Vaccine design via antigen reorientation

The rapid evolution of viruses like influenza and SARS-CoV-2 necessitates the development of universal vaccines. Current influenza vaccines target the variable HA-head, requiring annual updates. A more effective strategy is to focus the immune response on the conserved HA-stem region, which contains epitopes less prone to antigenic drift. Human monoclonal antibodies (mAbs) targeting HA-stem exhibit broad neutralizing activity and protection in animal models, correlating with protection in humans. Existing immunofocusing approaches, such as sequential immunization or mosaic display, involve complex regimens and don't fully address HA-head immunodominance. Other methods, including masking HA-head or designing HA-stem-only immunogens, have limitations in immunogenicity or require extensive design and optimization. This study explores antigen reorientation as a simple and effective immunofocusing strategy. The hypothesis is that an ‘upside-down’ HA configuration, where the HA-head is sterically occluded, would redirect the antibody response to the more exposed HA-stem. Stem-directed mAbs from vaccinated individuals exhibit reduced binding to whole viruses compared to recombinant HA proteins, suggesting steric occlusion of HA-stem during natural infection. This study aimed to achieve controlled antigen orientation via site-specific oligoD insertion to enhance alum binding, a well-established adjuvant, and apply this strategy to reorient HA for immunofocusing.

Extensive research has focused on developing universal influenza vaccines that overcome the limitations of current strain-specific vaccines. The immunodominance of the HA-head, which undergoes frequent antigenic drift, hinders the development of broadly protective vaccines. Several strategies have been explored to direct the immune response toward the conserved HA-stem, including sequential immunization with different HAs, mosaic display of heterologous HAs, and the design of HA-stem-only immunogens. While these approaches have shown some success, they often require complex vaccination regimens or result in reduced immunogenicity. This research builds upon the understanding that HA-stem is often sterically occluded by the HA-head, and therefore less immunogenic, on the surface of the virus. Methods using masking strategies or the generation of HA-stem-only proteins to enhance HA-stem immunogenicity have been previously described, but have inherent limitations. The present study proposes antigen reorientation as a novel approach to overcome these challenges.

The study first developed a method to control antigen orientation by inserting short stretches of aspartate residues (oligoD) into protein antigens. This oligoD insertion facilitates antigen binding to alum, a widely used vaccine adjuvant. The length of oligoD required for effective alum binding was determined using the Ebola glycoprotein (GP) as a model. Different lengths of oligoD (2D, 4D, 8D, 12D) were inserted at the C-terminus of GP, and their binding to alum was assessed using western blotting and ELISA. The effect of oligoD insertion on GP structure and immunogenicity was evaluated using biolayer interferometry (BLI), thermal melting analysis, and mouse immunization studies. The generalizability of the oligoD approach was demonstrated by inserting oligoD into SARS-CoV-2 spike protein and H1 HA. Following the demonstration of enhanced immunogenicity of these modified antigens using mouse immunization studies, the study focused on reorienting H2 HA by inserting oligoD into the HA-head to anchor it to alum in an 'upside-down' configuration. This reoriented H2 HA (reoH2HA) was then used to immunize mice, and the resulting antibody responses were analyzed using ELISA, competition ELISA, and negative-stain electron microscopy polyclonal epitope mapping (nsEMPEM). The nsEMPEM was used to determine the epitopes targeted by the elicited antibodies. Mouse immunization studies involved different immunization protocols and adjuvant combinations (alum alone, alum with CpG) to assess the impact on the immune response.

OligoD insertion significantly enhanced antigen binding to alum and increased immunogenicity in all three model antigens (Ebola GP, SARS-CoV-2 spike, and H1 HA). Insertion of 12D residues proved optimal. Mouse immunizations with oligoD-modified antigens resulted in significantly higher neutralizing antibody titers compared to wild-type antigens. Reorienting the H2 HA by inserting oligoD into its head resulted in an 'upside-down' configuration on alum, where the HA-head was anchored and the HA-stem was more exposed. Immunization with reoH2HA elicited a strong stem-directed antibody response with significantly increased cross-reactivity against both group 1 and group 2 influenza A subtypes compared to wild-type H2 HA. Competition ELISA demonstrated that reoH2HA-elicited antibodies competed more effectively with a known stem-specific broadly neutralizing antibody (MEDI8852) than antibodies elicited by wild-type H2 HA. nsEMPEM revealed that reoH2HA elicited cross-reactive antibodies that recognized stem epitopes of group 2 HAs, while wild-type H2 HA did not. Although cross-neutralizing activity was modest, IgG purified from reoH2HA immunized mice showed cross-neutralization against heterosubtypic H1N1 virus, while wild-type H2 HA did not.

This study demonstrates that antigen reorientation is a viable and generalizable strategy for immunofocusing. By anchoring the immunodominant HA-head to alum through oligoD insertion, the conserved HA-stem was made more accessible to the immune system, leading to a broader and more potent antibody response. This strategy offers several advantages over existing methods. It preserves the native structure and conformational epitopes of the antigen while simultaneously mitigating HA-head immunodominance. The simplicity of the oligoD insertion method makes it easily adaptable to other antigens. The observed cross-reactivity with both group 1 and 2 influenza A subtypes represents a significant advance toward developing a universal influenza vaccine. While the cross-neutralizing activity of reoH2HA-elicited antibodies was modest, this might still confer protection through Fc-mediated effector functions. Further research is needed to optimize adjuvant selection and to assess the protective efficacy of reoH2HA in animal challenge models.

This study successfully introduces antigen reorientation as a new immunofocusing strategy. The simple method of oligoD insertion enables controlled antigen orientation on alum, leading to enhanced immunogenicity and broadened cross-reactivity, particularly targeting the HA-stem in influenza. This approach holds significant promise for accelerating the development of epitope-focused vaccines against various infectious agents. Future work should focus on optimizing adjuvant formulations to enhance both neutralizing and effector functions and evaluating protective efficacy in animal challenge models.

The study's findings on cross-neutralization were modest. While the reoH2HA elicited a broader antibody response than wild-type H2 HA, the magnitude of cross-neutralization was limited. Further optimization of the antigen design, adjuvant selection, and immunization regimens may be necessary to achieve more potent cross-neutralization. The study primarily focused on influenza A viruses; the generalizability to other viral systems requires further investigation. The mechanism of action by which the cross-reactive antibodies induce protection (neutralization or Fc-mediated effector functions) warrants further investigation.