Fig 1.
Two different vaccine formulations were used. In the first vaccine formulation all three recombinant proteins OmpA, DnaK and Tul4 were conjugated to a single TMV virion (TMV-monoconjugate vaccine). The second vaccine formulation contained each recombinant protein of F. tularensis conjugated individually to TMV and then mixed in equal concentrations to generate a TMV-multiconjugate vaccine.
Fig 2.
Immunization Schedules I and II.
(A) C57BL/6 mice were immunized intranasally (i.n.) either with TMV-monoconjugate (60 μg/mouse) or TMV-multiconjugate vaccine formulations (20 μg each of OmpA-TMV; DnaK-TMV and Tul4-TMV conjugates. Total 60 μg/mouse) and booster vaccinations were administered i.n. using dosages similar to those for primary immunization on days 7 and 14 of the post-primary immunization (Schedule I). (B) Alternatively, mice were administered TMV-multiconjugate vaccines with booster immunizations i.n. on day 5 and 14 and subcutaneously (s.c.) on days 7 and 14 post-primary immunization (Schedule II). The dosages used were similar to those described for TMV-multiconjugate vaccine in A. Mice inoculated with TMV (30 μg/mouse) in a manner similar to the vaccinated groups were kept as controls.
Fig 3.
Expression and Purification of Recombinant DnaK, OmpA and Tul4 Proteins of F. tularensis SchuS4.
Purification of recombinant OmpA, DnaK and Tul4 proteins of F. tularensis SchuS4 proteins was confirmed by SDS-PAGE and western blot analysis using anti-His antibodies.
Fig 4.
Conjugation of DnaK, OmpA and Tul4 Proteins of F. tularensis SchuS4 to TMV.
Purified OmpA, DnaK and Tul4 proteins were combined with purified TMV and incubated with EDC and NHS for 0, 30 min, 1, or 2 hours as described in Methods section. Two μg of TMV or recombinant proteins DnaK, OmpA, Tul4 or 4 μg of the TMV-protein mixtures were resolved on an 8–16% SDS-PAGE gel to observe conjugation products indicated by changes in the molecular masses of the starting materials. (A) Conjugation of DnaK, OmpA and Tul4 to a single TMV virion to generate TMV-monoconjugate vaccine. The progress of conjugation process was observed over a period of time: Lane M = Precision Plus Dual Color standard (BioRad) Marker; Lane 1 = TMV-protein mix, 0 min; Lane 2 = TMV-protein mix, 30 min; Lane 3 = TMV-protein mix,1 hour; Lane 4 = TMV-protein mix, 2 hours. (B, C, D) Kinetics of DnaK, OmpA and Tul4 TMV-protein conjugations over a two hour incubation period to generate TMV-protein conjugates. The individual TMV-protein conjugates were then admixed to generate TMV-multiconjugate vaccine. Lane M = Precision Plus Dual Color standard (BioRad) Marker; Lane 1 = TMV; Lane 2 = Recombinant protein; Lane 3 = TMV-protein mix, 0 hour; Lane 4 = TMV-protein mix, 1 hour; Lane 5 = TMV-protein mix, 2 hours. In all cases, 2 hour time points were used for scale-up and vaccine preparation. Solid arrows indicate TMV-protein conjugate(s), dashed arrows indicate free TMV or free proteins.
Fig 5.
Immunization of Mice with TMV-Multiconjugate Vaccine Induces Antibody Responses Capable of Recognizing both Native and Recombinant OmpA, DnaK and Tul4 Proteins.
(A) Serum collected on day 28 post-immunization from C57BL/6 mice immunized with TMV-multiconjugate vaccine (Schedule II) was pooled (n = 4) and blotted against F. tularensis LVS and SchuS4 lysates. (B) Pooled serum from C57BL/6 mice (n = 4) immunized either with TMV-multiconjugate vaccine, or 100 CFU of F. tularensis LVS were collected on day 28 post immunization and blotted against purified recombinant OmpA, DnaK and Tul4 proteins. Sera from mice inoculated with TMV alone were used as controls.
Fig 6.
Antibody Responses in Mice Immunized with TMV-Monoconjugate and TMV-Multiconjugate Vaccines using Schedule I of Immunization.
Francisella specific total IgG, IgG1, IgG2a and IgG2b levels on day 28 in serum samples of C57BL/6 mice immunized with TMV-monoconjugate and TMV-multiconjugate vaccine using Schedule I were determined using an ELISA. Serum samples obtained from naïve mice or those inoculated with TMV alone were used as controls. The data are represented as Mean ±S.D. of absorbance values measured at 450nm. Table shows comparison of antibody titers between groups of mice vaccinated with these vaccine formulations.
Fig 7.
OmpA, DnaK and Tul4 Specific Antibody Responses in Mice Immunized with TMV-Monoconjugate and TMV-Multiconjugate Vaccines using Schedule I of Immunization.
F. tularensis SchuS4 recombinant proteins OmpA, DnaK and Tul4 specific IgG antibody levels on day 28 in serum samples of C57BL/6 mice immunized with TMV-monoconjugate and TMV-multiconjugate vaccine using Schedule I were determined by ELISA. Serum samples obtained from naïve mice or those inoculated with TMV alone were used as controls. The data are represented as Mean ±S.D. of absorbance values measured at 450nm. Table shows comparison of antibody titers between groups of mice vaccinated with these vaccine formulations.
Fig 8.
Antibody Responses in Mice Immunized with TMV-Multiconjugate Vaccines using Schedule I and II of Immunization.
Francisella specific total IgG, IgG1, IgG2a and IgG2b levels on day 28 in serum samples of C57BL/6 mice immunized with TMV-multiconjugate vaccine using Schedule II were determined by ELISA. The plates were coated with F. tularensis SchuS4 or LVS lysates. Serum samples obtained from naïve mice or those inoculated with TMV alone were used as controls. The data are represented as Mean ±S.D. of absorbance values measured at 450nm. The comparisons are shown with the data obtained from mice immunized with TMV-multiconjugate vaccine using schedule I (shown in Fig 6). Table shows comparison of antibody titers between groups of mice vaccinated with Schedule I and II vaccination regimens.
Fig 9.
OmpA, DnaK and Tul4 Specific Antibody Responses in Mice Immunized with TMV-Multiconjugate Vaccines using Schedule I and II of Immunization.
F. tularensis SchuS4 recombinant proteins OmpA, DnaK and Tul4 specific IgG, antibody levels on day 28 in serum samples of C57BL/6 mice immunized with TMV-multiconjugate vaccine using Schedule II were determined by ELISA. The plates were coated with recombinant F. tularensis SchuS4 proteins. Serum samples obtained from naïve mice or those inoculated with TMV alone were used as controls. The data are represented as Mean ±S.D. of absorbance values measured at 450nm. The comparisons are shown with the data obtained from mice immunized with TMV-multiconjugate vaccine using schedule I (shown in Fig 7). Table shows comparison of antibody titers between groups of mice vaccinated with Schedule I and II vaccination regimens.
Fig 10.
Protective Efficacy of TMV-Conjugate Vaccine.
(A) C57BL/6 mice (N = 8 per group) immunized with TMV-monoconjugate vaccine; (C) with TMV-multiconjugate vaccine (schedule I) or (E) with TMV-multiconjugate vaccine (Schedule II) were challenged i.n. with 10xLD100 of F. tularensis LVS on day 28 post-immunization. Mice vaccinated with TMV alone were used as controls. Challenged mice were observed for morbidity and mortality for a period of 21 days post-challenge. The survival results are expressed as Kaplan-Meier survival curves and statistical analysis was performed using Log-rank test. (B, D and F) Body weight of the challenged mice at the indicate time points. The data are represented as Mean ± S.D.