IntroductionLimited protective immunologic responses to natural N. gonorrhoeae infection and a lack of knowledge about mechanisms of protection have hampered development of an effective vaccine. Recent studies in humans and mice have found meningococcal outer membrane vesicle-containing vaccines (OMV) induce cross species immune responses against gonococci and are associated with protection. The exact mechanisms or how humoral and cellular immunity are related to protection, remain unclear.MethodsTo study this, we immunized mice with two meningococcal OMV-containing vaccines known to accelerate clearance of N. gonorrhoeae, 4CMenB and OMV from an engineered N. meningitidis strain lacking major surface antigens PorA, PorB, and Rmp (MC58 ΔABR). We assessed serologic and cellular immune signatures associated with these immunizations and assessed bacterial clearance in the mice using a vaginal/cervical gonococcal infection model.ResultsMice immunized with 4CMenB or MC58 ΔABR demonstrated shortened courses of recovery of vaginal N. gonorrhoeae compared to control mice immunized with alum alone. Vaccination with 4CMenB or MC58ΔABR OMV elicited serum and vaginal cross-reactive anti-Ng-OMV antibody responses that were augmented after vaginal challenge with N. gonorrhoeae. Further, splenocytes in 4CMenB and MC58 ΔABR immunized mice exhibited elevated cytokine production after restimulation with heterologous N. gonorrhoeae OMV when compared to splenocytes from Alum immunized mice. We further tested for correlations between bacterial burden and the measured anti-gonococcal immune responses within each vaccination group and found different immunologic parameters associated with reduced bacterial burden for each vaccine.DiscussionOur findings suggest the cross-protection against gonococcal infection induced by different meningococcal OMV vaccines is likely multifactorial and mediated by different humoral and cellular immune responses induced by these two vaccines.

BACKGROUND:Identification of immune correlates in murine gonorrhea models has been hampered by study-dependent differences in vaccine antigens and administration routes. We previously showed that detergent-detoxified outer membrane vesicles (dOMVs) isolated from a PorA-, PorB-, and RmpM-deficient meningococcal strain (ΔABR) elicit antibodies that cross-react with Neisseria gonorrhoeae and enhance gonococcal clearance in a mouse model of lower reproductive tract infection. In this study, we investigated whether (1) ΔABR dOMVs can protect mice from ascending gonococcal infection and (2) vaccination route influences immune responses. METHODS:Mice were vaccinated subcutaneously (SC) or intraperitoneally (IP) and then vaginally inoculated with gonococci. Bioburden of mice was measured and assessed relative to ΔABR dOMV-induced cellular and humoral immune responses. RESULTS:Subcutaneous and intraperitoneal vaccination accelerated gonococcal clearance from the lower and upper reproductive tract at similar rates. Probing of gonococcal protein microarrays with immune sera from the 2 groups identified multiple vaccine targets that were commonly immunogenic. Despite comparable clearance patterns in vaccinated mice, differences in immune induction were observed that were dependent on administration route. SC immunized mice demonstrated a neutrophil influx that correlated with decreased vaginal bioburden; higher serum bactericidal activity against nonsialylated gonococci was also noted. In contrast, IP immunization induced higher serum and vaginal IgA levels, serum bactericidal activity against sialylated gonococci, and antigonococcal opsonophagocytic killing activity of neutrophils. CONCLUSIONS:This work demonstrates that ΔABR dOMVs protect against ascending gonococcal infection and that cellular and functional antibody responses to the same candidate vaccine may vary depending on immunization route.

The recent entry of multiple vaccines targeting Neisseria gonorrhoeae (Ng) into the clinical development pathway has highlighted the need to establish methods of adequately assessing anti-gonococcal immune responses. Serum bactericidal activity (SBA) is utilized as a measurement of efficacy in licensure of meningococcal vaccines, but the importance of functional antibodies in preventing and/or clearing gonococcal infections remains largely unknown. In an effort to elucidate the utility of SBA as an immune correlate of protection, we sought to develop a standardized high-throughput human complement SBA (hSBA) assay for which any strain of interest could be tested under uniform conditions, with minimal screening of complement required. Utilization of IgG/IgM-depleted pooled human complement serum permitted testing of diverse serum resistant and sensitive gonococcal and unencapsulated meningococcal strains when bacteria were cultured to induce lipooligosaccharide sialylation; only one of nine randomly selected lots demonstrated intrinsic toxicity. The hSBA assay was well suited for use with multiple test serum sources, including rabbit, mouse, and human serum samples, suggesting widespread applicability.

[This corrects the article DOI: 10.3389/fimmu.2025.1539795.].

BACKGROUND:Despite decades of research efforts, development of a gonorrhea vaccine has remained elusive. Epidemiological studies suggest that detoxified outer membrane vesicle (dOMV) vaccines from Neisseria meningitidis (Nm) may protect against infection with Neisseria gonorrhoeae (Ng). We recently reported that Nm dOMVs lacking the major outer membrane proteins (OMPs) PorA, PorB, and RmpM induced greater antibody cross-reactivity against heterologous Nm strains than wild-type (WT) dOMVs and may represent an improved vaccine against gonorrhea.METHODS:We prepared dOMV vaccines from meningococcal strains that were sufficient or deleted for PorA, PorB, and RmpM. Vaccines were tested in a murine genital tract infection model and antisera were used to identify vaccine targets.RESULTS:Immunization with Nm dOMVs significantly and reproducibly enhanced gonococcal clearance for mice immunized with OMP-deficient dOMVs; significant clearance for WT dOMV-immunized mice was observed in one of two experiments. Clearance was associated with serum and vaginal anti-Nm dOMV immunoglobulin G (IgG) antibodies that cross-reacted with Ng. Serum IgG was used to identify putative Ng vaccine targets, including PilQ, MtrE, NlpD, and GuaB.CONCLUSIONS:Meningococcal dOMVs elicited a protective effect against experimental gonococcal infection. Recognition and identification of Ng vaccine targets by Nm dOMV-induced antibodies supports the development of a cross-protective Neisseria vaccine.

Detergent-extracted detoxified outer membrane vesicle (dOMV) vaccines are effective at preventing invasive serogroup B meningococcal (MenB) disease caused by the homologous Neisseria meningitidis strain from which they are produced, but offer limited protection from heterologous strains. Differences in vaccine efficacy are partially due to strain-specific variations in the antigenic sequence types and expression levels of outer membrane proteins (OMPs), including the immunodominant OMP PorA. In this study, dOMV vaccines deficient in major OMPs, including PorA, PorB, and RmpM were isolated and used to immunize rabbits and mice. Serum samples were obtained from each animal and tested for antibody responses against five MenB strains. Immunization with wild type dOMVs elicited antibodies to major antigens including PorA, PorB, RmpM, and lipooligosaccharide (LOS), and demonstrated limited bactericidal activity against heterologous strains. In contrast, OMP-deficient dOMV vaccines elicited broadly cross-reactive bactericidal antibodies, with PorA/PorB-dual deficient dOMVs inducing antibodies exhibiting the greatest cross-reactivity. Enhanced killing of heterologous strains correlated with binding to unique protein bands in immunoblots, suggestive of improved immunogenicity of antigens under-represented in the wild type vaccine.

Background With an incidence rate of 106 million infections a year, Neisseria gonorrhoeae has a significant effect on global morbidity. Rapid development of gonococcal antibiotic resistance, and reports of treatment failures with last-line cephalosporins, has caused the Centers for Disease Control and Prevention to label N. gonorrhoeae as an urgent threat and has sparked renewed interest in development of a gonococcal vaccine. Methods In this study, we immunized mice with detoxified outer membrane vesicles (dOMVs) isolated from the closely-related pathogen Neisseria meningitidis and examined the effect on gonococcal clearance in a murine vaginal colonization model. dOMV vaccines were derived from (1) wild type (WT) bacteria, (2) an isogenic strain (ΔABR) deleted for expression of the major outer membrane proteins PorA, PorB, and RmpM, or (3) an isogenic strain (OCh) deleted for PorA and expressing a varying PorB sequence type relative to the parental strain. ELISAs were used to evaluate anti-dOMV IgG and IgA antibody titers present in sera and vaginal washes. Sera were also used to identify potential gonococcal vaccine antigens using immunoblot and immunoprecipitation experiments. Results Although vaccination with WT dOMVs significantly enhanced gonococcal clearance relative to adjuvant-only controls, vaccination with ΔABR dOMVs resulted in clearance of a higher percentage of mice relative to WT dOMV-vaccinated mice one week post-immunization. Higher levels of clearance in ΔABR dOMV-immunized mice correlated with significantly increased vaginal IgA titers and enhanced immunogenicity of unique meningococcal protein antigens. Conclusion Immunization with meningococcal dOMVs deleted for PorA, PorB, and RmpM promotes gonococcal clearance in a murine model. Deletion of the major porins likely enhances immunogenicity of proteins that are less abundant on the meningococcal surface but exhibit a high degree of homology with corresponding gonococcal proteins, suggesting the potential utility of these dOMVs as a broadly cross-protective Neisseria vaccine. Disclosure No significant relationships.

The Neisseria meningitidis factor H binding protein (FHbp) is an important virulence factor and vaccine antigen contained in both USA licensed serogroup B meningococcal vaccines. Recent studies in human factor H (hFH) transgenic mice suggest that hFH-FHbp interactions lower FHbp-elicited immunogenicity. To provide tools with which to characterize and potentially improve FHbp immunogenicity, we developed an FHbp-cholera holotoxin-like chimera vaccine expression system in Escherichia coli that utilizes cholera toxin B (CTB) as both a scaffold and adjuvant for FHbp. We developed FHbp–CTB chimeras using a wild-type (WT) FHbp and a low hFH-binding FHbp mutant R41S. Both chimeras bound to GM1 ganglioside and were recognized by the FHbp-specific monoclonal antibody JAR4. The R41S mutant had greatly reduced hFH binding compared to the WT FHbp-CTB chimera. WT and R41S FHbp-CTB chimeric antigens were compared to equimolar amounts of FHbp admixed with CTB or FHbp alone in mouse immunogenicity studies. The chimeras were significantly more immunogenic than FHbp alone or mixed with CTB, and elicited bactericidal antibodies against a panel of MenB isolates. This study demonstrates a unique and simple method for studying FHbp immunogenicity. The chimeric approach may facilitate studies of other protein-based antigens targeting pathogenic Neisseria and lay groundwork for the development of new protein based vaccines against meningococcal and gonococcal disease.

PorB is a well-characterized outer membrane protein that is common among Neisseria species and is required for survival. A vaccine candidate, PorB induces antibody responses that are directed against six variable surface-exposed loops that differ in sequence depending on serotype. Although Neisseria meningitidis is naturally competent and porB genetic mosaicism provides evidence for strong positive selection, the sequences of PorB serotypes commonly associated with invasive disease are often conserved, calling into question the interaction of specific PorB loop sequences in immune engagement. In this report, we provide evidence that antibody binding to a PorB epitope can be altered by sequence mutations in non-epitope loops. Through the construction of hybrid PorB types and PorB molecular dynamics simulations, we demonstrate that loops both adjacent and non-adjacent to the epitope loop can enhance or diminish antibody binding, a phenotype that correlates with serum bactericidal activity. We further examine the interaction of PorB with outer membrane-associated proteins, including PorA and RmpM. Deletion of these proteins alters the composition of PorB-containing native complexes and reduces antibody binding and serum killing relative to the parental strain, suggesting that both intramolecular and intermolecular PorB interactions contribute to host adaptive immune evasion.

BACKGROUND:PsA-TT (MenAfriVac) is a conjugated polysaccharide vaccine developed to eliminate group A meningococcal disease in Africa. Vaccination of African study participants with 1 dose of PsA-TT led to the production of anti-A polysaccharide antibodies and increased serum bactericidal activity measured using rabbit complement (rSBA). Bactericidal responses measured with human complement (hSBA) are presented here. METHODS:Sera collected before and at 28 days and 1 year after vaccination with either PsA-TT or quadrivalent polysaccharide vaccine (PsACWY) from a random, age-distributed 360-subject subset of the Meningitis Vaccine Project study of PsA-TT in Africans aged 2-29 years were tested for hSBA. Geometric mean titer, fold-rise, and threshold analyses were compared between vaccine groups and age groups. hSBA, rSBA, and immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) results were compared and assay correlation and agreement determined. RESULTS:hSBA responses to PsA-TT were substantially higher than those to PsACWY at 28 days and 1 year following immunization, similar to previously reported rSBA and IgG results. The hSBA and IgG ELISA results identified differences between age groups that were not evident by rSBA. The rSBA data indicated sustained high titers 1 year after immunization, whereas hSBA GMTs at 1 year approached 4 in young children. CONCLUSIONS:The high level of protection following PsA-TT immunization campaigns is consistent with the strong hSBA immune responses observed here. Future implementation decisions will likely depend on immunologic data and their long-term correlation with disease and carriage prevention. Expanded immunologic and epidemiologic surveillance may improve the interpretation of differences between these immunoassays.

ABSTRACT A meningococcal group A polysaccharide (PS) conjugate vaccine (PsA-TT) has been developed for African countries affected by epidemic meningitis caused by Neisseria meningitidis . Complement-mediated serum bactericidal antibody (SBA) assays are used to assess protective immune responses to meningococcal vaccination. Human complement (hC′) was used in early studies demonstrating antibody-mediated protection against disease, but it is difficult to obtain and standardize. We developed and evaluated a method for sourcing hC′ and then used the SBA assay with hC′ (hSBA) to measure bactericidal responses to PsA-TT vaccination in 12- to 23-month-old African children. Sera with active complement from 100 unvaccinated blood donors were tested for intrinsic bactericidal activity, SBA titer using rabbit complement (rSBA), and anti-group A PS antibody concentration. Performance criteria and pooling strategies were examined and then verified by comparisons of three independently prepared hC′ lots in two laboratories. hSBA titers of clinical trial sera were then determined using this complement sourcing method. Two different functional antibody tests were necessary for screening hC′. hSBA titers determined using three independent lots of pooled hC′ were within expected assay variation among lots and between laboratories. In African toddlers, PsA-TT elicited higher hSBA titers than meningococcal polysaccharide or Hib vaccines. PsA-TT immunization or PS challenge of PsA-TT-primed subjects resulted in vigorous hSBA memory responses, and titers persisted in boosted groups for over a year. Quantifying SBA using pooled hC′ is feasible and showed that PsA-TT was highly immunogenic in African toddlers.

Gonorrhea occurs at high incidence throughout the world and significantly impacts reproductive health and the spread of human immunodeficiency virus. Current control measures are inadequate and seriously threatened by the rapid emergence of antibiotic resistance. Progress on gonorrhea vaccines has been slow; however, recent advances justify significant effort in this area. Conserved vaccine antigens have been identified that elicit bactericidal antibodies and, or play key roles in pathogenesis that could be targeted by a vaccine-induced response. A murine genital tract infection model is available for systematic testing of antigens, immunization routes and adjuvants, and transgenic mice exist to relieve some host restrictions. Furthermore, mechanisms by which Neisseria gonorrhoeae avoids inducing a protective adaptive response are being elucidated using human cells and the mouse model. Induction of a Th1 response in mice clears infection and induces a memory response, which suggests Th1-inducing adjuvants may be key in vaccine-induced protection. Continued research in this area should include human testing and clinical studies to confirm or negate findings from experimental systems and to define protective host factors.

BACKGROUNDQuinolone-resistant Neisseria gonorrhoeae (QRNG) arise from mutations in gyrA (intermediate resistance) or gyrA and parC (resistance). Here we tested the consequence of commonly isolated gyrA(91/95) and parC86 mutations on gonococcal fitness.METHODSMutant gyrA(91/95) and parC86 alleles were introduced into wild-type gonococci or an isogenic mutant that is resistant to macrolides due to an mtrR(-79) mutation. Wild-type and mutant bacteria were compared for growth in vitro and in competitive murine infection.RESULTSIn vitro growth was reduced with increasing numbers of mutations. Interestingly, the gyrA(91/95) mutation conferred an in vivo fitness benefit to wild-type and mtrR(-79) mutant gonococci. The gyrA(91/95), parC86 mutant, in contrast, showed a slight fitness defect in vivo, and the gyrA(91/95), parC86, mtrR(-79) mutant was markedly less fit relative to the parent strains. A ciprofloxacin-resistant (Cip(R)) mutant was selected during infection with the gyrA(91/95), parC86, mtrR(-79) mutant in which the mtrR(-79) mutation was repaired and the gyrA(91) mutation was altered. This in vivo-selected mutant grew as well as the wild-type strain in vitro.CONCLUSIONSgyrA(91/95) mutations may contribute to the spread of QRNG. Further acquisition of a parC86 mutation abrogates this fitness advantage; however, compensatory mutations can occur that restore in vivo fitness and maintain Cip(R).

Neisseria meningitidis serogroup B has been predominant in Brazil, but no broadly effective vaccine is available to prevent endemic meningococcal disease. To understand genetic diversity among serogroup B strains in Brazil, we selected a nationally representative sample of clinical disease isolates from 2004, and a temporally representative sample for the state of São Paulo (1988-2006) for study (n = 372).We performed multi-locus sequence typing (MLST) and sequence analysis of five outer membrane protein (OMP) genes, including novel vaccine targets fHbp and nadA.In 2004, strain B:4:P1.15,19 clonal complex ST-32/ET-5 (cc32) predominated throughout Brazil; regional variation in MLST sequence type (ST), fetA, and porB was significant but diversity was limited for nadA and fHbp. Between 1988 and 1996, the São Paulo isolates shifted from clonal complex ST-41/44/Lineage 3 (cc41/44) to cc32. OMP variation was associated with but not predicted by cc or ST. Overall, fHbp variant 1/subfamily B was present in 80% of isolates and showed little diversity. The majority of nadA were similar to reference allele 1.A predominant serogroup B lineage has circulated in Brazil for over a decade with significant regional and temporal diversity in ST, fetA, and porB, but not in nadA and fHbp.

Capsular polysaccharide (CP) plays an important role in the pathogenicity and immunogenicity of Staphylococcus aureus, yet the common serotypes of S. aureus isolated from US pediatric patients have not been reported. We investigated capsular serotype as well as methicillin susceptibility, presence of Panton-Valentine leukocidin (PVL), and clonal relatedness of pediatric S. aureus isolates. Clinical isolates were tested for methicillin susceptibility, presence of mecA, lukS-PV and lukF-PV, cap5 and cap8 genes by PCR, and for capsular or surface polysaccharide expression (CP5, CP8, or 336 polysaccharide) by agglutination. Genetic relatedness was determined by pulsed-field gel electrophoresis. All S. aureus isolates encoded cap5 or cap8. Sixty-nine percent of 2004-2005 isolates were methicillin-susceptible (MSSA) and most expressed a detectable capsule. The majority of MRSA isolates (82%) were unencapsulated, exposing an expressed cell wall techoic acid antigen 336. Pulsed-field type USA300 were MRSA, PVL-positive, unencapsulated strains that were associated with deep skin infections and recurrent disease. Over half (58%) of all isolates from invasive pediatric dermatologic infections were USA300. All pediatric isolates contained either capsule type 5 or capsule type 8 genes, and roughly half of the S. aureus clinical disease isolates from our population were diverse MSSA-encapsulated strains. The majority of the remaining pediatric clinical disease isolates were unencapsulated serotype 336 strains of the PVL(+) USA300 community-associated-MRSA clone.

The genus Neisseria includes both pathogenic and commensal species. N. meningitidis and N. gonorrhoeae are obligate human pathogens with no reservoir outside of the human host. N. lactamica, N. sicca, N. subfl ava (biovars subfl ava, fl ava, and perfl ava), N. mucosa, N. fl avescens, N. cinerea, N. polysaccharea, and N. elongata subspecies elongata, glycolytica, and nitroreducens are human commensal organisms that are rarely associated with disease. Commensal organisms found in animal respiratory tract or oral fl ora include N. canis and N. weaveri in dogs, N. dentrifi cans in guinea pigs, N. macacae in rhesus monkeys, N. dentiae in cows, and N. iguanae in iguanid lizards.