Effect Of Binding Human Factor H On The Immune Activities Of Gonorrhea Vaccine Candidate NspA

Gonorrhea caused by Neisseria gonorrhoeae is one of the most prevalent sexually transmitted diseases. N. gonorrhoeae can cause repeat infection in humans, and gonococcal infections also facilitate HIV transmission. The treatment of gonorrhea becomes problematic while more and more clinical isolates are found to be resistant to normally used antibiotics. Therefore, development of effective vaccines becomes a key to prevent gonococcal infections.Preliminary researches in our laboratory suggested that NspA (Neisseria surface protein A) was a gonorrhea vaccine candidate since the protein induced specific bactericidal and opsonic antibodies in mice. NspA of N. meningitidis can bind human complement regulatory protein factor H (fH). Binding of meningococcal fHBP (factor H-binding protein) to fH decreases its immunogenicity and bactericidal activity of the Abs against it. Based on the fact that gonococcal NspA is95%homologous to meningococcal NspA, we postulate that gonococcal NspA could bind fH. To research the effect of binding human fH on the immune activities of gonococcal NspA, we have performed the following studies.1. Prokaryotic expression of fH SCR5-8and the preparation of polyclonal antibodies against itBinding of meningococcal NspA to fH occurred through fH domains SCR6-7. Therefore, we designed forward and reverse primers based on the published fH gene sequence. The gene fragment of fH SCR5-8was cloned by using pCAGGS-fH gifted by Prof. Dan M. Granoff as template. The constructed recombinant prokaryotic expression vector pCold-fHSCR5-8was submitted to sequence analysis. BLAST result demonstrated that the cloned fH SCR5-8gene was100%homologous to that published in GenBank. The expressed His-TF-fH SCR5-8fusion protein of76kDa was isolated by SDS-PAGE. The93%purity of the recombinant fH SCR5-8(rfH SCR5-8) was obtained following purification by Bio-Rad protein purifier. To prepare antibodies for recognition fH, the eukaryotic expression vector pCAGGS-fH were used to immunize mice. ELISA assay revealed that the fH specific antibody titer was between1:6400and1:12800. Western blotting assay suggested that the rfH SCR5-8reacted with the antiserum.2. Prokaryotic expression of NspA and the preparation of polyclonal antibody against gonococcal WHO-A strain To prepare rNspA for binding rfH, the gene encoding NspA was amplified by PCR using pVAX1-NspA as template. The PCR product was inserted into pCold-TF vector. The resultant prokaryotic expression vector pCold-NspA was used to express recombinant His-TF-NspA (68kDa) in E. coli BL21by adding IPTG as an inducer. The rNspA was purified by Bio-Rad protein purifier, obtaining a purity of95%. To prepare antibodies that can react with NspA, mice were immunized by gonococcal WHO-A strain; and, ELISA assay suggested that the NspA specific antibody titer was between1:1600and1:3200. Western blotting assay revealed that the rNspA could bind the WHO-A antiserum.3. Binding of gonococcal NspA to fH SCR5-8WHO-A cells were incubated with rfH SCR5-8and fH gene immune sera. Flow cytometry assay indicated the binding of rfH SCR5-8to WHO-A cells, which suggested that there was one or more substances in the surface of WHO-A cells could bind rfH SCR5-8. A reaction band of68kDa was detected by Western blot following that rNspA was transferred to PVDF membrane and then incubated with rfH SCR5-8and fH gene immune sera, which suggested that rNspA could bind fH SCR5-8. ELISA assay indicated the binding of rNspA to rfH SCR5-8as well by incubating the enveloped rNspA with rfH SCR5-8.The gene fragment of fH SCR5-8was inserted into pEGFP-Nl eukaryotic expression vector, and the resultant vector pEGFP-fH was transfected into CT-26cells. The transfection cells stably expressing interest gene were obtained by G418screening. Total proteins were extracted from transfection cells, transferred to PVDF membrane, incubated with rNspA and WHO-A antiserum. A reaction band of76kDa suggested the binding of rfH SCR5-8to rNspA.4. Effect of binding fH on the immune activities of candidate vaccine NspATo study the effect of binding fH on the immune activities of gonorrhea vaccine candidate NspA, mice were immunized with rNspA and rNspA-rfH compound. ELISA assay indicated that the antibody titer of immunization with rNspA-rfH compound was similar to that of rNspA, the ability of the antibody bind to Neisseria gonorrhoeae decreased slightly. Antibody-dependent complement mediated cytotoxicity assay suggested that bactericidal activities of antiserum against rNspA-rfH compound had slight decrease compared with that of rNspA antiserum.