Oral administration of recombinant Neisseria meningitidis PorA genetically fused to H. pylori HpaA antigen increases antibody levels in mouse serum, suggesting that PorA behaves as a putative adjuvant

Abstract

The Neisseria meningitidis outer membrane protein PorA from a Chilean strain was purified as a recombinant protein. PorA mixed with AbISCO induced bactericidal antibodies against N. meningitidis in mice. When PorA was fused to the Helicobacter pylori HpaA antigen gene, the specific response against H. pylori protein increased. Splenocytes from PorA-immunized mice were stimulated with PorA, and an increase in the secretion of IL-4 was observed compared with that of IFN-γ. Moreover, in an immunoglobulin sub-typing analysis, a substantially higher IgG₁ level was found compared with IgG_2a levels, suggesting a Th2-type immune response. This study revealed a peculiar behavior of the purified recombinant PorA protein per se in the absence of AbISCO as an adjuvant. Therefore, the resistance of PorA to proteolytic enzymes, such as those in the gastrointestinal tract, was analyzed, because this is an important feature for an oral protein adjuvant. Finally, we found that PorA fused to the H. pylori HpaA antigen, when expressed in Lactococcus lactis and administered orally, could enhance the antibody response against the HpaA antigen approximately 3 fold. These observations strongly suggest that PorA behaves as an effective oral adjuvant.

Keywords:

• AbISCO ^TM-100® adjuvant
• Helicobacter pylori antigen
• hybrid PorA antigens
• meningococcal B porin antigen
• oral immunization
• recombinant PorA

Abbreviations:

• AbISCO ^TM-100®, adjuvant based on saponin
• immunostimulant complex matrix
• LAL, limulus amebocyte lysate

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We acknowledge Claudio Figueroa, Bárbara Riveros and América Abarca from the ISP laboratory for helpful discussions and technical assistance and Shelton Wright and Hans Jensen for critical review of the manuscript.

Funding

This research was funded by FONDEF DO2I-1067 (CONICYT-CHILE), FONDECYT 1085232 (CONICYT-CHILE) and BMRC CTU-06 Area 5 (Pontificia Universidad Católica, Laboratorio Recalcine, CONICYT-CHILE), granted to A. Venegas and an institutional grant to A. E. Vásquez from the Instituto de Salud Pública de Chile.

Authors’ Contributions

Conception and design of the experiments: AEV, AV; performance of PorA and PorB cloning and expression experiments: AEV, AA, EB, MM and RM; purification of proteins and protease degradation assays: AA, MJB, and JHT; PorA hybrid constructions and transfer to pSEC vector: MM; expression of PorA hybrid in L. lactis: MM and EB; mice immunization assays: RM, EB, DS, MM and JHT; bactericidal assays: AEM; statistical analysis and manuscript writing: AEV, PRH and AV.