Tackling immunosuppression by Neisseria gonorrhoeae to facilitate vaccine design

Abstract

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.

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.

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.

Fig 3. Murine antibody responses after immunisation with Ng- or Nm-PorB expressing OMVs.

A-C) Endpoint IgG antibody titres of pooled sera recognising the model antigen fHbp, and the OMV antigens MtrE and MetQ, determined by ELISA. D-F) Endpoint IgG1 and IgG2a titres recognising the model antigen fHbp, and the OMV antigens MtrE and MetQ, determined by ELISA. Data are the mean ± standard deviation. *** p < 0.001, **** p < 0.0001. G-I) Antibody deposition on whole cell N. gonorrhoeae strains FA1090, G97687 and 60755 determined by flow cytometry showing the mean fluorescence intensity.

Fig 4. Murine antibody responses to gonococcal lipooligosaccharide (LOS) after immunisation with Ng- or Nm-PorB expressing OMVs.

A-B) Western blots probing gonococcal LOS with murine sera after immunisation with Ng PorB OMVs (left blot) or Nm PorB OMVs (right blot) (A) or with 2C7 mAb (B). C) LOS ELISA probing with murine sera from Ng- or Nm-PorB OMV immunised mice or control groups, with or without pre-blocking with the 2C7 mAb. * p < 0.05.

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.

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.

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.

Fig 8. Cytokines produced by murine splenocytes after immunisation with Ng- or Nm-PorB OMVs, when re-stimulated with concanavalin A (ConA, positive control), Ng PorB OMVs, Nm PorB OMVs or no stimulation.

A) interferon-ɣ (IFNɣ), B-F) interleukin (IL)-4, IL-10, IL-17A, IL-6 and IL-2. Data are the mean ± standard error of the mean. * p < 0.05, *** p < 0.001, **** p < 0.0001, ns: not significant.