Group B streptococcus capsular polysaccharide - Cholera toxin B subunit conjugate vaccine for mucosal immunisation : development and preclinical evaluation of experimental conjugate vaccines

Abstract

Group B streptococci (GBS) can colonize the female genital and rectal tracts and cause invasive infection in susceptible newborns. An optimally effective GBS vaccine should induce both mucosal and systemic immunity. We prepared GBS capsular polysaccharide (CPS) - recombinant cholera toxin B subunit (rCTB) conjugate vaccines by different methods and evaluated their systemic and mucosal immunogenicity in mice. Specific IgM, IgG and IgA antibodies to the CPS were tested in serum and IgG and IgA antibodies in lungs, intestinal, rectal and vaginal extracts by ELISA.We found, as tested with GBS type III CPS, that generally CPS III conjugates were superior to unconjugated CPS in eliciting CPS-specific immune responses in serum and mucosal tissue extracts. The conjugates when administrated subcutaneously induced only IgG responses in serum, lung and vagina, while intranasal (i.n.) vaccination also elicited specific anti-CPS IgA responses in the lungs and vagina. A CPS-tetanus toxoid conjugate administrated i.n. induced a strong serum IgG but only a weak mucosal IgA response, while CPS-rCTB conjugates elicited high IgG as well as IgA antibodies in both serum and the lungs after i.n. immunization. Both large and small molecular size fractions of CPS III-rCTB conjugates induced high, almost comparable levels of CPS specific IgG in serum, lungs and vagina that were generally superior to those obtained with a mixture of CPS III and rCTB. However, the smaller conjugates in most cases elicited a lower anti-CPS IgA immune response as compared with the larger conjugates, and the strongest anti-CPS responses in both tissues and serum were obtained with the large fraction of a CPS III-rCTB conjugate prepared by reductive amination.The CPS specific immune responses in various organs were dependent on the route of immunization. Generally, as tested with four routes of mucosal vaccination, including i.n. oral, vaginal and rectal immunizations, the highest levels of IgA and IgG anti-CPS antibody responses were generated in the regions or sites of conjugate exposure. All four mucosal routes also induced antibody responses to CPS in distant mucosal tissues. Rectal vaccination induced high specific IgA in the vagina and the intestine, and oral administration in the lungs and rectum. All four routes of vaccination with the conjugate elicited similar, high levels of anti-CPS IgG in serum. I.n. immunization was usually the most efficient way to obtain strong anti-CPS responses in both serum and mucosal tissues including both the lungs and the vagina.Priming with pure GBS CPS III alone or with GBS CPS III-rCTB conjugate induced comparable levels of specific IgG and IgA in serum, lungs and vagina. However, the mice primed with CPS III and boosted with CPS III-rCTB conjugate by i.n. route failed to produce significant levels of IgG2a, IgG2b and IgG3 in serum in contrast to mice primed with the conjugate, which produced high levels of all these IgG sub-classes. Pre-immunization with rCTB did not suppress specific serum IgG response induced by GBS CPS III-rCTB conjugate intranasally, but did inhibit mucosal IgA responses.I.n. immunization with a combination of CPS type III-rCTB and CPS type Ia-rCTB conjugates was shown to induce anti-CPS III and Ia immune responses in serum and lungs that were fully comparable with the responses to immunization with the monovalent Ia-rCTB or III-rCTB conjugates.These results demonstrate that (1) use of rCTB as a carrier protein for GBS III CPS could markedly improve the mucosal immune response; (2) rectal and vaginal vaccination and slightly less consistently i.n. immunization appear to be the most efficient ways of stimulating local anti-CPS immune responses in the rectal and vaginal tracts; (3) GBS CPS III-rCTB and Ia-rCTB conjugates prepared by reductive amination may serve as a basis for development of effective bivalent or multivalent mucosal vaccines to prevent mucosal colonization and invasive infection caused by GBS.