Enhancement of protective efficacy following intranasal immunization with vaccine plus a nontoxic LTK63 mutant delivered with nanoparticles

Abstract

Most vaccines are still given parenterally. Mucosal vaccination would offer different advantages over parenteral immunization, including blocking of the pathogens at the portal of entry. In this paper, nontoxic Escherichia coli heat-labile enterotoxin (LT) mutants and Supramolecular Biovector systems (SMBV) were evaluated in mice as mucosal adjuvants and delivery systems, respectively, for intranasal immunization with the conjugated group C meningococcal vaccine. The conjugated vaccine formulated together with the LT mutants and the SMBV induced very high titers of serum and mucosal antibodies specific for the group C meningococcal polysaccharide. This vaccination strategy also induced high titers of antibodies with bactericidal activity, which is known to correlate with efficacy. Importantly, the mucosal vaccination, but not the conventional parenteral vaccination, induced bactericidal antibodies at the mucosal level. These data strongly support the feasibility of development of intranasal vaccines with an enhanced protective efficacy against meningococci and possibly against other encapsulated bacteria.

FIG. 1.

Serum and mucosal anti-MenC antibody responses. Groups of six to eight BALB/c mice were immunized three times with the different formulations shown, containing the conjugated CRM-MenC vaccine (2.5 μg of oligosaccharide per dose) and the LTK63 mutant (1 μg per dose) with or without the SMBV (100 μg per dose). In group 7, conjugated CRM-MenC vaccine and the LTK63 mutant were in the same particles, whereas in group 6, they were in different particles. Serum samples were taken before (pre) and after (post-1, -2, and -3) each immunization and tested individually to quantitate MenC-specific IgG and IgA antibody titers. Nasal washes were taken only after the third immunization at the moment of sacrifice. Each column represents the mean of each group at each time point plus 1 standard deviation.

FIG. 2.

Serum and mucosal anti-LT antibody responses. Groups of six to eight BALB/c mice were immunized three times with the different formulations shown, containing the conjugated CRM-MenC vaccine (2.5 μg of oligosaccharide per dose) and the LTK63 mutant (1 μg per dose) with or without the SMBV (100 μg per dose). In group 7, conjugated CRM-MenC vaccine and the LTK63 mutant were in the same particles, whereas in group 6, they were in different particles. Serum samples were taken before (pre) and after (post-1, -2, and -3) each immunization, and tested individually to quantitate LT-specific IgG and IgA antibody titers. Nasal washes were taken only after the third immunization at the moment of sacrifice. Each column represents the mean of each group at each time point plus 1 standard deviation.

FIG. 3.

Bactericidal activity in serum samples and nasal washes of mice immunized three times with the different formulations shown in Table 1, containing the conjugated CRM-MenC vaccine (2.5 μg of oligosaccharide per dose) and the LTK63 mutant (1 μg per dose) with or without the SMBV (100 μg per dose). Bactericidal antibody titers from pooled serum samples taken before (pre) and after (post-1, -2, and -3) each immunization are shown. Bactericidal titers from individual serum samples did not differ from the data obtained with pooled samples (not shown). Results are expressed as the last dilution of the serum samples giving 50% bacterial killing. Bactericidal activity was also tested in nasal washes taken after the third immunization and pooled. Each pool was tested, starting from a 1:2 dilution. Considering the dilution factor inherent in the nasal wash, results are expressed as the actual numbers of surviving bacterial colonies.