(M0930-04-19) Investigational Vaccine Induces Immune Correlates of Protection and Resistance to Infection against Neisseria gonorrhoeae in Mice

Purpose: Gonorrhea is a sexually transmitted disease caused by the strict human pathogen Neisseria gonorrhoeae. According to the Centers for Disease Control and Prevention (CDC), approximately 1,568,000 new gonococcal infections occur in the United States every year, and gonorrhea is the second most reported bacterial infection. The control of this infectious disease depends upon the effective diagnosis and antibiotic treatment. The most effective antimicrobial agents are fluoroquinolones and broad-spectrum cephalosporins. However, gonorrhea has developed resistance to fluoroquinolones. The emergence of resistance to cephalosporins will soon complicate this disease's successful treatment. Despite the considerable burden of this disease, there is currently no vaccine available for gonorrhea. An effective vaccination strategy for treating gonorrhea is imminent. In this regard, our aim was 1) to formulate a dissolving microneedle (MN) based adjuvanted inactivated gonorrhea microparticle (MP) vaccine, 2) to evaluate the in vitro safety, immunogenicity, autophagy, and antigen presentation, 3) to investigate the humoral immune responses, correlates of protection, resistance to infection, and cellular immune responses following in vivo immunization of mice.

Methods: N. gonorrhoeae strain CDC-F62 was grown and inactivated using formalin. This whole-cell inactivated gonorrhea was encapsulated into a biodegradable pre-crosslinked albumin matrix. Gonococci vaccine MP (Gc-MP) and adjuvant MP (Alum and AddaVax™) were prepared by a method previously developed in our laboratory using the Buchi Mini Spray Dryer B-290. The MP were analyzed using a dynamic light scattering (DLS) particle size analyzer for measuring the particle size, polydispersity index, and zeta potential. The MP with 20% antigen-loading were mixed with hyaluronic acid and trehalose to form an aqueous solution. This solution was then loaded into the MN molds (10x10 array) and the MN were created using spin casting method. The MN were characterized by their appearance, needle length, mechanical strength, dissolving time, and formation of microchannels into an excised mouse skin. The MP were then tested in vitro for safety using cytotoxicity assay, immunogenicity using nitric oxide assay, autophagy, and antigen presentation. The in vivo efficacy of this vaccine formulation was assessed using female mice immunized with one prime and two booster doses on days 0, 14 and 28. The serum samples were collected every alternate week. Enzyme-linked immunosorbent assay (ELISA) was used to measure IgG levels, its sub-types, and IgM in the collected mice sera. Bactericidal antibodies can confer protection against N. gonorrhoeae. The immunized mice sera were assessed for their bactericidal correlates of protection using serum bactericidal assay. Following vaccination, at week 8, the mice were challenged with a live Neisseria gonorrhoeae strain, CDC-F62 (murine lower genital tract infection model), at 10⁶ CFU/mL. The ability of gonorrhea-specific antibodies to kill or inhibit the growth of live N. gonorrhoeae parent strain CDC-F62 used in the vaccine formulation was observed. At the end of the study, mice were sacrificed. The immune organs were isolated and harvested for assessing the cellular immune response, CD4, and CD8 T cell response using flow cytometry.

Results: Effective vaccine MP encapsulating the whole-cell of Neisseria gonorrhoeae were formulated. Gc-MP were spherical with a particle size of 3-5 µm, polydispersity index of 0.34, and zeta potential of -25.1 ± 5.79 mV. The MN were sharp, strong, and individual needles were uniform in length. The average length of the MN was found to be 350 µm. The MN dissolved within ten minutes when applied to an excised mouse skin, creating microchannels. In vitro assays demonstrated the safety of the MP as they were non-cytotoxic within the tested concentration range. Nitrite production and MHC I and MHC II expression was significantly higher in the vaccine MP group than in the untreated control group (p < 0.001). Gonococci MP combined with adjuvant MP (Gc-MP+ Alum MP and Gc-MP+ AddaVax™ MP) induced significantly (p < 0.001) higher autophagy in macrophages than unstimulated macrophages. ELISA demonstrated significantly higher levels of total serum IgG, IgM, IgG1, and IgG2a in the group receiving adjuvanted gonorrhea vaccine when compared to the control group. The antibodies generated in mice after immunization were bactericidal towards the gonorrhea antigen at 1:1000 dilution titer. The control group cleared the infection in 10-12 days, whereas those immunized with our novel vaccine in 6-9 days. Thus, significant protection was seen in the immunized mice. The mice receiving adjuvanted vaccine expressed significantly higher CD4 and CD8 levels when compared to the control group in the spleen and lymph nodes.

Conclusion: This study demonstrates a potential vaccine for gonorrhea. Dissolving MN-based adjuvanted inactivated gonococci vaccine formulation enhanced antigen uptake, processing, and antigen presentation in vitro. We also explored the in vivo immunogenicity, correlates of protection, and resistance to infection of our novel gonorrhea vaccine. Correlates of protection of a gonorrhea vaccine model have not been extensively explored thus far. Our findings indicate the type of immune responses needed to be induced to generate protective immunity and imply that a vaccine against gonorrhea may be feasible.

References: 1. Gala, R. P., Zaman, R. U., D’Souza, M. J. & Zughaier, S. M. Novel Whole-Cell Inactivated Neisseria Gonorrhoeae Microparticles as Vaccine Formulation in Microneedle-Based Transdermal Immunization. Vaccines 6, 60 (2018).
2. Song, W. et al. Local and humoral immune responses against primary and repeat Neisseria gonorrhoeae genital tract infections of 17beta-estradiol-treated mice. Vaccine 26, 5741–5751 (2008).
3. Menon, I. et al. Microneedles: A New Generation Vaccine Delivery System. Micromachines 12, 435 (2021).

Acknowledgements:The project is funded by an R15 grant from the NIH- 1R15AI133473-01A1.


Figure 1. Schematic representation of the immune cells activation post skin vaccination. Adjuvanted gonococci microparticles (Gc-MP+ Alum MP+ AddaVax™ MP) are effectively taken up by the antigen-presenting cells (APCs), processed inside the cytoplasm of the APCs or inside the autophagosomes, and presented to the antigen presentation molecules (MHC I and II) which further stimulate the cells of adaptive immunity.


Figure 2. Nitric oxide release from macrophages pulsed with gonococci vaccine microparticles (Gc-MP) with or without adjuvant MP. Macrophages were adjusted to 1x106 cells/mL and pulsed with various doses of vaccine microparticles for 18 hours. Gonorrhea vaccine microparticles Gc-MP-100 µg/mL, Gc-MP with Alum microparticles (Gc-MP+ Alum MP) 50 µg/mL of each, Gc-MP with AddaVax™ microparticles (Gc-MP+ AddaVax™ MP) 50 µg/mL of each. Unstimulated control cells alone were used. Nitrite accumulation in supernatants was measured using the Griess reaction. Dendritic cell (DC 2.4) viability after exposure to different amounts of gonococci vaccine microparticles measured using MTT assay. DC 2.4 cells (2.5x104 cells/well) were exposed to increasing doses of Gc-MP (50 to 500 µg/well) or DMSO (50 µL) and incubated at 37°C for 48 hours. All the experiments were repeated three times. Data is expressed as mean ± SD, Ordinary one-way ANOVA test, post-hoc Tukey’s multiple comparison test. * p < 0.05 significant, ** p < 0.01 very significant, *** p < 0.05 extremely significant, **** p < 0.001 extremely significant.


Figure 3. A. Serum Bactericidal Assay: In vivo microneedle immunization with inactivated whole-cell gonorrhea antigen generated bactericidal antibodies to N. gonorrhoeae. Colony Forming Units recovered (CFU/mL) of N. gonorrhoeae (mean ±SEM, N=5 mice), * p < 0.05 significant, ** p < 0.01 very significant. B. Protection in mice following live challenge. % Of infected mice at each day after inoculation for 12 days. C. Resistance to infection in mice following live challenge. CFU/mL recovered of N. gonorrhoeae.