Recombinant CTB Research Articles

Effectiveness of liposomes possessing surface-linked recombinant B subunit of cholera toxin as an oral antigen delivery system.

Liposomes appear to be a promising oral antigen delivery system for the development of vaccines against infectious diseases, although their uptake efficiency by Peyer's patches in the gut and the subsequent induction of mucosal immunoglobulin A (IgA) responses remain a major concern. Aiming at targeted delivery of liposomal immunogens, we have previously reported the conjugation via a thioether bond of the GM1 ganglioside-binding subunit of cholera toxin (CTB) to the liposomal outer surface. In the present study, we have investigated the effectiveness of liposomes containing the saliva-binding region (SBR) of Streptococcus mutans AgI/II adhesin and possessing surface-linked recombinant CTB (rCTB) in generating mucosal (salivary, vaginal, and intestinal) IgA as well as serum IgG responses to the parent molecule, AgI/II. Responses in mice given a single oral dose of the rCTB-conjugated liposomes were compared to those in mice given one of the following unconjugated liposome preparations: (i) empty liposomes, (ii) liposomes containing SBR, (iii) liposomes containing SBR and coadministered with rCTB, and (iv) liposomes containing SBR plus rCTB. Three weeks after the primary immunization, significantly higher levels of mucosal IgA and serum IgG antibodies to AgI/II were observed in the rCTB-conjugated group than in mice given the unconjugated liposome preparations, although the latter mice received a booster dose at week 9. The antibody responses in mice immunized with rCTB-conjugated liposomes persisted at high levels for at least 6 months, at which time (week 26) a recall immunization significantly augmented the responses. In general, mice given unconjugated liposome preparations required one or two booster immunizations to develop a substantial anti-AgI/II antibody response, which was more prominent in the group given coencapsulated SBR and rCTB. These data indicate that conjugation of rCTB to liposomes greatly enhances their effectiveness as an antigen delivery system. This oral immunization strategy should be applicable for the development of vaccines against oral, intestinal, or sexually transmitted diseases.

A nontoxic mutant of cholera toxin elicits Th2-type responses for enhanced mucosal immunity.

We have characterized a nontoxic mutant of cholera toxin (CT) as a mucosal adjuvant in mice. The mutant CT was made by substitution of serine with phenylalanine at position 61 of the A subunit (S61F), which resulted in loss of ADP ribosyltransferase activity and toxicity. Mice were intranasally immunized with ovalbumin, tetanus toxoid, or influenza virus either alone or together with mutant CT S61F, native CT, or recombinant CT-B. Mice immunized with these proteins plus S61F showed high serum titers of protein-specific IgG and IgA antibodies that were comparable to those induced by native CT. Further, high protein-specific IgA antibody responses were observed in nasal and vaginal washes, saliva, and fecal extracts as well as increased numbers of IgG and IgA antibody forming cells in cervical lymph nodes and lung tissues of mice intranasally immunized with these proteins and S61F or native CT, but not with recombinant CT-B or protein alone. Both S61F and native CT enhanced the induction of ovalbumin-specific CD4(+) T cells in lung and splenic tissues, and these T cells produced a Th2-type cytokine pattern of interleukin 4 (IL-4), IL-5, IL-6, and IL-10 as determined by analysis of secreted proteins and by quantitation of cytokine-specific mRNA. These results have shown that mutant CT S61F is an effective mucosal adjuvant when administrated intranasally and induces mucosal and systemic antibody responses which are mediated by CD4(+) Th2-type cells.

Intranasal immunization with liposomes induces strong mucosal immune responses in mice

BALB/c mice were immunized intranasally with either soluble ovalbumin (OVA) or OVA entrapped in liposomes. The effect of adding Sigma cholera toxin B subunit (sCT-B), which contained low amounts of cholera holotoxin (CT), or recombinant CT-B (rCT-B) which was free from CT, as mucosal adjuvants was also investigated. The mucosal [lung enzyme-linked immunospot assay (ELISPOT), lung washing] and systemic (serum antibody and spleen ELISPOT) responses of immunized mice to OVA and CT-B were determined. Results showed that soluble OVA and liposome-entrapped OVA were poor inducers of mucosal or systemic responses unless CT-B was added as adjuvant. The types of responses augmented by sCT-B and rCT-B were different. CT-B containing low levels of CT (i.e. sCT-B) boosted both mucosal and systemic IgA and IgG responses, whereas rCT-B only increased IgG responses, unless antigen was entrapped in liposomes. Although rCT-B was unable to adjuvant IgA responses against soluble OVA, it was able to induce IgA responses against itself. These data show that mucosal responses can be increased by addition of CT-B containing low levels of CT to antigen preparations given intranasally, suggesting a direct role for CT-A in isotype switching. Furthermore, the ability of CT-B to adjuvant IgA responses against added antigens and its ability to induce responses against itself appear to be separate phenomena. The results from this study should assist the rational formulation of mucosal vaccines which induce potent mucosal and systemic immune responses.

The adjuvant effect of Vibrio cholerae and Escherichia coli heat-labile enterotoxins is linked to their ADP-ribosyltransferase activity.

This study addressed the question of whether the mucosal adjuvant property of cholera toxin (CT) and the structurally closely related Escherichia coli heat-labile toxin (LT) requires the enterotoxic and adenylate cyclase/cAMP activating property of these molecules. Therefore, we investigated the cytotoxic and adjuvant abilities of the enterotoxins and compared the results with those obtained with the non-toxic CT and LT derivatives; recombinant CTB (rCTB) and a mutated LT (mLT), which had a single amino acid substitution in position 112 (Glu----Lys) of the A subunit. Detailed functional studies revealed that, in contrast to the enterotoxins, both rCTB and mLT lacked ADP-ribosylating and cAMP-stimulating abilities. However, similar membrane ganglioside GM1-receptor binding ability of all the putative adjuvants was demonstrated. When the probe antigen, keyhole limpet hemocyanin (KLH), was given perorally together with CT or LT strong gut mucosal anti-KLH immune responses were stimulated, whereas no or very low anti-KLH responses were seen in the groups which received antigen admixed with rCTB or the mLT. Moreover, the specific serum antibody responses to the various immunization protocols closely paralleled the local anti-KLH response in the gut. From these results it appears that the adjuvant mechanism of LT, and probably also of CT, is linked to the ability to ADP-ribosylate and to stimulate cAMP formation. However, this study does not unequivocally rule out other possibilities such as interactions by the A1 fragment of CT or LT with other G-proteins than Gs alpha or events that parallel or precede the effects on the adenylate cyclase/cAMP system. Thus, the levels of ADP-ribosylation and cAMP-induction that are required and the key event or target cell that is responsible for the adjuvant effect of CT and LT remain to be elucidated. Studies are underway to address these issues.

Delivery of the cholera toxin B subunit by using a recombinant Yersinia enterocolitica strain as a live oral carrier

The gene ctxB encoding the cholera toxin B subunit was subcloned to design its production by Yersinia enterocolitica. It was joined in two ways to yopH, a gene of the virulence plasmid pYV specific to this genus. This gene encodes one of the major Yop proteins (YopH) secreted by bacteria incubated at 37°C in a Ca2+-deprived medium. In a first construction, an operon fusion was obtained between ctxB and yopH so that CT-B and a truncated YopH protein were produced. The recombinant CT-B from Y. enterocolitica was structurally and antigenically similar to CT-B produced by Vibrio cholerae. In another construction, the fusion gene obtained directed the production of YopH′/CT-B hybrid proteins that were secreted by Y. enterocolitica. In both cases, Y. enterocolitica directed the production of the recombinant proteins only when the bacteria were incubated in conditions of Yops production. When bacteria carrying the operon fusion were given orally to mice, a clear serum antibody response against CT-B was detected by ELISA. According to immunoblot analysis, this response was only directed against the polymeric form of the B subunit.