶1 correspondence, the 2F5 peptide probe binds nearly 100% of the hybridoma secreted antibodies.
Figure 6
Figure 6. B cell ELISpots measuring epitope-specific or antigen-specific cells.
(A) 2F5 peptide-specific B cell responses induced by a homologous or a heterologous regimen after each immunization. Bars represent the mean and SEM values corresponding to measurements of five mice. Colored bars represent B cell responses from the heterologous prime∶boost immunization regimen and are color-coded to indicate the immunogen inoculated prior to collection of the B cells. As a negative control we measured anti-β-Gal protein responses, which are depicted in this graph as an average of responses of all animals participating in this analysis after 3 inoculations and is plotted as the horizontal dotted line across the bars. (B) B cell ELISpot measuring peptide, anti-ES5 responses in the one-time inoculated ES5 control mice after 34 days, the length of the entire regimen. (C) 2F5 peptide-specific (left) and protein control β-Gal (right) memory B cell responses after 6 days in vitro culture in the presence (stimulated) or absence (unstimulated) of LPS stimulation. Similarly, we compare the heterologous regimen (ES5-ES1-ES2) to the one inoculation ES5 control to show that the 2F5 peptide specific memory B cells in the LPS stimulated experiment were generated via an effective cross-priming heterologous ES boost.
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