Immunogenicity of a malaria parasite antigen displayed by Lactococcus lactis in oral immunisations

Abstract

A putative protective protein from Plasmodium falciparum merozoites, MSA2, was expressed in two different ways on the cell surface of the Gram-positive food-grade bacterium, Lactococcus lactis. The first display format exploits an LPXTG-type anchoring motif of the lactococcal proteinase PrtP to covalently anchor MSA2 to the genetically modified producer cells. In a second display format, MSA2 was fused to the peptidoglycan-binding domain (Protein Anchor) of the lactococcal cell wall hydrolase AcmA and was non-covalently rebound to the surface of non-genetically modified, non-living high-binder L. lactis cells, termed Gram-positive enhancer matrix (GEM) particles. The L. lactis recombinants carrying covalently bound MSA2 were used to immunise rabbits through nasal and oral routes. The highest levels of IgG antibodies reacting with near-native MSA2 on merozoites was elicited by oral administration. Intestinal antibodies to MSA2 were produced only after oral immunisation. MSA2-specific T(h)-cell activation could be demonstrated. Based on these results, the immunogenicity in oral immunisations of MSA2, bound non-covalently to non-genetically modified L. lactis GEM particles, was compared with MSA2 that was bound covalently to genetically modified L. lactis. These two forms elicited similar titres of serum antibodies. The results illustrate the potential of using non-genetically modified L. lactis as a safe vaccine delivery vehicle to elicit systemic antibodies, thereby avoiding the dissemination of recombinant DNA into the environment.

Fig. 1

Schematic drawings of the most relevant parts of pNG3041 and pNG3043. The indicated domains contain the nucleotide sequences encoding. Open box: MSA2 without signal sequence and GPI anchor sequence. Black box: native signal sequence of MSA2. GPI anchor sequence of MSA2. The arrow represents the nisin-inducible promoter of L. lactis. The lollypop represents a lactococcal transcriptional terminator sequence. signal sequence of PrtP. prosequence of PrtP. one repeat of the AcmA cell wall anchor. The three repeats constitute the entire cell-wall anchor domain (PA). cell wall anchor of PrtP (cP).

Fig. 2

Panel A: SDS-PAGE analysis of Coomassie-stained proteins in cell lysates (5 × 10⁸ cells) prepared from—lane 1: L. lactis-pNZ8048, empty cloning vector; lane 2: L. lactis-pNG3043(MSA2-Cov); lane 3: the equivalent of 1 ml culture supernatant of L. lactis-pNZ8048; lane 4: the equivalent of 1 ml culture supernatant of L. lactis-pNG3041(MSA2-nCov); lane 5: L. lactis GEM particles; lane 6: L. lactis GEM particles with MSA2-nCov absorbed from 1.25 ml L. lactis-pNG3041(MSA2-nCov) cell-free culture supernatant. M: molecular weight marker (precision Plus Prestained Marker, BioRad), the sizes of which are indicated in kDa in the left margin. The asterisk and the arrow indicate the positions of MSA2-Cov and MSA2-nCov, respectively. Panel B: Immunoblot decorated with anti MSA2-GST antibodies to detect MSA2-containing fusion proteins. Content of the lanes is the same as in panel A.

Fig. 3

Immunofluorescence assay (IFA) showing reaction of rabbit anti-MSA2 antibodies on: (A) L. lactis-pNG3043 induced for 4 h with nisin to express MSA2-Cov; (B) L. lactis GEM particles after absorption with MSA2-nCov from the cell-free culture supernatant of L. lactis-pNG3041 induced for 4 h to express MSA2-nCov. The negative controls did not show any fluorescence (data not shown).

Fig. 4

Electron micrographs showing, by immunogold labelling using rabbit anti-MSA2 antiserum and gold-labelled goat anti-rabbit IgG, MSA2 on the surface of: (A) L. lactis(MSA2-Cov), 20 min after induction with nisin A; (B) L. lactis GEM particles with attached MSA2-nCov. The black bars indicate a size of 1 μm. For detection, secondary antibodies labelled with 15 nm gold particles were used, both in (A) and (B). Because of the used whole mount sample preparation procedure (see Section 2), the GEM particles shrink and the photograph was more enlarged in order to obtain similar sized bacterial particles.

Fig. 5

Immunoblots showing reaction of sera of immunised rabbits with total protein extracts of 5 × 10⁸L. lactis cells per lane—lane 0: preimmune serum; lane 2: serum after the 2nd immunisation; lane 3: serum after the 3rd immunisation. Serum dilution 1:100. Panel A: Serum of a rabbit immunised i.m. with L. lactis(MSA2-Cov). Panel B: Serum of a rabbit orally immunised with L. lactis(MSA2-Cov). Panel C: Serum of a rabbit orally immunised with L. lactis GEM particles with bound MSA2-nCov.