(W1230-08-48) Beyond Specificity: Evaluating the Cross-Protective Potential of a Whole-Cell Inactivated Microparticulate Gonorrhea Vaccine

Purpose: Neisseria gonorrhoeae is the bacterium responsible for Gonorrhea, the second most prevalent sexually transmitted infection in the United States, with over 600,000 cases annually. The growing public health threat from Gonorrhea is fueled by the bacterium’s rapid acquisition of multidrug resistance and its ability to evade adaptive immunity, which frequently leads to recurring infections and complicates treatment. Currently, there is no vaccine against Gonorrhea, and previous vaccine development efforts have largely failed due to their inability to prevent reinfection and address the bacteria’s extensive antigenic variation. To address these longstanding obstacles, we have developed a novel adjuvanted whole-cell inactivated gonococcal microparticulate vaccine, delivered painlessly via microneedle patches, and specifically formulated using the CDC F62 strain. The CDC F62 strain was chosen as a vaccine candidate due to its well-characterized genetic background. More importantly, CDC F62 displays representative antigenic profiles found in clinically relevant isolates, making it a strong model for eliciting broadly reactive immune responses and facilitating cross-protection studies. Building on encouraging protective efficacy observed against the homologous F62 strain, this study aims to evaluate the cross-protective capabilities of our vaccine formulation against two clinically significant heterologous strains: the disseminated FA1090 and the serum-resistant FA19. By testing the breadth of protection, this research seeks to generate essential preclinical evidence in support of developing a widely effective gonorrhea vaccine.

Methods: The Neisseria gonorrhoeae was first grown and then formalin-inactivated. Then, using a method developed in our lab, we encapsulated the whole cell inactivated gonorrhea into a biodegradable pre-crosslinked albumin microparticles along with the adjuvants Alum and AddaVax™ using spray drying method. Next, these microparticles were loaded into dissolving microneedles made from hyaluronic acid, deionized water, and trehalose. Then, five groups were tested: a naïve control (A), a group vaccinated with CDC F62 vaccine and challenged with CDC F62 (B), a group vaccinated with adjuvanted CDC F62 vaccine and challenged with CDC F62 (C), a group vaccinated with adjuvanted CDC F62 vaccine and challenged with the heterologous FA19 strain (D), and a group vaccinated with adjuvanted CDC F62 vaccine and challenged with the heterologous FA1090 strain (E). Every two weeks, we collected blood samples. Then, at week 8, we challenged the groups with the different strains of gonorrhea to evaluate the cross-protective potential of our vaccine. Then, at week 10, the mice were sacrificed, and their spleens and lymph nodes were harvested to assess cellular responses. Flow cytometry was used to quantify the cellular response based on CD4⁺ and CD8⁺ expression, while the IgG response was measured using ELISA.

Results: After the IgG antibodies were measured, we observed a robust immune response in all groups, especially when compared to the control group (****p < 0.0001). These findings suggest that our vaccine not only elicited a humoral response against the CDCF62 strain but also against the other two strains. Analysis of the spleen CD8+ T cell responses revealed a significant increase in the percentage of CD8+ cells in groups C (*p < 0.05) and D (*p < 0.05) compared to the naïve group (A), while groups B (ns p >0.05) and E (ns p >0.05) did not show statistically significant differences from the naïve group. For the spleen CD4+ T cells, all vaccinated and challenged groups B(***p < 0.0002), C(****p < 0.0001), D(***p < 0.0002), E(*p < 0.05) exhibited a robust and significant increase in CD4+ cell percentages compared to the naïve group, with the highest responses observed in groups C and D. In the lymph nodes, CD8+ T cell responses were highest in group E (*p < 0.05), which was significantly elevated compared to the naïve group. CD4+ T cell responses in the lymph nodes were significantly higher in all vaccinated and challenged groups (B(*p < 0.05), C(**p < 0.0021), D (***p < 0.0002), E (****p < 0.0001)) compared to naïve group, with the strongest responses in groups D and E.

Conclusion: Overall, these results demonstrate that vaccination with the adjuvanted CDC F62 formulation could elicit a robust IgG response against all the strains of gonorrhea that were tested, induced strong CD4+ T cell responses in both spleen and lymph nodes, and could elicit cross-reactive cellular immunity against heterologous gonococcal strains, particularly when combined with adjuvant. CD8+ T cell responses were more modest but showed significant enhancement in select groups, supporting the potential of this approach for the development of a broad gonococcal vaccine.

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A graphical representation of the methodology of the study. 1. Growth and Inactivation of bacteria 2. Spray drying of inactivated bacteria into microparticles. 3. Loading microparticles into microneedle patches. 4. Administration of the vaccine to the mice. 5. Sacrifice and serum and organ collection from the mice. 5. ELISA analysis of serum samples and Flow cytometry analysis of organ samples.

Total Serum IgG Levels

Percentage of cells in the spleen and lymph node populations expressing CD4 and CD8 markers.