Evaluation of TcpF-A2-CTB chimera and evidence of additive protective efficacy of immunizing with TcpF and CTB in the suckling mouse model of cholera

Abstract

The secreted colonization factor, TcpF, which is produced by Vibrio cholerae 01 and 0139, has generated interest as a potential protective antigen in the development of a subunit vaccine against cholera. This study evaluated immunogenicity/protective efficacy of a TcpF holotoxin-like chimera (TcpF-A2-CTB) following intraperitoneal immunization compared to TcpF alone, a TcpF+CTB mixture, or CTB alone. Immunization with the TcpF-A2-CTB chimera elicited significantly greater amounts of anti-TcpF IgG than immunization with the other antigens (P<0.05). Protective efficacy was measured using 6-day-old pups reared from immunized dams and orogastrically challenged with a lethal dose of El Tor V. cholerae 01 Inaba strain N16961. Protection from death, and weight loss analysis at 24 and 48 hours post-infection demonstrated that immunization with TcpF alone was poorly protective. However, immunization with TcpF+CTB was highly protective and showed a trend toward greater protection than immunization with CTB alone (82% vs 64% survival). Immunization with the TcpF-A2-CTB chimera demonstrated less protection (50% survival) than immunization with the TcpF+CTB mixture. The TcpF-A2-CTB chimera used for this study contained the heterologous classical CTB variant whereas the El Tor CTB variant (expressed by the challenge strain) was used in the other immunization groups. For all immunization groups that received CTB, quantitative ELISA data demonstrated that the amounts of serum IgG directed against the homologous immunizing CTB antigen was statistically greater than the amount to the heterologous CTB antigen (P≤0.003). This finding provides a likely explanation for the poorer protection observed following immunization with the TcpF-A2-CTB chimera and the relatively high level of protection seen after immunization with homologous CTB alone. Though immunization with TcpF alone provided no protection, the additive protective effect when TcpF was combined with CTB demonstrates its possible value as a component of a multivalent subunit vaccine against Vibrio cholerae 01 and 0139.

Figure 1. Schematic representation of the dual promoter TcpF-A2-CTB expression plasmid pGAP22A2.

The IPTG inducible T7 promoter controls the mature tcpF-a2 gene product in frame and down-stream of the pelB leader sequence. The arabinose inducible pBAD promotor controls the mature ctb gene in frame with the ltIIB leader sequence.

Figure 2. Coomasie blue stained SDS-PAGE gel of purified recombinant TcpF-A2-CTB chimera and TcpF proteins.

Samples were reduced and boiled prior to loading on a 15% SDS-PAGE gel, causing the TcpF chimera to separate into the larger TcpF-A2 (∼42 kDa) fusion protein and the monomeric CTB proteins (11.5 kDa).

Figure 3. G_M1 ganglioside ELISA demonstrates functional receptor binding by the TcpF-A2-CTB chimera and CTB.

Several dilutions from stock solutions containing equimolar amounts of TcpF chimera, CTB, or TcpF were loaded onto ELISA plates coated with G_M1 ganglioside and serially diluted. Plates were probed with either rabbit anti-CTB (A) or TcpF antibody (B) followed by secondary probing with HRP-goat anti-rabbit IgG. Plates were read at OD₄₉₀ and optical densities were recorded. As controls duplicate antigen samples were assayed on empty plates (−G_M1) to demonstrate that CTB binding to immobilized G_M1 ganglioside is required to generate a signal in this assay.

Figure 4. Quantitative ELISA analysis of serum samples.

Day 21 and 42 serum samples were assayed for anti-TcpF (A) or anti-CTB (B) IgG amounts. Each data point represents an individual mouse within a group and the horizontal bars indicate the geometric mean of each group. Statistical differences between groups were analyzed using ANOVA with the Tukey-Kramer post-test analysis (*P<0.05 versus TcpF+CTB, and P<0.001 versus TcpF; ^# P<0.001 versus TcpF+CTB, and P<0.01 versus TcpF).

Figure 5. Anti-TcpF and anti-CTB amounts as percentages of total fecal IgA.

Fecal extracts from day 21 and 42 were assayed using quantitative ELISA for anti-TcpF (A) or anti-CTB (B) IgA. In order to normalize the data total IgA levels in each extract were also determined using quantitative ELISA. The data presented in the graphs represent the percentage specific IgA antibody amounts to the total IgA amounts in each sample. Each data point represents one mouse and the horizontal bars represent the geometric mean of each group.

Figure 6. Average weight loss of pups at 24 and 48 hours post-infection with V. cholerae.

Pups were weighed immediately before, and 24 and 48 hours post-infection with 15LD₅₀ of V. cholerae. Weight losses at 24 hours (A) and 48 hours (B) were compared to their initial weights at time  = 0. Error bars represent the SEM. Statistical analysis was performed using ANOVA followed by the Tukey-Kramer post-test. Symbols 7A, *statistically different from all groups (P<0.05) except TcpF+CTB (P>0.05); ^#statistically different from all groups groups (P<0.05) expect TcpF (P>0.05); ^∧statistically different from TcpF chimera and TcpF only groups (P<0.05); ^@statistically different from all groups (P<0.05) except the PBS group (P>0.05). Symbols 7B, *statistically different from all groups (P<0.05); ^#statistically different from TcpF+CTB and CTB only groups (P<0.001); ^∧statistically different from all goups (P<0.05) except the CTB only group (P>0.05).

Figure 7. Quantitative measurements of serum IgG antibodies specific for CTB_ET or CTB_cl in mice immunized with TcpF-A2-CTB_cl chimera, TcpF+CTB_ET, or CTB_ET.

Day 42 serum samples were assayed for anti-CTB IgG amounts to either CTB_ET or CTB_cl. Each data point represents the anti-CTB IgG amount for an individual mouse. Anti-CTB IgG amounts specific for CTB_ET and CTB_cl in each individual mouse are connected with a line. Horizontal lines per group represent the geometric mean titer to either CTB_ET or CTB_cl. Above each grouping are the antigens that were used for immunization and the corresponding P-value obtained for within group comparisons. Statistical differences were analyzed using a two-tailed paired t-test, and P-values less than 0.05 were considered significant.