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. 2006 Mar;74(3):1612-20.
doi: 10.1128/IAI.74.3.1612-1620.2006.

Vaccination of mice with gonococcal TbpB expressed in vivo from Venezuelan equine encephalitis viral replicon particles

Affiliations

Vaccination of mice with gonococcal TbpB expressed in vivo from Venezuelan equine encephalitis viral replicon particles

Christopher E Thomas et al. Infect Immun. 2006 Mar.

Abstract

We investigated the immunogenicity of gonococcal transferrin binding protein B (TbpB) expressed with and without a eukaryotic secretion signal from a nonpropagating Venezuelan equine encephalitis virus replicon particle (VRP) delivery system. TbpB was successfully expressed in baby hamster kidney (BHK) cells, and the presence of the eukaryotic secretion signal not only apparently increased the protein's expression but also allowed for extracellular localization and glycosylation. Mice immunized with VRPs produced significant amounts of serum antibody although less than the amounts produced by mice immunized with recombinant protein. The response of mice immunized with VRPs encoding TbpB was consistently more Th1 biased than the response of mice immunized with recombinant protein alone. Boosting with recombinant protein following immunization with TbpB VRPs resulted in higher specific-antibody levels without altering the Th1/Th2 bias. Most of the immunization groups produced significant specific antibody binding to the intact surface of the homologous Neisseria gonorrhoeae strain. Immunization with TbpB VRPs without a eukaryotic secretion signal generated no measurable specific antibodies on the genital mucosal surface, but inclusion of a eukaryotic secretion signal or boosting with recombinant protein resulted in specific immunoglobulin G (IgG) and IgA in mucosal secretions after TbpB VRP immunization. The TbpB VRP system has potential for an N. gonorrhoeae vaccine.

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Figures

FIG. 1.
FIG. 1.
Western blot showing expression of TbpB from TbpB VEE replicons electroporated into BHK cells. Lane 1 contains 20 μg of total membranes from iron-stressed FA1090 as a size control for full-length native TbpB, lane 2 contains 50 μg of crude cell lysate from BHK cells electroporated with green fluorescent protein-expressing VEE replicon (negative control), lane 3 contains 50 μg of crude cell lysate from BHK cells electroporated with TbpB VEE replicon pUNCH698 (tbpB), and lane 4 contains 50 μg of crude cell lysate from BHK cells electroporated with tPA-TbpB VRP replicon pUNCH696 (tPA-tbpB). Numbers to the right of the blot mark the relative mobilities of molecular size markers in kDa. The blot was probed with rabbit anti-rrHis-TbpB.
FIG. 2.
FIG. 2.
Western blot showing the effect of deglycosylation on TbpB expressed from BHK cells infected with TbpB VRPs. Lane 1 contains 10 μg of total membranes from iron-stressed FA1090 as a size control for full-length native TbpB. Each of the remaining lanes contains 10 μg crude BHK cell lysate treated identically, except that PNGase F was added to samples shown in lanes 3 and 5. Lysates in lanes 2 and 3 were derived from BHK cells infected with TbpB VRPs lacking the tPA signal sequence. Lysates in lanes 4 and 5 were derived from BHK cells infected with tPA-TbpB VRPs (containing the tPA signal sequence). Numbers to the right of the blot mark the relative mobilities of molecular size markers in kDa. The blot was probed with rabbit anti-His TbpB.
FIG. 3.
FIG. 3.
Western blot showing TbpB present in the supernatant of BHK cells electroporated with tPA-TbpB VEE replicon RNAs. Lane 1 contains 10 μg of total membranes from iron-stressed FA1090 as a size control for full-length native TbpB. Lanes 2 and 3 contain 10 μl of culture supernatant, while lanes 4 and 5 contain the equivalent of 600 μl of supernatant that had been immunoprecipitated. The samples in lanes 2 and 4 came from cultures electroporated with TbpB VEE replicon RNAs lacking the tPA signal sequence, pUNCH698 (tbpB). The samples in lanes 3 and 5 came from cultures electroporated with tPA-TbpB VEE replicon RNAs containing the tPA signal sequence, pUNCH696 (tPA-tbpB). Numbers to the right of the blot mark the relative mobilities of molecular size markers in kDa. The blot was probed with rabbit anti-His-TbpB.
FIG. 4.
FIG. 4.
Serum IgG response against gonococcal antigens, as measured by quantitative ELISA. Open bars indicate the average amounts of TbpB-specific IgG at 2 weeks after the third immunization (week 10), and filled bars indicate the average amounts of TbpB-specific IgG at 2 weeks after the fourth immunization (week 13). Late rrTbpB indicates the group receiving a single injection of rrTbpB at week 13. The error bars indicate the standard deviations for individual mice from each group. Specific IgG levels were significantly higher than those of the age-matched, mock-immunized group controls for all experimental groups with the exception of the late rrTbpB group. Symbols: †, samples that significantly increased after final recombinant protein boost (week 10 versus week 13); ‡, samples that significantly increased compared with their age-matched, non-tPA-containing partner (TbpB VRPs with and without tPA signal) (applies to samples from both week 10 and week 13). The significance threshold was P of <0.05 by Student's t test.
FIG. 5.
FIG. 5.
Serum IgG1 and IgG2a antibody responses to TbpB. The number at the top of each bar is the ratio of IgG1/IgG2a for each sample. In each bar, the top open portion indicates the amount of IgG1 recognizing TbpB, while the lower filled portion indicates the amount of IgG2a recognizing TbpB in the same sample. The error bars indicate the standard deviations for individual mice from each group. Symbols: *, ratios significantly different from age-matched rrTbpB samples (recombinant protein versus TbpB VRPs); †, ratios in TbpB VRP groups that were significantly different from age-matched, tPA-containing partner (TbpB VRPs with and without tPA signal). The significance threshold was P of <0.05 by Student's t test comparing individual-mouse ratios of IgG1 and IgG2a.
FIG. 6.
FIG. 6.
TbpB-specific IgG generated in mouse vaginal secretions. Vaginal wash samples were collected 2 weeks after the fourth immunization. Each bar presents the average amount of TbpB-specific IgG generated by each immunization group. Late rrTbpB indicates the group that received a single injection of rrTbpB in Ribi adjuvant at week 10. The error bars indicate the standard deviations for individual mice from each group. *, samples significantly different from age-matched, mock-immunized samples. The significance threshold was P of <0.05 by Student's t test.
FIG. 7.
FIG. 7.
TbpB-specific IgA generated in mouse vaginal secretions. Vaginal wash samples were collected 2 weeks after the fourth immunization. Each bar presents the average amount of TbpB-specific IgA generated by each immunization group. Late rrTbpB indicates the group that received a single injection of rrTbpB in Ribi adjuvant at week 10. The error bars indicate the standard deviations for individual mice from each group. Specific IgA levels were significantly higher than those of mock-immunized group controls for all experimental groups with the exception of the TbpB VRP group. Symbols: †, samples that significantly increased after final recombinant protein boost; ‡, samples that significantly increased compared with their age-matched, non-tPA-containing partner. The significance threshold was P of <0.05 by Student's t test.
FIG. 8.
FIG. 8.
Whole-cell binding activity of antibodies recognizing surface-exposed epitopes of TbpB. Antibody bound to the surface of iron-stressed FA1090 after subtraction of antibody bound to FA6916 (ΔtbpB-tbpA) is shown. Late rrTbpB indicates the group that received a single injection of rrTbpB in Ribi adjuvant at week 10. The error bars indicate the standard deviations of between three and eight determinations on between 2 and 4 days. Symbols: *, surface binding significantly different from age-matched, mock-immunized samples (immunized versus mock); †, surface binding that increased significantly after final recombinant protein boost (week 10 versus 13); ‡, TbpB VRP-immunized groups that bound significantly more antibodies than their age-matched, tPA-TbpB VRP-immunized partner. The significance threshold was P of <0.05 by Student's t test.

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