Recombinant hybrid proteins from pertactin type 1 and 2 of Bordetella pertussis are more immunogenic in mice than the original molecules

The present study explores the concept of hybrid pertactin (PRN) molecules for immunizing against Bordetella pertussis. New molecules were designed using an additive/inclusive approach that comprehends the complete sequences/ regions of two different types of pertactin (Prn). PRN molecules bear the two variable R1 regions from Prn1 and Prn2. The genes of Prn1, Prn2 and six variants of PRN were cloned in Escherichia coli, and PRN proteins were over-expressed at 25-30 % of total protein concentrations using the pET28a/BL21 Codonplus RP expression system. The proteins were purified (> 90 % purity) using the His-tag /Ni-NTA affinity method with amounts of 8-10 mg/g of wet biomass. After refolding, the PRNs were recognized by anti-Prn monoclonal antibodies that bind protective conformational and linear epitopes/regions. Moreover, a panel of ten sera from individuals boosted with a commercial vaccine reacted with the PRN molecules without differences from the P.69 protein. The PRN proteins were highly immunogenic in Balb/c mice, with the induction of the IgG2a and IgG2b subtypes. Particularly, two PRNs (PRN2-lc-1 > PRN2-1) induced highly significant anti-Prn1 antibody levels (p < 0.001). Moreover, the PRN2-lc-1 induced higher levels of antibodies (p < 0.05) against epitopes located at the immunodominant N-terminus region and the variable region R1. The PRN2-lc-1 and PRN2-1 molecules exhibited an enhanced immunological profile in Balb/c mice in terms of level of whole anti-Prn IgG antibodies in respect to natural Prn controls. These two molecules constitute valuable candidates for further evaluation in vivo in acellular vaccine formulations.