Mammalian cell expression of malaria merozoite surface proteins and experimental DNA and RNA immunisation

Abstract

The gene for a 45 kDa merozoite surface protein (MSA-2) of the human malaria parasite Plasmodium falciparum was PCR amplified and cloned into eukaryotic expression vectors VR1012 and pcDNA3 to yield plasmids P1 and P2, respectively. The coding sequences for two N-terminal fragments of the 185 kDa merozoite surface protein (MSA-1) gene were similarly PCR amplified and cloned into vectors VR1020 and VR1012 to yield plasmids P3 and P4, respectively. The MSA-1 signal peptide sequence, present in P4, was replaced with the human tissue plasminogen activator signal sequence in P3. The four plasmids expressed the cloned genes under the control of the cytomegalovirus promoter and carried 3' bovine growth hormone termination/poly A signals. P1, P3 and P4 also contained the cytomegalovirus intron A enhancer sequence. MSA-1 expression was more readily detected than MSA-2 in Cos cells transfected with P3/P4 and P1/P2 respectively. The MSA-2 gene was also cloned into the phagemid pBluescript IISK+ with and without a 3' poly A tail composed of 35 A residues. MSA-2 was synthesised in HeLa cells infected with a recombinant vaccinia virus carrying T7 RNA polymerase when MSA-2 recombinant pBluescript was transfected into the cells. Inoculation with P1 intramuscularly or intradermally and with P2 intradermally into rabbits led to the production of antibodies to MSA-2 detectable by immunofluorescence and Western blotting. Antibodies were also produced against MSA-1 after intramuscular/intradermal inoculation with P3 and P4. Inoculation of rabbits with MSA-2 mRNA yielded better antibody titres when a poly A tail was present. Antibody levels were maintained for > 9 weeks after the final immunisation. However the immune sera failed to inhibit in vitro parasite growth.

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

“…Therefore, the find-ings suggest that foreign proteins expressed in L. lactis can also be used in oral vaccination procedures to elicit protective secretory antibodies in the gut. Antibody titres achieved by using both L. lactis display formats of MSA2 are better than those achieved by immunisation with plasmid DNA [34] or peptide-diphtheria toxoid conjugates [35] through the intramuscular and intradermal routes. Intramuscular immunisation with synthetic peptide polymers based on MSA2 B-cell epitopes elicited high-titre antibodies against the immunising peptide, which reacted only poorly with near-native MSA2 on merozoites [36].…”

Antibodies to a merozoite surface protein promote multiple invasion of red blood cells by malaria parasites

“…However, the present results show that MSA2 synthesized in the prokaryote Escherichia coli migrates as a 45-kDa, and not as a 28-kDa protein predicted from the DNA sequence. Recombinant MSA2 produced in mammalian COS cells, where post-translational modifications may differ from P. falciparum, also migrated as a 45-kDa protein on SDS-PAGE (Ramasamy et al 1999). Therefore it is possible that the presence of repeats and flanking sequences with unusual amino acid composition in extensive b turn or loop structures (Ramasamy 1998) and the acidic nature of MSA2 (predicted pI ¼ 5·25) may be responsible for the retarded migration of MSA2 and GST-MSA2 in SDS-PAGE.…”

Genetic and antigenic analysis of Babesia bigemina isolates from five geographical regions of Brazil

“…Hines et al (1992) demonstrated that polyclonal antibodies against these antigens were able to neutralize B. bovis infectivity. Rotman et al (1999) and Ramasamy et al (1999) showed respectively that Plasmodium yoelli MSP-1 recombinant and DNA vaccine with Plasmodium falciparum genes which codes MSA-1 and MSA-2 were able to protect against these Plasmodium species.…”