United States Patent:  6,030,624

Assignee:  UAB Research Foundation (Birmingham, AL)

Filed:  August 15, 1997

The present invention provides chimeric proteins such as Salivary Binding Protein (SBR) coupled to the B subunit of cholera toxin. Such a chimeric protein, when expressed in attenuated Salmonella typhymurium produces significant increases in serum IgG and salivary IgA antibody levels after oral immunization. In another embodiment of the present invention, the recombinant plasmid contains a salivary binding protein-cholera toxin A2/B chimeric protein expressed in E. coli. Intragastric immunization of SBR coupled to CTB in this chimeric protein form leads to increased antigen responsive T cells. In another embodiment of the present invention, the recombinant plasmid contains a salivary binding protein-cholera toxin^.DELTA.A1 chimeric protein expressed in Salmonella typhimurium. Oral immunization using this recombinant plasmid results in increased serum IgG responses to antigen. Oral immunization using this recombinant plasmid also resulted in increased salivary IgA antibody responses to antigen.

SUMMARY OF THE INVENTION

The present invention demonstrates that primary oral immunization of mice with a bacterial protein antigen genetically coupled to the A2/B subunits of cholera toxin induced specific secretory immunoglobulin A and serum IgG antibodies that persisted at substantial levels for at least 11 months. A subsequent single booster immunization did not further enhance the antibody responses. Long-term antibody persistence may be especially important in infections caused by common pathogens for which continuous immunity would be advantageous.

The present invention further shows that a major adhesin from the oral pathogen Streptococcus mutans is mucosally immunogenic upon genetic fusion with the cholera toxin A2/B subunits. To take advantage of the ability of Salmonella typhimurium to deliver cloned antigens to the mucosal inductive sites that would obviate the need for antigen purification, this chimeric construct was expressed in an attenuated S. typhimurium strain under the control of bacteriophage T7 transcription. Residual expression of the temperature-regulated T7 RNA polymerase at 30^oC. allowed production of the chimeric protein at 2-3% of the total soluble protein, but it was increased 5-6 times following induction at 37^oC. Oral administration of a single dose of 10⁹ recombinant Salmonella to mice resulted in serum IgG and salivary IgA antibody responses to Salmonella, cholera toxin, and the streptococcal adhesin, which were generally enhanced after a booster immunization.

The present invention also discloses an avirulent Salmonella typhimurium vaccine strain expressing a streptococcal protein adhesin, and a similar clone which produces the same streptococcal antigen linked to the cholera toxin A2/B subunits, which were compared for their ability to induce antibody responses to the expressed heterologous antigen after oral or intranasal immunization of mice. Expression of cloned immunogens in these systems is temperature-regulated, being optimal at 37^oC., and the two clones produced similar levels of the streptococcal antigen. Both clones were found to stimulate high levels of serum IgG and mucosal IgA antibodies to the cloned immunogen. A consistent trend was observed towards higher mucosal IgA but lower serum IgG responses in the case of the S. typhimurium vector that co-expressed CTA2/B, a potential mucosal adjuvant, regardless of the route of administration. Also noteworthy was the capacity of these antigen-delivery systems to induce anamnestic mucosal and systemic responses to the cloned immunogen 15 weeks after the primary immunization, despite pre-existing immunity to the Salmonella vectors. Although the serum IgG response against the Salmonella vector was characterized by a high IgG2a/IgG1 ratio (indicative of the Th1/Th2 profile), a mixed IgG1 and IgG2a pattern was observed for the carried heterologous antigen, which displayed a dominant IgG1 response when administered as a purified immunogen. The present invention indicates that the recombinant streptococcal antigen and CTA2/B are strong immunogens when expressed by the antigen-delivery system, and that CTA2/B may have an additional immunoenhancing activity in the mucosal compartment besides its ability to target antigen uptake into the mucosal inductive sites and, therefore, may be useful as a S. typhimurium-cloned adjuvant for co-expressed protein Ags.

Claim 1 of 11 Claims

1. A method of producing an immune response by administration of an attenuated strain of bacteria, wherein said attenuated bacteria express an antigen of interest as a fusion protein from a plasmid which comprises in operable linkage:

a) an origin of replication;

b) a promoter; and,

c) DNA sequences encoding the antigen of interest, wherein said DNA sequences are fused in frame to the A2 subunit of cholera toxin.

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