Overproduction of SM28GST in a baculovirus expression vector and its use to evaluate the in vivo immune responses of mice vaccinated against Schistosoma mansoni with naked DNA encoding the SM28GST gene.
To evaluate the immune responses of mice vaccinated intramuscularly with naked DNA encoding a single parasite-derived gene, sufficient quantities of protein are necessary for use in the immunological assays. A plasmid carrying the cDNA encoding the entire sequence for the 28-kDa Schistosoma mansoni glutathione S-transferase (Sm28GST) was used as a source of naked DNA to vaccinate mice. Using polymerase chain reaction employing custom primers to add Eco RI and Hind III restriction sites at the 5' and 3' ends, respectively, a 651-bp fragment was amplified from the vaccine plasmid. This product was isolated, ligated into the pFastBac HTb donor plasmid containing a 6X histidine (6X-his) tag, and transposed into the baculovirus expression vector system. Following blue white selection screening, high molecular weight DNA was isolated and transfected in Sf21 insect ovary cells using a liposomal preparation. Culture medium containing infective virus particles was used to infect a series of Sf21 cultures and the cells were lysed after 3-5 days. The lysates were subjected to immobilized metal (Ni-NTA) affinity chromatography from which the 6X-his-tagged recombinant Sm28GST was eluted in 250 mM imidazole. The eluted protein was probed with a polyclonal rabbit antibody specific for the Sm28GST and subsequently recognized using a monoclonal antibody specific for the 6X-his tag following concentration of the pooled fractions. Mice were vaccinated intramuscularly with purified plasmid DNA encoding either the Sm28GST or firefly luciferase. Skin tests performed using recombinant Sm28GST were positive in only those mice vaccinated with naked DNA encoding the Sm28GST gene. In a different group of experimental mice, only sera from mice vaccinated with naked DNA encoding Sm28GST contained IgG-specific anti-Sm28GST antibodies at 14 days postvaccination, and at 42 days the levels were suggestive of an anamnestic response. These results suggest that naked DNA vaccination of mice is capable of inducing both antigen-specific cell-mediated and humoral immune responses against Sm28GST and further strengthen the case for this antigen being a vaccine candidate.
Department of Structural and Cellular Biology, College of Medicine, University of South Alabama, Mobile 36688-0002, USA.
S G
SG
Kayes
R C
RC
Shaneyfelt
C
C
Monteiro
J J
JJ
O'Brien
D000818:Animals; D000909:Antibodies, Helminth / Q000097:blood; D000947:Antigens, Helminth / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D016367:Baculoviridae / Q000235:genetics; D018509:DNA, Helminth / Q000008:administration & dosage / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D015967:Gene Expression Regulation, Viral; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D015801:Helminth Proteins; D007111:Immunity, Cellular; D007273:Injections, Intramuscular; D051379:Mice; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D012550:Schistosoma mansoni / Q000201:enzymology / Q000235:genetics / Q000276:immunology*; D012555:Schistosomiasis mansoni / Q000517:prevention & control*; D014611:Vaccination / Q000379:methods; D019444:Vaccines, DNA / Q000008:administration & dosage / Q000276:immunology*
D000909:Antibodies, Helminth; D000947:Antigens, Helminth; D018509:DNA, Helminth; D015801:Helminth Proteins; C052358:P28 antigen, Schistosoma; D011994:Recombinant Proteins; D019444:Vaccines, DNA; D005982:Glutathione Transferase