Construction And Immunological Effect Observation For Nucleic Acid Vaccine Of Pys48, A Transmission Blocking Candidate Antigen Of Plasmodium Yoelii

ObjectiveMalaria is a kind of infectious diseases with high concern all over the world.A recent survey from WHO has shown that,over 3.3 billion people were at risk of malaria, causing nearly 2 million deaths.Anti-malaria therapy has reduced the mortality and predominantly suppressed the parasite transmission,however,the widespread emergency of drug or insecticide resistant malaria parasites,sole-antimalaria therapy can not control the disease effectively.In order to prevent or eliminate the spread of the malaria disease,it is urgent to create effective anti-malaria vaccine.Currently the researchers not only dedicate to the infection blocking vaccine developing but focus on Transmission Blocking Vaccine(TBV) study.Transmission blocking vaccine can inhibit the transmission between the mosquito and human by blocking the development of the malaria sexual stage parasites.That could be emerging during the course of anti-malaria drug treatment or other prophylactic vaccines targeting asexual stages of the parasites,as well as preventing the spread of drug-resistant parasites.The most important is that,considering the people in the malaria endemic region,the transmission blocking vaccine is of great value.P48 is a target protein expressed on the surface of malaria sexual stage parasites. For Plasmodium falciparum,Pfs48 plays a key role in parasite fertilization and antibodies that exclusively target conformational epitopes of Pfs48 protein prevent fertilization.Furthermore,anti Pfs48 antibodies are present in human sera from endemic areas and correlate with transmission blocking activity.The high level antibody responses elicited by Pfs48 depend on the correct conformation.The immunogenicity is the important premise in evaluating vaccine availability. Nucleic acid vaccines,or DNA vaccines,offer several advantages over conventional methods of immunization in that they are easy to develop safe and well tolerated by laboratory animals,as well as in humans and are capable of stimulating the cellular and humoral immune responses.More recently,it has been shown that immunization with nucleic acid encoding malaria Pre-blood and blood stage antigens followed by primary/boosting strategy conferred complete protection in mice.Thus,Nucleic acid vaccines may offer the best transmission blocking activity with several advantages, compared with conventional methods of immunization.This study is the first time to demonstrate a construction,based on nucleic acid vaccine with genes encoding an antigen present on the sexual stages of P.yoelii,Pys48, to immunize the mice and induce biologically important antibodies that can block development of the sexual stage parasite and thus transmission of the disease.Materials and Methods1.Extraction P.yoelii17XL genomic DNA and construction of Pys48 nucleic acid vaccineGenomic DNA of P.yoelii17XL was isolated from PRBC.The entire coding sequence of Pys48 was amplified by PCR and sub-cloned into pcDNA3.1+.The constructed plasmid was analyzed by sequencing using vector-specific primers.After further culture and positive clone selection,the plasmid were double digested and analyzed with 1%agarose gel.2.Determination of the vectored DV/Pys48 nucleic acid vaccinePositive recombinant was cultured in LB liquid medium overnight.A small amount of plasmid was extracted and single or double enzyme digested by HindⅢand/or EcoRV,1%agarose gel electrophoresis analysis.3.Determination of the specific antibody level in mice immunized with Pys48 nucleic acid vaccine at different time by indirect ELISABriefly,flat-bottom,96-well microtiter plates were coated with antigen overnight at 4℃.The saturating concentration of antigen was determined to be 200ng per well. The plates were blocked for one hour at room temperature.Mouse sera were diluted in blocking buffer.One hundred microliters of diluted serum was added to antigen-coated wells in duplicate and incubated for two hours at room temperature.After extensive washing with TBST,the plates were incubated with 100μl of HRP-conjugated goat anti-mouse IgG+IgM for one hour.Bound antibodies were visualized by adding 100μl of the substrate solution.The absorbance at 492nm was read with a microplate reader.4.Recognition of anti serum by Immuno-blotting(1)Gametocyte samples were lysed and separated by electrophoresis in 5-12% gradient SDS-PAGE and then electroblotted onto a PVDF membrane.The membrane was blocked with TBS plus 5%skimmed milk and assayed against individual sera. Primary antibodies were detected with HRP-conjugated goat anti mouse Ig.(2) Air-dried parasites on Multitest slides were fixed with ice-cold acetone and then blocked with PBS containing 5%nonfat dry milk for 30 min at 37℃.The slides were incubated with diluted anti-serum for 60 min at 37℃and rinsed with PBS.After incubation with FITC-conjugated goat anti-mouse immunoglobulinG(IgG) plus IgM for 30 min at 37℃,then DAPI stained.After rinsed with PBS the sliders were mounted under a coverglass in bicarbonate buffered glycerin and observed with a fluorescence microscope.5.Observation of transmission blocking effects on Pys48 nucleic acid vaccineThe mice blood were harvested on day 3 after infection,ookinete culture fluid were added into the blood after centrifugalization.The blood were incubated with culture fluid for 24h at 24℃.The suspension was added to the slide with fluorescence and blocked for 30min.The slides were incubated with 100 u 1 of FITC-conjugated goat ant-mouse IgG for 30min.The numbers of ookinetes were counted by fluorescence microscope.Results1.The identification of target gene and digestion of positive cloneThe sequencing result was according to the Pys48 sequence on GenBank.And the target EB-band digested Dv/Pys48 by two different restriction enzymes was consisted with Pys48 gene.2.The levels of specific antibody in serum from BALB/c mice immunized with Pys48 nucleic acid vaccineThe level of specific antibody increased on the 4th week after first vaccination. After two booster immunization antibody levels remained high level,especially after the last immunization,serum antibody titer maintain at the level of 5000.3.Immuno-blotting and immunofluorescence assayWestern blot hybridization experiments showed that,Pys48 nucleic acid vaccine induced serum antibodies in mice to identify the natural gametophyte-specific antigen, immunofluorescence analysis showed that specific proteins at the surface of gametocyte.4.Transmission blocking effects of the specific serum to Pys48 on gametocytesThe specific serum to Pys48 can block the development of the P.yoelii17XL ookinetes effectively.Compared with the control group,1:3 diluted serum can block the ookinetes development more completely than 1:9 diluted serum.ConclusionSuccessfully constructed Pys48 nucleic acid vaccine,immunization BALB/c mice can induce high level specific antibody response,which significantly inhibited the sexual stage of parasite development.