Immune Effects And Mechanisms Of Transmission Blocking Vaccines In Malaria

Malaria is a kind of infectious diseases with high concern all over the world. The prevention and therapy of such infectious diseases as Malaria, AIDS and tuberculosis have been taken as the focus of researches by WHO. However, Malaria is one of the most prevalent infectious diseases worldwide, with over three billion people at risk of infection and 500 million clinical cases reported annually.Anti-malaria therapy has reduced the death rate and suppressed the plasmodial transmission predominantly ,but because of the widespread drug or insecticide resistant to malaria parasites, sole-antimalaria threpy can't containment the disease epidemic effectivly.Now the researchers not only dedicate to the infection blocking vaccine developing but foucus on the transmission blocking vaccine(TVB) study. Transmission blocking vaccine can inhibite the transmission between the mosquito and human by blocking the development of the malaria parasites in the mosquito midgut. The forte of TVB is that the mutation is more less than that of infection blocking vaccine because it can escape the stress of the host't immune system.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.Transmission blocking proteins can be classified to two families. Those were P25 and P21/P28. Although Many study show P25 or P28 induced strong transmission-blocking antibody response in mice, the systematic study of their immune response mechainism haven't been developed. The immune effecs of the vaccines are based on the abilities to induce the specific T or B cells. To determine the immunogencity is the important premise in evluating vaccine avialibility. We used yaest-produced Pvs25 and Pvs28 recombinent proteins to immunize the mice in order to find the immune response features and determine the innune activites.Rodents have the similar biological character with that of human, so we used Pys25 recombiant proteins to immunize the mice and observed the transmission blocking effects in order to find the effector molecules and the mechainism.Material and Methods1. Determination of contents of IgG in DBA/2 mice immunized with Pvs25 and Pvs28 by ELISABriefly, flat-bottom, 96-well microtiter plates were coatedwith antigen overnight at 4℃. The saturating concentrationof antigen was determined to be 200 ng of Pvs25 or 100 ng of Pvs28 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, perature. After extensive washing with TBS-T, the plates were incubated with 100μl of HRP-conjugated goat anti-mouse IgG for one hour. Bound antibodies were visualized by adding 100μl of the substrate solution. The absorbance at 490 nm was read with a microplate reader.2. Determination of contents of IgG1 and IgG2a in DBA/2 mice immunized with Pvs25 and Pvs28 by ELISABriefly, fiat-bottom, 96-well microtiter plates were coatedwith antigen overnight at 4℃. 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. perature. After extensive washing with TBS-T, the plates were incubated with 100L of biotin-conjugated rat anti-mouse IgG1 or IgG2a for one hour. Bound antibodies were visualized by adding 100μl of the substrate solution. The absorbance at 450 nm was read with a microplate reader.3. Determination of the contents of IFN-γ,IL-4 and IL-10 in DBA/2 mice immunized with Pvs25 and Pvs28 by double antibody sandwich ELISAAll cells used were spleen cells harvested from mice on14,28,42,56,70,84,98 and 112 days after immunization. Concentration of cells were adjusted to 1×10⁷/ml and 500μl cells per well were plated on the plastic 24-well plates with RPMI 1640 medium supplemented with 10% heat-inactivated FCS in 95% air-5% CO₂ at 37℃for 48 hours. Supernatants were collected after centrifugation at room temperature, 350g. Supernatants above were tested in duplicate using ELISA kits for IFN-γ,,IL-4 and IL-10.4. Determination of the numbers of IFN-γ⁺and IL-4⁺ in DBA/2 mice immunized with Pvs25 and Pvs28 by ELISPOTAll cells used were spleen cells harvested from mice on 70, 98 days after immunization. Concentration of cells were added into PVDF-plates which were coated with anti-IFN-γ,IL-4 antibody 4℃overnight. At the same time added the Pvs25 or Pvs28 recombinent proteins. The cells were cultured with 10% heat-inactivated FCS in 95% air-5% CO₂ at 37℃for 24, incubited with the anti-IFN-γ,anti-IL-4 antibody conjucted with biotin for 1hours. Bound antibodies were visualized by adding 100L of the substrate solution. The number of IFN-γ⁺ and IL-4⁺ were counted by ELISPOT analysator.5. Determination of the contents of IgG in DBA/2 mice immunized with Pys25 by double antibody sandwich ELISABriefly, flat-bottom, 96-well microtiter plates were coatedwith antigen overnight at 4℃. The saturating concentrationof antigen was determined to be 200 ng of Pys25 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. perature. After extensive washing with TBS-T, the plates were incubated with 100L of HRP-conjugated goat anti-mouse IgG for one hour. Bound antibodies were visualized by adding 100μ1 of the substrate solution. The absorbance at 490 nm was read with a microplate reader.6. Determination of the contents ofcytokines in DBA/2 mice immunized with Pys25 by ELISAAll cells used were spleen cells harvested from mice on 0,2 weeks alter first immunization and 2 weeks after booster. Concentration of cells were adjusted to 1 x 10⁷/ml and 500μ1 cells per well were plated on the plastic 24-well plates with RPMI 1640 medium supplemented with 10% heat-inactivated FCS in 95% air-5% CO₂ at 37℃for 48 hours. Supernatants were collected after centrifugation at room temperature, 350g. Supernatants above were tested in duplicate using ELISA kits for IFN-γand IL-4.7. Determination of the numbers of IFN-γ⁺and IL-4⁺ in DBA/2 mice immunized with Pys25 by ELISPOTAll cells used were spleen cells harvested from mice on 2 weeks after booster. Concentration of cells were added into PVDF-plates which were coated with anti-IFN-γ,IL-4 antibody 4℃overnight. At the same time added the Pvs25 or Pvs28 recombinent proteins. The cells were cultured with 10% heat-inactivated FCS in 95% air-5% CO₂ at 37℃for 24, incubited with the anti-IFN-γ,anti-IL-4 antibody conjucted with biotin for lhours. Bound antibodies were visualized by adding 100μ1 of the substrate solution. The number of IFN-γ⁺and IL-4⁺ were counted by ELISPOT analysator.8. Observation of transmission blocking effects on Pvs28 recombinant proteinsThe mice blood were harvested on day 3 after infection, ookinete culture fluid were added into the blood after centrifugalization. The blood were incubited with culture fluid for 16h at 24℃. The suspension was added to the slide with fluorescence and blocked for 30min. The slides were incubated with 100μ1 of FITC-conjugated goat anti-mouse IgG for 30min. The numbers of ookinetes were counted by fluorescence microscope.Results1. The levels of IgG,IgG1 and IgG2a in serum from DBA/2 mice by Pvs25 and Pvs28The level of IgG, IgG1 and IgG2a increased on the day28 after first vaccination, after this point the level of IgG and IgG1 were persisting on a very high level until day 112 and there was no evidence to decrease; although the level of IgG2a decresed rapidly after this peak, its level from the recombinant protein group was higher than that from the control group till the day 112 after first vaccination.2. The level of cytokines in spleen cell supernatant from DBA/2 mice in different time after vaccination and the numbers of IFN-γ⁺ and IL-4⁺ cells in spleen lymphocytes from DBA/2 mice vaccinated by Pvs25 and Pvs28The level of IFN-γelevated sharply at day 14 and then dropped to the level of control group. Levels of IL-4 and IL-10 increased from day 28 after first vaccination and got further higher henceforth. Despite of its later decrease, the levels kept higher till day 112 compared with the control. Number of IL-4⁺ cells from mice spleen elevated on day 70 and day 98, while that of IFN-γ⁺ cells didn't change, compared with the control.3. The levels of IgG in serum from DBA/2 mice immunized by Pys25The level of IgG increased on the 2 weeks after first vaccination, after this point the level of IgG were persisting on a very high level until 3 weeks after booster. although the level of IgG decresed rapidly after this peak, its level from the recombinant protein group was higher on 20 weeks after booster.4. The level of cytokines in spleen cell supernatant from DBA/2 mice in different time and the numbers of IFN-γ⁺ and IL-4⁺ cells in spleen lymphocytes from DBA/2 mice vaccinated by Pys25The levels of IFN-γand IL-4 increased on 2 weeks after first vaccination and got further higher henceforth. Despite of this the level of IL-4 kept higher after booster, the level of IFN-γdecreased to that of the control.Number of IL-4⁺ cells from mice spleen elevated on 2 weeks , while that of IFN-γ⁺ cells didn't change, compared with the control.5. Transmission blocking effects of the specific serum to Pys25 on P. yoelii 17XL ookinetesThe specific serum to Pys25 can block the development of the P. yoeliil7XL ookinetes effectively on 2 weeks after booster and the transmission blocking effect is charactrized with tite-dependent, 4-fold diluted serum can block the ookinetes development compeletly.Conclusion1. The rPvs25 and rPvs28 can induce the high level IgG, and the predominant subclass is IgG1.2. The rPvs25 and rPvs28 possess the ideal immunocompetence.3. The rPvs25 and rPvs28 can induce murine immune response, predominantly the Th2 response.4. The rPvs25 and rPvs28 can induce the strong routine immune response and make mice keep the immune memory.5. The rPys25 can induce murine immune response, predominantly the Th2 response.6. The rPys25 possess the ideal immunocompetence and can induce specific serum, which can block the development of ookinetes.