Adjuvants Derived from Neisseria meningitidis Serogroup B Induce a Cross Reactive Response against Neisseria gonorrhoeae in Mice

Introduction: Neisseria is a large genus of bacteria that colonize mucosal surfaces. Of the 11 species that colonize humans, only two are pathogens, N. meningitidis and N. gonorrhoeae. Both are obligate human pathogens; the last is causal agent of gonorrhea disease. Although curable with timely antibiotic treatment, an annual incidence of more than 62 million cases is estimated by the World Health Organization and there are no successful vaccines available. In contrast, several prophylactic vaccine options for Neisseria meningitidis meningitis do exist. Of note, there is trace of cross parenteral response induced between N. meningitidis and N. gonorrhoeae, and Proteoliposome (PL, also named outer membrane vesicles, OMV) vaccine has shown high impact on this response. Objective: To determine effect of VAMENGOC-BC™ and its derivates (AFPL1 and AFCo1) at mucosal and systemic level and possible cross response against Neisseria gonhorroeae in Balb/c and C57Bl/6 mice. Methods: We evaluated cross response against N. gonorrhoeae in mouse serum IgG and saliva IgA after mucosal immunization with VA-MENGOC-BC or its derivatives (AF, Adjuvant Finlay PL1 or AFCo (cochleate) 1). Results: Immunizations with AFPL1 or AFCo1 induce anti N. gonorrhoeae at saliva IgA and serum IgG responses similar to VA-MENGOC-BC™ vaccine. Conclu-sions: Such data confirms a new possible window of prime-boost vaccination strategy against gonorrhea and extends our knowledge regarding the effect of PL vaccines on cross responses.


Introduction
Neisseria is a large genus of bacteria that colonize mucosal surface. Of the 11 species that colonize humans, only two are pathogens, N. meningitidis and N. gonorrhoeae. Both are obligate human pathogens; the last is the causal agent of gonorrhea disease. Although curable with timely antibiotic treatments, high numbers of resistant strains had an annual incidence of more than 62 million cases estimated by the World Health Organization and there are no successful vaccines available [1]. This is also a neglected disease that can be associated with human immunodeficient virus (HIV)-co-infection. However, there are reports of some potential vaccine candidates against N. gonorrhoeae [2] [3] and cross IgG response between N. meningitidis and N. gonorrhoeae has been reported [4]. This is mainly consequence of the similar structural proteins important for immunogenicity. In addition, proteoliposome (PL, also named outer membrane vesicles, OMV) vaccines against N. meningitidis have been demonstrated to reduce the incidence of human Gonorrhea infection as first reported using VA-MENGOC-BC TM vaccination in Cuba [5] followed by MeNZB vaccination in New Zealand [6]. While N. gonorrhoeae infects and colonizes at mucosal surfaces where IgA is the main protecting antibody, both VA-MENGOC-BC TM and MeNZB vaccines are parenteral vaccines mainly generating systemic specific IgG protective responses. However, evaluation of mucosal immunization has been limited due to its lower immunogenicity and higher dose requirement as compared to parenteral routes and the absence of mucosal adjuvants [7].
We have developed a PL-derived cochleate (AFCo1), which contains similar proteins and pathogen-associated molecular patterns as PL. It induces specific systemic IgG and mucosal IgA in mice and is thus a promissory mucosal adjuvant. AFCo1 applied by nasal route induces mucosal (regional and distal) and systemic immune responses (specific saliva IgA, serum IgG, and IgG2a subclass), and IFN-γ polarizing to a Th1 pattern [8] [9] [10]. Successful results were obtained using nasal immunization of AFCo1 containing recombinant glycoprotein 2 from Herpes simplex virus type 2 [11]. It also demonstrated adjuvant effect with N. meningitidis and Salmonella typhi polysaccharides inducing memory B cells, Th1 cytokines and specific IgA production with similar anti polysaccharide C response to the bivalent VA-MENGOC-BC™ vaccine without the requirement of covalent conjugation [12].
As noted above, one of the shortcomings of parenteral immunization is its relatively poor ability to induce genital-tract-specific IgA [13] [14]. IgA is considered important in protecting the genital tract from infection, as its presence is correlated with a protective role against Chlamydia and human immunodeficiency virus (HIV) [15] [16]. In mice, intranasal immunization has been promising in terms of eliciting genital-tract antigen-specific IgA and IgG [17] [18], primates [19], and humans [20] [21]. Therefore, we conducted this study of gonococcal cross protection by meningococcal vaccination to obtain more information on this subject. Animal immunization and sample collection. In the pathogen free mice: two groups of seven female C57Bl/6 mice each were inoculated (i.n) with AFCo1 or AFPL1m respectively, using 3 doses of 100 µg with 7 days-intervals in 12.5 µL per nostril per dose per mouse. One group was inoculated (i.m) with AFPL1 absorbed onto Al(OH) 3 using 2 doses of 25 µg with 7 days-intervals in 100 µL.

Material and Methods
Another group was used as control. In the conventional mice: three groups of seven female BALB/c mice each were inoculated i.m with AFCo1, AFPL1 or VA-MENGOC-BC™ using 2 doses of 12 µg with 14 days-intervals in 100 µL per mouse. Another two similar age and sex groups were immunized i.n with AFCo1 or AFPL1 using 3 doses of 100 µg with 7 days-intervals in 12.

Results
Parenteral immunizations with VAMENGOC-BC™ or its derivatives (AFPL1 and AFCo1) induced systemic specific IgG response without a mucosal mucosal specific IgA response, but intranasal AFPL1 and AFCo1 did induce salivary specific IgA in BALB/c mice We confirmed that i.m applications of VA-MENGOC-BC™ vaccine, AFPL1 or AFCo1 induce specific systemic IgG response in BALB/c mice. VA-MENGOC-BC™ and AFCo1 induced similar specific IgG response to each other, but they were higher than that induced by AFPL1 ( Figure 1A). IgA ( Figure 1B). Nevertheless, i.n applications of AFPL1 or AFCo1 induced a systemic specific IgG and mucosal specific IgA in BALB/c mice. Both immune responses were higher in AFCo1 than AFPL1 immunized mice ( Figure 1C and Figure 1D). Overall, the mucosal routes induced specific IgA but parenteral administration did not.
Parenteral VA-MENGOC-BC™ did not induce salivary IgA but a mucosal boost did In order to further evaluate the prime-boost strategies using two routes of immunization to explore the possible influence of mucosal challenge after a pre-   Evaluation of IgG1 and IgG2c subclasses as a Th2 or Th1 polarized response, respectivelly, was also conducted. Serum anti PL IgG1 was higher than anti N. gonorrhoeae, in concordance with their specific serum IgG. The anti N. gonorrhoeae IgG induced by AFPL1 was less than 0.4 OD value. This negative value did not mean a negative response but a response lower than the cut off value of the assay ( Figure 3B). Anti PL IgG2c responses were similar among all formulations but significantly higher than anti N. gonorrhoeae antigens which were below the cut off ( Figure 3C).
Anti PL and N. gonorrhoeae vaginal IgG responses were both induced. Note that these results are presented in OD because there were below the stringent cut off but still significant induced ( Figure 3D) as also represented above for anti N. gonorrhoeae IgG2c ( Figure 3C)

Discussion
Parenteral immunization with VAMENGOC-BC™ and its derivatives (AFPL1 and AFCo1) which conserve core protective antigens induced systemic specific IgG response, but not mucosal specific IgA responses. In contrast, intranasal AFPL1 and AFCo1 induce mucosal specific IgA in BALB/c mice. This is in concordance with the well-known dictum that mucosal administration is required to induce mucosal response. This route induces similar IgG responses to the parenteral route but also induced potent mucosal IgA responses [22] [23]. This is in contrast to VAMENGOC-BC™ applied by parental route which requires a mucosal boost (AFPL1 or AFCo1) to induce a specific IgA response. AFCo1, with a multilayer microparticle containing calcium is more stable and more potent than the AFPL1 nanoparticle administered by the mucosal route as was previously described [7] [24].
VA-MENGOC-BC™ and its derivatives, AFPL1 or AFCo1, by i.n or i.m immunizations induce anti Neisseria gonorrhoeae IgG cross reactive responses in C57Bl/6 mice. Specific IgG1 was higher than IgG2c against both antigens, PL and N. gonorrhoeae; but the former antigens induced higher specific IgG1 levels than the later one. IgG2c is also induced against both antigens but more so for PL than Ng antigens. In addition, mucosal specific IgA responses against both antigens were poorly detected in C57Bl/6 mice.
Cross reactive responses of genus Neisseria has been investigated, mainly between pathogenic species N. meningitidis and N. gonorrhoeae [4] [25]. These studies followed demonstration that VA-MENGOC-BC™ human vaccination decreased the gonococcal incidence in Cuba as reported by us as early as 2009 [5], later referred by others authors in 2018 [26] and 2019 [27] and extended to other similar vaccines in 2017 [6]. Certainly, PL (OMV) vaccines have a high impact in this type of cross reactive response.
The complex constitution of the PL [28] [29] [30] as protective antigenic core of VA-MENGOC-BC™ with its adjuvant properties [8] [24] [31] are certainly involved in the induction of cross protection. These proteins have a structural homology with N. gonorrhoeae and have been described as capable of producing a cross-response [26]. Another possibility is based on the structural homology that exists between commensal species of the genus; such is the case of N. lactamica since it is closely related to N. meningitidis and N. gonorrhoeae as it colonizes the upper respiratory tract [32]. In addition, genetic analyses of both pathogenic and non-pathogenic Neisseria show they are closely related, with evolutionary studies suggesting frequent exchange of genetic material including virulence genes [33]. Of note is that a decline of gonococcal incidence was also observed in New Zealand using MeNZB which also contains PL (OMV) [6].
The induction of more IgG1 (Th2 polarizing) than IgG2c (Th1 polarizing) against Ng formulations seems to be more related to the antigens per se than the adjuvant because this adjuvant present in VAMENGOC-BC™, AFPL1, or AFCo1 induces mainly a Th1 response in mice and human [7] [24] [25] [26]. Neverthe- Open Journal of Immunology less, the induction of a Th2 pattern is important for IgA mucosal response but the initial selections of C57Bl/6 for the experiment seem to be wrong. The use of BALB/c mice induces this specific IgA response against PL and N. gonorrhoeae.

Conclusion
In conclusion, we have shown that VAMENGOC-BC™ and its derivatives (AFPL1 and AFCo1) induce systemic specific IgG response without a mucosal one, but intranasal immunization with AFPL1 and AFCo1 induces both systemic and mucosal responses in BALB/c mice. The induction of cross reactive IgG response with Ng antigens was demonstrated in C57Bl/6 mice and extended to specific IgA in BALB/c mice.