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Tackling immunosuppression by Neisseria gonorrhoeae to facilitate vaccine design [1]

['Rebekah A. Jones', 'Sir William Dunn School Of Pathology', 'University Of Oxford', 'South Parks Road', 'Oxford', 'United Kingdom', 'Fidel Ramirez-Bencomo', 'School Of Biological Sciences', 'Faculty Of Biology', 'Medicine']

Date: 2024-12

Gonorrhoea, caused by Neisseria gonorrhoeae, is a common sexually transmitted infection. Increasing multi-drug resistance and the impact of asymptomatic infections on sexual and reproductive health underline the need for an effective gonococcal vaccine. Outer membrane vesicles (OMVs) from Neisseria meningitidis induce modest cross-protection against gonococcal infection. However, the presence of proteins in OMVs derived from N. gonorrhoeae that manipulate immune responses could hamper their success as a vaccine. Here we modified two key immunomodulatory proteins of the gonococcus; RmpM, which can elicit ‘blocking antibodies’, and PorB, an outer membrane porin which contributes to immunosuppression. As meningococcal PorB has adjuvant properties, we replaced gonococcal PorB with a meningococcal PorB. Immunisation with OMVs from N. gonorrhoeae lacking rmpM and expressing meningococcal porB elicited higher antibody titres against model antigens in mice compared to OMVs with native PorB. Further, a gonococcal protein microarray revealed stronger IgG antibody responses to a more diverse range of antigens in the Nm PorB OMV immunised group. Finally, meningococcal PorB OMVs resulted in a Th1-skewed response, exemplified by increased serum IgG2a antibody responses and increased IFNɣ production by splenocytes from immunised mice. In summary, we demonstrate that the replacement of PorB in gonococcal OMVs enhances immune responses and offers a strategy for gonococcal vaccine development.

Neisseria gonorrhoeae is the bacterium that causes the sexually transmitted infection gonorrhoea. Gonorrhoea is a public health concern due to the bacterium developing resistance to numerous antibiotics, leading to the prospect of untreatable gonorrhoea infections. Untreated gonorrhoea causes severe complications, particularly impacting reproductive health. So far, no gonorrhoea-specific vaccine is available. N. gonorrhoeae vaccine efforts focus on optimising outer membrane vesicles (OMVs). However, the success of this approach may be hampered by the presence of immunomodulatory proteins in N. gonorrhoeae OMVs. Here we show that two key immunomodulatory proteins; the major outer membrane porin, PorB, and its stabilising protein, RmpM likely contribute to the lack of success of a gonorrhoea OMV-based vaccine. We improved immune responses to gonorrhoea OMVs by genetically modifying N. gonorrhoeae to produce OMVs lacking RmpM and containing a PorB replacement. Moving forwards, the modified OMVs represent a promising platform for a vaccine against gonorrhoea.

Funding: This work was supported by Wellcome Trust Collaborative and Investigator Awards (214374/Z/18/Z and 221924/Z/20/Z to CMT). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files.

This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

Our objective was to generate a gonococcal OMV-based vaccine that lacks key immunomodulatory proteins which would otherwise suppress protective immune responses. As PorB is essential for the viability of N. gonorrhoeae, it has proven difficult to study the role of this protein during infection, including its influence on immune responses. To circumvent the immunosuppressive properties of PorB and RmpM, we replaced N. gonorrhoeae porB with a meningococcal porB gene in a N. gonorrhoeae strain lacking rmpM; the introduction of meningococcal porB rescued the viability of gonococcus lacking its native porB. We show that mice immunised with OMVs derived from N. gonorrhoeae expressing meningococcal PorB elicit significantly higher antibody titres against a range of antigens compared to OMVs containing gonococcal PorB. We also show that immunisation with OMVs derived from N. gonorrhoeae expressing meningococcal PorB enhanced Th1-mediated responses compared to OMVs containing gonococcal PorB, exhibiting increased IgG2a antibody responses against OMV antigens and eliciting increased IFNɣ from murine splenocytes. Our data suggest that N. gonorrhoeae PorB significantly impairs immune responses following administration as an OMV-based vaccine against this bacterium, and this can be circumvented by its replacement with meningococcal PorB, paving the way for the development of gonococcal vaccines derived from OMVs from the bacterium itself.

N. meningitidis expresses two major porins, PorA and PorB, in contrast to other Neisseria species which only express a single major porin, related to PorB [ 38 , 39 ]. Importantly, meningococcal PorB displays broad immunostimulatory properties and has been used as a vaccine adjuvant due to its ability to induce antigen-specific B- and T-cell responses [ 40 – 42 ]. This is distinct from the properties of N. gonorrhoeae PorB that contribute significantly to immunosuppression [ 30 ]. PorB proteins from N. meningitidis and N. gonorrhoeae share 60–70% amino acid sequence homology, depending on the strains being compared [ 22 , 43 ].

Two key antigens have proposed roles in N. gonorrhoeae immune evasion. The porin protein PorB is the most abundant outer membrane protein and facilitates ion exchange across the outer membrane. PorB is essential for the viability of N. gonorrhoeae and represents a large portion (~60%) of both outer membrane and OMV proteomes [ 28 , 29 ]. N. gonorrhoeae PorB has several immunomodulatory properties [ 30 ]. Gonococcal PorB contributes to resistance against the complement system by recruiting human negative complement regulators [ 31 , 32 ]. Gonococcal PorB also influences innate and adaptive immune responses by repressing cellular killing mechanisms and influencing apoptosis in neutrophils and macrophages, and by inhibiting T cell proliferation during antigen presentation [ 29 , 33 ]. Additionally, gonococcal PorB would not be an ideal vaccine antigen because it is extremely variable, with 1,229 unique PorB amino acid sequences reported across 5,000 gonococcal strains [ 34 , 35 ]. A second immune suppressive protein is RmpM; the N-terminus of RmpM binds to trimeric PorB in the bacterial outer membrane and links it to peptidoglycan through its C-terminal domain [ 36 ]. Antibodies against RmpM have been reported to block the recognition of other surface antigens, thereby preventing immune killing of bacteria [ 37 ].

A critical outstanding question is why N. gonorrhoeae-derived OMVs have been relatively unsuccessful as a vaccine platform. In Bexsero OMVs, only 25 outer membrane proteins common to N. meningitidis NZ98/254 and N. gonorrhoeae are present, of which only 12 are abundantly and consistently expressed in different batches [ 22 ]. Together with other fundamental differences in antigen expression, this suggests that N. gonorrhoeae-derived OMVs should have a greater efficacy against gonococcal infection than meningococcal OMVs. However, the presence of antigens within N. gonorrhoeae OMVs that suppress immune responses might undermine this approach. Importantly, N. gonorrhoeae suppresses the development of T helper (Th)1- and Th2-mediated adaptive immune responses, instead priming an innate immune response governed by Th17 cells, leading to neutrophil recruitment and promoting gonococcal survival [ 23 ]. Indeed, the most promising subunit and OMV vaccines for N. gonorrhoeae utilise adjuvants that drive a Th1 response, such as CpG and microencapsulated IL-12 [ 24 , 25 ]. Promoting a Th1 response is currently considered a key factor for a successful gonococcal vaccine, exemplified by intravaginal addition of IL-12 leading to faster clearance of murine gonococcal infection [ 26 ]. In mice, a Th1 response is characterised by interferon-ɣ (IFNɣ) production, which stimulates the expression of the immunoglobulin (Ig) G2a antibody isotype, whereas a Th2 response is characterised by interleukin-4 (IL-4) production and stimulates the expression of IgG1 antibody isotype [ 27 ].

Immunosuppression induced by N. gonorrhoeae might be a significant factor when comparing the host response to infection with the gonococcus and other species of Neisseria. Asymptomatic colonisation of the nasopharynx by Neisseria meningitidis leads to the induction of an appropriate humoral response that prevents invasive meningococcal disease [ 14 ]. Equally, colonisation with the commensal species Neisseria lactamica can induce protection against infection with N. meningitidis [ 15 ]. However, infection with N. gonorrhoeae does not appear to elicit natural immunity against itself except in some highly exposed individuals [ 16 ]. In contrast, immunisation with N. meningitidis outer membrane vesicle (OMV)-based vaccines conferred a degree of cross-protection against gonorrhoea [ 17 – 19 ]; OMVs are naturally shed from the surface of Gram-negative bacteria and thereby contain a multitude of cell surface proteins [ 20 ]. The effectiveness estimates of OMV-based meningococcal vaccines against N. gonorrhoeae range from 31% to 46%, which persists for up to three years post-vaccination [ 21 ]. Given the capacity of Neisseria species, other than N. gonorrhoeae, to elicit adaptive immune responses, it is perhaps unsurprising that meningococcal vaccines confer a degree of cross-protection against the gonococcus due to cross-reactive antigens.

There are several challenges faced in developing vaccines against N. gonorrhoeae. Importantly, natural infection does not appear to provide immunity, as N. gonorrhoeae suppresses adaptive immune responses and the killing mechanisms of innate immune cells [ 9 ]. N. gonorrhoeae is a human-specific pathogen, representing an additional challenge in developing physiologically relevant animal models of infection. Together these factors mean that there are no known correlates of protection against the gonococcus. Further challenges to vaccine development include the extraordinary capacity of N. gonorrhoeae to vary the composition of its cell surface through phase variation and antigenic variation [ 10 ], and the ability of the gonococcus to evade patrolling immune cells by residing intracellularly within epithelial cells, neutrophils, and macrophages [ 11 , 12 ]. Finally, N. gonorrhoeae utilises numerous mechanisms to manipulate the host environment to promote its survival, including suppressing host immune responses [ 13 ].

Neisseria gonorrhoeae causes the sexually transmitted infection gonorrhoea and has developed resistance against all known treatments [ 1 ]. When untreated, infection with N. gonorrhoeae can lead to severe complications, including pelvic inflammatory disease, infertility, ectopic pregnancy, and neonatal blindness [ 2 ]. Additionally, gonorrhoea facilitates the acquisition and spread of HIV [ 3 ]. Gonococcal infection is highly prevalent in low- and middle-income countries (LMICs), with the African WHO region estimated to have the highest annual incidence of gonorrhoea [ 4 ]. Therefore, the control of gonococcal infection would have a major impact on female sexual and reproductive health of those living in impoverished circumstances, exemplified by the disproportionate number of quality-adjusted life-years lost by women due to gonococcal infection [ 5 ]. N. gonorrhoeae has a remarkable ability to develop resistance to antibiotics through the acquisition of plasmids and chromosomal mutations [ 6 ]. Therefore, attention has turned towards preventative measures, including the development of vaccines [ 7 ]. Importantly, vaccines can play a key role in tackling AMR by decreasing the number of infections, thereby reducing antibiotic use and the emergence and spread of resistant bacteria [ 8 ].

Results

Replacement of porB We genetically engineered N. gonorrhoeae to remove the immunosuppressive properties of PorB and RmpM from our gonococcal vaccine. To generate N. gonorrhoeae FA1090 expressing a meningococcal porB, N. meningitidis MC58 porB (“Nm PorB”) was amplified by PCR, fused to a kanamycin resistance cassette and introduced into FA1090 by transformation (Fig 1A). N. meningitidis MC58 porB is a class 3 porB allele while N. gonorrhoeae FA1090 porB is a PorB.IB allele [44]; the proteins share 68% amino acid identity (S1 Fig). Insertion of Nm porB was confirmed by PCR and sequencing, demonstrating that the gonococcal porB gene had been entirely replaced with the meningococcal gene. The resultant strain, FA1090 MC58 PorB , had a reduced growth rate compared to wild type (WT) FA1090 (S2 Fig). Western blot analysis using PorB-specific typing monoclonal antibodies confirmed that FA1090 MC58 PorB expressed Nm PorB alone (Fig 1B). In addition, the rmpM gene was replaced with ermC’, and the deletion confirmed by PCR and Western blot analysis (Fig 1D). The deletion of rmpM exacerbated the growth defect of FA1090 MC58 PorB (S2 Fig). PPT PowerPoint slide

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TIFF original image Download: Fig 1. A) Generation of N. gonorrhoeae (Ng) FA1090 expressing MC58 Nm porB. Schematic representation of replacing the Ng porB gene with Nm porB, under the control of the native Ng promoter. Promoters shown in grey. Kanamycin resistance cassette (kanR) under its own promoter is present downstream of the open reading frame for selection. B) Western blots confirming the replacement of Ng PorB with Nm PorB, using mAbs against specific PorBs. C) Schematic representation of the deletion of rmpM in Ng FA1090 by replacement with an erythromycin resistance cassette (eryR). D) Western blot confirming deletion of rmpM. Arrows denote open reading frames. Dotted lines represent regions of homologous recombination. Figure not to scale. https://doi.org/10.1371/journal.ppat.1012688.g001

Ng PorB OMVs and Nm PorB OMVs have additional proteome differences OMVs were generated from FA1090ΔrmpM (Ng PorB OMVs) and FA1090 Nm porB ΔrmpM (Nm PorB OMVs). Upon analysis of the OMVs by denaturing SDS-PAGE, we observed different protein profiles of the Ng PorB OMVs and Nm PorB OMV preparations, in addition to the change in PorB. Differences in the OMV proteomes were consistent between independent batches (S3 Fig). To compare the OMV proteomes in detail, we analysed them by mass spectrometry. A total of 174 proteins were identified across three biological replicates of Ng PorB OMVs and Nm PorB OMVs (S2 Table). Proteins identified in only one sample, with low peptide number (< 2), or low overall coverage (< 7%), were eliminated from the analysis, leaving 86 proteins identified with high confidence (FDR of < 1%, S3 Table). Proteomic analysis confirmed that Ng PorB OMVs only contained FA1090 PorB, and Nm PorB OMVs only contained MC58 PorB. Other than PorB, one protein was found solely in Nm PorB OMVs, NGO_09965, an Opacity (Opa) family protein, and one protein was found solely in Ng PorB OMVs (NEIS0210, unknown function). A total of 31 out of the 86 proteins identified exhibited similar abundance in Nm PorB OMVs and Ng PorB OMVs, while 46 had a higher abundance (ratio ≥ 2) in Nm PorB OMVs compared to Ng PorB OMVs, with only four proteins found to have lower abundance (ratio ≤ 0.5) (Fig 2 and S3 Table). PPT PowerPoint slide

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TIFF original image Download: Fig 2. The proteomes of OMVs derived from N. gonorrhoeae FA1090ΔrmpM and FA1090 MC58 PorB ΔrmpM differ. Proteins with an abundance ratio (Nm PorB OMVs/Ng PorB OMVs) ≥ 2 are indicated in green, and those with an abundance ratio ≤ 0.5 are indicated in red. Proteins with an abundance ratio between 0.5 and 2 are indicated in blue. https://doi.org/10.1371/journal.ppat.1012688.g002 Several groups of proteins with related functions exhibited a greater than two-fold increase in Nm PorB OMVs compared with Ng PorB OMVs. Iron acquisition and storage proteins transferrin binding protein B (TbpB), transferrin binding protein A (TbpA) and bacterioferritin (BrfB) were in higher abundance in Nm PorB OMVs. Furthermore, Nm PorB OMVs had a higher abundance of other nutrient acquisition proteins, including zinc-acquisition proteins TonB-dependent function protein-H and -J (TdfH, TdfJ), zinc-binding protein A (ZnuA, also known as MntC), and the methionine transporter MetQ. Potentially related to the increase in nutrient acquisition proteins, surface lipoprotein assembly modulator proteins 1 and 2 (Slam1 and Slam2) also had higher abundances; Slam1 is involved in the translocation of TbpB to the outer membrane [45]. The abundance of metabolism-related proteins ethanol–active dehydrogenase (AdhP), dihydrolipoamide acetyltransferase (AceF), and carbonic anhydrase (Cah) were also increased in Nm PorB OMVs. A final group of interest were proteins related to host-pathogen interactions, including Neisseria surface protein A (NspA), IgA1 protease (NEIS1959), macrophage infectivity potentiator (Ng-MIP), and Neisseria heparin binding antigen (NHBA), which were all increased in Nm PorB OMVs compared to Ng PorB OMVs. Two Opa-related proteins, NGO_05420 and OpaD (NEIS0903) had the largest differences when comparing Nm PorB OMVs and Ng PorB OMVs, with abundance ratios (AR, Nm/Ng) of 74 and 44, respectively. Such a large difference in AR is consistent with ON:OFF phase variation, which is known to affect Opa expression [46]. An additional Opa54-related protein, NGO_06725, also had a higher abundance in Nm PorB OMVs, with an AR of 6.2. Additionally, NGO_07725, an Opa54-related protein, was identified with slightly lower abundance in Nm PorB OMVs with an AR of 0.84. Together these data suggest that phase variation and/or mutations occurred within the Opa coding regions during the construction of the OMV-producing strains, resulting in the expression of multiple Opa54 proteins. Overall, replacing the porB gene in N. gonorrhoeae FA1090 with meningococcal porB resulted in additional differences in the OMV proteomes with changes in factors involved in nutrient acquisition, metabolism, and host-pathogen interactions. As a significant number of proteins were more abundant in Nm PorB OMVs, we used quantitative mass spectrometry to determine whether the abundance of PorB differed between Nm and Ng PorB OMVs. PorB represented 70.2% (± 9.3%) of the Ng PorB OMV proteome, compared to 36.5% (± 3.9%) of the Nm PorB OMV proteome. Quantitatively, PorB measured 506 μg/mL (± 191 μg/mL) in Ng PorB OMVs and 235 μg/mL (± 67 μg/mL) in Nm PorB OMVs (Table 1). In a 12.5 μg vaccine dose, PorB would average 8.8 μg (± 1.2 μg) in Ng PorB OMVs and 4.6 μg (± 0.5 μg) in Nm PorB OMVs. In summary, the amount of PorB is reduced in Nm PorB OMVs compared to Ng PorB OMVs, potentially reflecting the increased abundance of other proteins. PPT PowerPoint slide

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TIFF original image Download: Table 1. Absolute quantification of PorB in Ng- and Nm-PorB OMVs using quantitative mass spectrometry. Data are the mean ± standard deviation. https://doi.org/10.1371/journal.ppat.1012688.t001

Immunoprofiling of murine antibody responses against gonococcal antigens To further characterise antibody responses after immunisation with OMVs, sera from individual immunised mice were used to probe microarrays containing 91 gonococcal surface proteins. The serum IgG reactivity to each individual gonococcal antigen was determined for mice immunised with i) PBS alone, ii) fHbp alone, iii) fHbp with Ng PorB OMVs, or iv) fHbp with Nm PorB OMVs and shown as a heat map or volcano plots (Figs 5 and S5, respectively). Low background responses for the PBS and fHbp-immunised groups are apparent. For sera derived from mice immunised with OMVs, reactivities against multiple gonococcal antigens were detected. When comparing the Ng PorB OMV and Nm PorB OMV immunised groups, several antigens exhibit reactivities in both groups. Antigens with stronger reactivities indicated by higher mean fluorescence intensity (MFI) included MtrE, SliC, Lipoprotein 2 (NEIS0906), GNA2091 (NEIS2071), NEIS1462, Lipoprotein 1 (NEIS1063), PilQ and NEIS1487. Of these eight proteins, the antibody reactivity was stronger in the Nm PorB OMV immunised group for seven of the antigens. PilQ was the exception, where reactivity in Ng PorB OMV immunised mice was stronger. A relatively lower MFI was observed in both OMV immunised groups for BamE, PilE, and Ton2, where again the reactivity was higher in the Nm PorB OMV immunised group for three of the four antigens; for Slam1, reactivity levels were similar between Nm PorB OMV and Ng PorB OMV immunised mice. A subset of antigens showed reactivity in only one group; NEIS2647 (NGO554) displayed reactivity in only Ng PorB OMV immunised mice, whereas a larger number of antigens, including Ape1, IgA protease, Maf1, MetQ, NspA, Potf3, NEIS1125 and NEIS1405, displayed reactivity only in Nm PorB OMV immunised mice. The stronger antibody responses to MtrE and MetQ in Nm PorB OMV immunised mice, compared to Ng PorB OMV immunised mice, shown by the protein microarray are consistent with the ELISA data (Fig 3). Overall, microarray data analysis demonstrated that total murine IgG antibody responses were higher and more diverse after immunisation with Nm PorB OMVs compared to immunisation with Ng PorB OMVs. PPT PowerPoint slide

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TIFF original image Download: Fig 5. Antigen-specific total IgG responses in sera from individual mice immunised with Ng PorB OMVs (third section) or Nm PorB OMVs (fourth section), compared to PBS alone (first section) and fHbp alone (second section); each column is the result for an individual mouse. Individual antigens are in indicated in each row. Colour represents the mean fluorescence intensity (MFI) value for antibody binding to each antigen. https://doi.org/10.1371/journal.ppat.1012688.g005 The gonococcal protein microarray revealed strong reactivities to PorB and Opa variants. The microarray included PorB from FA1090 and, as expected, mice immunised with Ng PorB OMVs generated a strong antibody response to FA1090 PorB, the variant present in these OMVs. In contrast, Nm PorB OMVs elicited very weak antibody responses to FA1090 PorB. For Opa proteins, both Nm PorB OMV and Ng PorB OMV immunised mice exhibited widespread reactivity against Opa variants. The reactivity profiles to Opa variants are remarkably similar for both OMV immunised groups, particularly given the differences in Opa expression profiles revealed by proteomic analysis of the OMVs, consistent with responses elicited against epitopes shared between Opas. To further assess murine polyclonal IgG responses after immunisation with Ng or Nm PorB OMVs, the gonococcal protein microarrays were also used to analyse the reactivity of IgG1 and IgG2a subclasses (Fig 6). Similar prominent profiles for IgG1 and IgG2a reactivity to the Opa variants were observed for both Ng- and Nm-PorB OMVs. Interestingly, some antigens tended to elicit IgG2a over IgG1 antibody responses; for example, BamE, SliC, Lipoprotein 1 (NEIS1063) and Lipoprotein 2 (NEIS0906) have relatively stronger MFI signals for IgG2a. However, this observation was independent of the Ng/Nm PorB OMV immunisation group. Overall, when examining individual antigens, reactivity in Nm PorB OMV immunised mice was stronger for both IgG1 and IgG2a, compared to Ng PorB OMV immunised mice, suggesting that increases in both subclasses contributed to the overall higher IgG responses shown in Fig 5. PPT PowerPoint slide

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TIFF original image Download: Fig 6. Antigen-specific IgG1 and IgG2a responses in sera from mice immunised with Ng PorB OMVs or Nm PorB OMVs. Individual antigens are in indicated in each row; each column is the result for an individual mouse. Each colour block represents the mean fluorescence intensity (MFI) value for antibody binding to each antigen. https://doi.org/10.1371/journal.ppat.1012688.g006 Principal Component Analysis (PCA) was applied to all four immunisation groups in order to capture the variance between each serum sample and group samples with similar reactivity profiles. As both OMV groups responded strongly to nearly all Opa proteins and only Ng PorB OMVs elicited PorB reactivity, we removed PorB and Opa variants to ascertain which other antigens were contributing to the different responses after immunisation with Ng or Nm PorB OMVs. PCA of individual serum samples showed that Ng PorB OMV and Nm PorB OMV immunised mice were well separated from the fHbp alone and PBS control groups in the PC1 dimension (Fig 7A; each point is a serum sample from a single mouse). Even with the PorB and Opa variants removed, sera from Nm PorB OMV immunised mice were separated from Ng PorB OMV serum samples, further from the PBS controls. This observation is attributable to a greater amplitude of antigen responses overall in the Nm PorB OMV serum samples. The antigens contributing most strongly to this separation were Lipoprotein 2 (NEIS0906), outer membrane protein H.8, Potf3, Lipoprotein 1 (NEIS1063), SliC, MtrE, GNA2091 (NEIS2071) and NEIS1487 (Fig 7B). In summary, PCA showed that differences in murine IgG antibody responses to Ng PorB or Nm PorB OMVs are attributable to several different gonococcal antigens. PPT PowerPoint slide

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TIFF original image Download: Fig 7. PCA applied to antigen-specific total IgG responses. A) Separation by individual serum sample, including all antigens. The biplot is generated using the squared coordinates (cos2) for PC1 and PC2, calculated as the squared coordinates of the eigenvalues. B) Contributions of individual antigens, excluding PorB and Opa variants, to PC1 and PC2 separation. https://doi.org/10.1371/journal.ppat.1012688.g007

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