Expression of a Functional Single-Chain Variable-Fragment Antibody against Complement Receptor 1 inStreptococcus gordonii

Abstract

Streptococcus gordonii, an oral commensal organism, is a candidate vector for oral-vaccine development. Previous studies have shown that recombinant S. gordonii expressing heterologous antigens was weakly immunogenic when delivered intranasally. In this study, antigen was specifically targeted to antigen-presenting cells (APC) in order to potentiate antigen-APC interactions and increase the humoral immune response to the antigen. To achieve this goal, a single-chain variable-fragment (scFv) antibody against complement receptor 1 (CR1) was constructed. Anti-CR1 scFv purified from Escherichia coli was able to bind to mouse mixed lymphocytes and bone marrow-derived dendritic cells. The in vivo function of the anti-CR1 scFv protein was assessed by immunizing mice intranasally with soluble scFv and determining the immune response against the hemagglutinin (HA) peptide located on the carboxy terminus of the scFv. The serum anti-HA immunoglobulin G (IgG) immune response was dose dependent; as little as 100 ng of anti-CR1 scFv induced a significant IgG immune response, while such a response was minimal when the animals were given an unrelated scFv. The anti-CR1 scFv was expressed in S. gordonii as a secreted protein, which was functional, as it bound to dendritic cells. Mice orally colonized by the anti-CR1-secreting S. gordonii produced an anti-HA IgG immune response, indicating that such an approach can be used to increase the immune response to antigens produced by this bacterium.Streptococcus gordonii is a commensal bacterium found in the oral cavities of humans. The organism is considered to be an attractive vector as a live-oral-vaccine vehicle (14,23). A number of heterologous antigens have been expressed in this organism as either secreted proteins (15, 27) or cell wall-anchored proteins (16,17,19,20,25,26). In the murine oralcolonization model, the recombinant S. gordonii was able to establish long-term colonization (16,20). However, there are difficulties in stimulating a strong protective immune response against recombinant antigens following oral colonization.Antigen targeting to immune cells has the potential to manipulate the immune system and elicit an immune response more efficiently. Monoclonal immunoglobulin G (IgG) antibodies have long been used as specific targeting vehicles. A number of reports have indicated success in achieving enhanced immune responses using antibodies to complement receptor 1 (CR1) and CR2 (3,8,30), Fc receptors (1, 2), and dendritic cell DEC205 receptor (5, 6). However, there are limitations in using intact IgG as a targeting vehicle; these limitations include a weak extravascular transport ability for IgG and difficulties with expressing whole IgG by bacteria. Single-chain variable-fragment (scFv) antibodies, however, offer a number of advantages, e.g., they can be readily produced by bacteria and can be easily engineered genetically as fusion proteins carrying polypeptide antigens. In the context of antigen targeting, scFvs against CR1 and -2 (21, 24), DEC205 (9), CD3 (31), a...

Mutation of the Thiol-Disulfide Oxidoreductase SdbA Activates the CiaRH Two-Component System, Leading to Bacteriocin Expression Shutdown in Streptococcus gordonii

“…Experiments were carried out using S. gordonii SecCR1 as the parent strain. S. gordonii SecCR1 is a derivative of S. gordonii DL-1 Challis that secretes a single-chain variablefragment antibody (scFv) against complement receptor 1 (CR1), which is a protein that requires disulfide bonds for stability (30). This strain was used in our previous studies of disulfide bond formation in S. gordonii (26), and the S. gordonii SecCR1 ⌬sdbA mutant has the same phenotype as the ⌬sdbA mutant of S. gordonii DL-1 Challis (see Fig.…”

“…PCR was carried out using Phusion high-fidelity DNA polymerase (New England BioLabs, Whitby, ON, Canada) to amplify 425 bp of the upstream gene sgo_1071 and 525 bp of the downstream gene sgo_1074, using the SL1178/SL1222 and SL1220/SL1221 primer pairs, respectively (see Table S1 in the supplemental material). The PCR products were digested with restriction enzymes, as indicated in Table S1 in the supplemental material, gordonii DL1 Challis, as described previously (30). Transformants were selected on BHI medium containing the appropriate antibiotics, and insertion of aphA3 and deletion of ciaRH were confirmed by PCR.…”

Functional Analysis of Paralogous Thiol-disulfide Oxidoreductases in Streptococcus gordonii

“…S. gordonii SecCR1 is a recombinant strain of S. gordonii Challis DL-1 that secretes a single-chain variable fragment antibody (scFv) against complement receptor 1 (CR1) (19). Production of recombinant SpaP-S1 was tested using S. gordonii RJM4 as the parent strain (25).…”

“…PCR products were digested with restriction enzymes as indicated in supplemental Table S1 and ligated together with T4 DNA ligase (New England Biolabs). The ligation products were amplified using the outside primers, and the resulting constructs were used to transform S. gordonii SecCR1 and S. gordonii RJM4 as described previously (19). Transformants were selected on BHI containing the appropriate antibiotics, and insertion of the ermAM cassette was confirmed by PCR.…”

“…SecCR1 secretes scFv against CR1 fused to the signal peptide of Streptococcus mutans SpaP (19). Single-chain antibodies contain two intramolecular disulfide bonds, one in each of the V H and V L domains, and disruption of these bonds prevents stable folding, resulting in aggregation and proteolysis (46).…”