Protective immune response against disseminated neumococal infection elicited by recombinant Lactococcus lactis expressing pneumococcal PppA protein

MEDINA, M.; VILLENA, J.C.; VINTIÑI, E.; HEBERT, E.M.; ALVAREZ, S.; RAYA, R.R.

San Miguel de Tucumán

Simposio; II SIMPOSIO INTERNACIONAL DE BACTERIAS LACTICAS; 2006

CERELA-CONICET

Streptococcus pneumoniae is the main cause of bacterial meningitis, pneumonia and otitis media. Current vaccination strategies rely on multivalent pneumococcal conjugate vaccine, which can only be effective against disease caused by serotypes contained in the vaccine. Here, we report the development of a mucosal live vaccine using the food-grade lactic acid bacterium, Lactococcus lactis, as an attractive antigen delivery vector against S. pneumoniae, in particular against serotypes not covered by the present conjugate vaccine which may still cause pneumococcal disease. The pppA gene was amplified by PCR, subcloned into the NcoI and SalI sites of pET28c, and transformed into Escherichia coli BL21(DE3) cells for expression. After induction of the recombinant BL21(DE3) cells with 1mM IPTG (isopropyl-â-d-thiogalactopyranoside) during 2 hours, expression of the desired recombinant protein (approx. 21 kDa) was confirmed by electrophoresis on a SDS-PAGE gel. The pppA was also subcloned as a SalI-EcoRV into plasmid pVE5547 and the resulting plasmid was introduced by electroporation into L. lactis NZ9000. Recombinant L. lactis cells, after induction with nisin, were charged on its surface with the 354- aminoacids recombinant PppA protein and used to study the survival of S. pneumoniae intraperitoneally challenged mice. Differences in median survival time between groups were analyzed by the Mann-Whitney U test (one tailed). Mice were challenged with 108 cells/mouse of S. pneumoniae T 14. The median survival time for adult mice that received PBS or L. lactis PppA- was less than 4.5 days and all mice in these groups died in just over 7 days. The young mice in control group showed a lower survival than adults and all of them died in 3.5 days. In contrast, the mice immunizated with L. lactis PppA+ stayed alive during all the assayed period (closely monitored during 21 days). Our data suggest that the recombinant L. lactis strain secreting PppA has potential for in vivo immunization.