Characterisation of Cytochrome c peroxidase: A vaccine candidate for Neisseria gonorrhoeae

Abstract

Neisseria gonorrhoeae is an obligate human pathogen and the causative agent of gonorrhoea, a sexually transmitted infection. Over 100 million cases of gonorrhoea are reported globally each year. Antibiotics have been widely used to treat gonorrhoea. However, with the recent emergence of multi-drug resistance strains, gonorrhoea has been recognised as a major public health challenge and a vaccine for N. gonorrhoeae is urgently required. Previously, vaccine development has been challenging due to the bacteria’s ability to avoid and modulate the hosts immune response. Conserved surface components of N. gonorrhoeae have been identified and due to the bacteria’s variability, the use of a combination of antigens has been proposed as the optimal approach for development of a N. gonorrhoeae vaccine. This study aims to characterise Cytochrome c peroxidase (Ccp) as a potential vaccine candidate for N. gonorrhoeae. We have characterised Ccp by evaluating this outer membrane proteins expression and cell localisation in a panel of N. gonorrhoeae strains, and the immunogenicity and functional activity of antibodies raised to a recombinant Ccp protein. In Western blot and whole cell enzyme linked immunosorbent assay (ELISA) analyses, it was found that Ccp was expressed in all N. gonorrhoeae strains analysed in this study. However, the surface exposure of Ccp was variable across this panel of N. gonorrhoeae strains. In a whole cell ELISA, we found there was a higher level of absorbance detected for Ccp in N. gonorrhoeae strain 1291 at the highest serum dilutions, compared to a Ccp mutant strain. In a flow cytometric analysis, we also found that there was a higher level of fluorescence detected for Ccp in N. gonorrhoeae strain 1291, compared to a Ccp mutant strain. N. gonorrhoeae strain 1291 was then analysed via trypsin digestion. After multiple repeats the results were inconclusive, where although the intensity of Ccp did decrease, inconsistencies were observed in the level of decrease seen. A panel of 14 N. gonorrhoeae strains were then analysed via trypsin digestion. The signal for Ccp decreased in 11 strains, indicating cell surface exposure of Ccp. However, the signal for Ccp did not decrease in three strains, indicating that Ccp was not surface exposed in all the N. gonorrhoeae strains analysed. The ccp sequences from the panel of N. gonorrhoeae strains were then analysed to determine if there were any differences between the strains that could impact the cellular localisation of Ccp in these strains. A single base pair difference was present in nearly half of the N. gonorrhoeae strains analysed in this study. However, no correlation was found between the surface exposure of Ccp in N. gonorrhoeae, as predicted by the trypsin digestion analysis, and the presence of this single base pair difference. Antibodies raised to the recombinant Ccp protein were then analysed in Western blot and whole cell ELISA analyses, with N. gonorrhoeae strain 1291 and the panel of N. gonorrhoeae strains. The recombinant protein was found to be immunogenic in murine models with a dominant Immunoglobulin (Ig) G1 response, and the antibodies raised to recombinant Ccp protein were cross-reactive with all N. gonorrhoeae strains analysed. Antibodies raised to the recombinant Ccp protein were also analysed in a serum bactericidal activity (SBA) assay with N. gonorrhoeae strains 1291 and World Health Organisation (WHO) K. We found that antibodies raised against recombinant Ccp protein did not promote complement mediated killing via serum bactericidal activity. Our data indicated that Ccp would not be suitable in a single protein vaccine due to the variability of its surface expression, since it would only be recognised by vaccine induced antibodies in strains that express this protein on the surface of the bacterium. However, the proteins conservation and ability to induce an immune response may make it a candidate for a small component in a multicomponent vaccine for N. gonorrhoeae, if antibodies were found to be able to neutralise the function of Ccp. The multicomponent nature would prevent vaccine escape by strains that do not express Ccp on the surface of this bacterium.