Figure 1.
rBmHAT specific IgG antibodies in the sera of macaques.
Macaques were immunized three times at four weeks interval with 200 µg of rBmHAT combined with alum (AL007) adjuvant. Sera collected one month after the final immunization was analyzed for IgG titer by ELISA. Approximately, 100 ng of each recombinant protein (100 ng/100 µl/well) were coated onto the wells of an ELISA plate and bound serum IgG was detected using a HRP labeled anti-monkey IgG secondary antibodies. Each bar represents mean ±S.D of 5 animals. Significant **P<0.001 titer of antibodies observed compared to other antigens.
Table 1.
Titer of IgG antibodies.
Figure 2.
rBmHAT specific antibodies isotypes in the sera of macaques.
Levels of IgG1, IgG2, IgG3, IgA and IgE antibodies against A) rBmHSP, B) rBmALT-2, C) rBmTSP and D) rBmHAT were determined in the sera (collected one month after the final dose of vaccine) of each rhesus macaque using an indirect ELISA. IgG1 antibodies were predominantly present in the immunized animals against all the four antigens tested. Each bar represents mean ±S.D of 5 animals. Significant **P<0.001 titer of antibodies observed compared to other animals.
Figure 3.
T cells proliferative responses in rBmHAT vaccinated macaques.
PBMC from immunized macaques isolated at 4 weeks post final immunization were stimulated with rBmHAT or medium only (negative control). Flow cytometry analysis for the loss of CFSE labeling (indicating cell proliferation) was performed for cells within the live T cell (CD3+) or CD4+ or CD8+ T cell subsets. Shown are the mean ± S.I and results (% proliferating cells) of the five macaques in each group. The frequency for each sample is the value obtained following subtraction of the medium control. Significant **P<0.001 and *P<0.05 proliferation of cells observed compared to controls.
Table 2.
Increase in IFN- γ production following rBmHAT antigen stimulation.
Table 3.
Sera from macaques immunized with rBmHAT participated in the killing of B. malayi L3 in an ADCC mechanism.
Figure 4.
All 10 monkeys challenged with B. malayi L3 were screened for the appearance of Mf in the peripheral blood circulation by Knott technique and PCR analysis.
A) Results from Knott technique showed the presence of mf in the blood that was stained with methylene blue. B) Mf specific Hha I gene was amplified and analyzed in 1% agarose gel.
Table 4.
Detection of Mf in the macaques challenged with B. malayi L3.
Figure 5.
Percent of eosinophils in the peripheral blood of control and vaccinated macaques.
Percent of eosinophils were evaluated in the peripheral blood of macaque pre- and post- challenge. Following challenge there was an increase in the frequency of eosinophils in animals that were microfilaremic as determined by Knott’s technique. Each line represents the eosinophil count of individual macaque on weeks −13, −9, −5, −1 before challenge, on day of challenge (week 0) and at weeks 1, 5, 10, and 14 post challenges. The levels of eosinophils in Mf positive animals are represented by a solid line. Levels of eosinophils in Mf negative animals are represented as a dotted line. # Macaques immunized with rBmHAT. Significant at **P<0.001 high number of eosinophils.
Figure 6.
Antigen specific IFN-γ and IL-10 secreting cells in macaques.
To determine the antigen-specific IFN-γ and IL-10 secreting cells ELISPOT was performed in the PBMC collected 20 weeks post challenge from vaccinated and control macaques. 1×106 cells/ml were stimulated with 100 ng/well of B. malayi adult soluble antigen (BmA) for 48 hrs at 5% CO2 and used in the ELISPOT assays. Spot forming units were counted using a dissecting microscope. Antigen-specific cells were counted by subtracting the number of spots in the negative control wells from the wells containing antigens. Results are shown as the mean spot forming units obtained from triplicate wells. Each bar represents mean ± S.D of 5 animals. Significant **P<0.001 number of spots compared to other groups.
Table 5.
PBMC were isolated 10 weeks post challenge from each macaque and cultured at 2×105 cells per well in triplicate wells of a 96 wells plate.
Table 6.
Correlates of protection in macaques vaccinated with rBmHAT.