Differential immunogenicity of Vibrio cholerae O139 variants expressing different combinations of naturally occurring and atypical forms of the serogroup polysaccharide.

Field testing of an inactivated bivalent O1/O139 cholera vaccine suggests that Vibrio cholerae O1 is more immunogenic than V. cholerae O139. To investigate whether this might be partly attributable to the production of capsular polysaccharide (CPS) by O139 isolates, we have compared the immunogenicity of variant strains expressing different combinations of lipopolysaccharide (LPS) and CPS. These studies indicate that the core-linked LPS structure is of paramount importance for induction of antibodies to the serogroup antigen. By contrast CPS was minimally immunogenic. Significantly the presence of CPS did not modulate the immunogenicity of the underlying LPS. To examine whether differences in LPS structure might contribute to the differing immunogenicities of the O1 and O139 serogroups, an attempt was made to modify the normal O139 LPS structure by provision of one of several heterologous wzz genes. The resulting variants displayed additional, atypical surface polysaccharide, whose modal length was characteristic for the particular wzz gene. By immunoblotting this novel material showed a ladder-like banding pattern typical of LPS, but its failure to be stained by silver indicated that it was not core-associated and was therefore more like truncated CPS. Consistent with our earlier findings, studies using systemic or mucosal routes of immunization failed to demonstrate any consistent enhancement of antibody responses associated with production of these aberrant polysaccharide polymers.

Effect of pre-existing immunity for systemic and mucosal immune responses to intranasal immunization with group B Streptococcus type III capsular polysaccharide-cholera toxin B subunit conjugate.

The effects of priming with a group B Streptococcus type III capsular polysaccharide (GBS CPS III)-recombinant cholera toxin B subunit (rCTB) conjugate, purified GBS CPS III or rCTB alone on the systemic and mucosal immune responses to CPS III after intranasal (i.n.) immunization were investigated in mice. Priming with purified GBS CPS III followed by boosting with GBS CPS III-rCTB conjugate or priming with the conjugate followed by boosting with free CPS induced comparable levels of specific IgG and IgA in both serum and in lungs and vagina. However, i.n. immunization comprising both priming and boosting with conjugate was superior to priming with CPS and boosting with conjugate or the reverse, especially with regard to inducing mucosal IgA anti-CPS responses. All the immunization schemes, except priming and boosting with free CPS, induced high and similar levels of IgG1 in serum. In contrast, mice primed with free CPS III and then boosted with CPS III-rCTB conjugate by the i.n. route failed to produce significant levels of IgG2a, IgG2b and IgG3 in serum, at difference from mice primed with the conjugate and boosted with either conjugate or free CPS. Pre-immunization with rCTB either i.n. or i.p. did not suppress specific serum IgG responses induced by GBS CPS III-rCTB conjugate intranasally, but did inhibit serum and especially mucosal IgA responses. Our findings suggest that priming with CPS affects the distribution of IgG subclasses to GBS CPS and that pre-existing anti-carrier rCTB immunity can have an inhibitory effect on mucosal immune responses elicited by the conjugate vaccine given by the i.n. route.

A comparative serological study of antigenic glycolipids from Mycobacterium tuberculosis.

Two fractions of antigenic diacyl trehaloses (DAT1 and DAT2) were isolated from the type strain of Mycobacterium tuberculosis (H37Rv). Phenolic glycolipid (PGL) and polar glycolipid antigens (C1-C4) were isolated from an unusual smooth so-called 'Canetti' strain of M. tuberculosis. These lipids were analyzed by enzyme-linked immunosorbent assay (ELISA) using antisera against a range of mycobacteria and sera from 50 tuberculosis patients and 25 healthy blood donors. All the lipids gave strong reactions with homologous mycobacterial antisera, except the least polar 'Canetti' polar glycolipid (C4). The phenolic glycolipid (PGL) from the 'Canetti' strain gave only a very weak response with antisera against M. tuberculosis H37Rv. The diacyl trehaloses (DAT) from H37Rv gave only weak reactions with the antisera against the 2 Canetti strains of M. tuberculosis. The 3 most polar glycolipid antigens (C1-C3) from the Canetti strain gave strong responses with serum against M. tuberculosis H37Rv. None of the lipids was able to discriminate between patient and control sera at a level suitable for a serodiagnostic test. A combination of results from several lipids appears to be of greater value in this respect. Thus, PGL, DAT2 and C2 were the best combination, reacting with all but 4 of the patient sera and with only 1 of the control sera.

Comparative studies of antigenic glycolipids of mycobacteria related to the leprosy bacillus.

The leprosy bacillus, Mycobacterium leprae, is a member of a small group of mycobacteria comprising the species Mycobacterium bovis, Mycobacterium marinum, Mycobacterium kansasii, Mycobacterium tuberculosis, Mycobacterium ulcerans and related taxa. This relationship is based on the similarity of the characteristic lipid types in the cell envelope. Mycobacterium leprae produces a phenolic glycolipid antigen which is species specific. This communication reports a comparison of the specificity of the lipid antigens of other members of this group of mycobacteria. Mycobacterium kansasii, in accordance with previous studies, produces phenolic glycolipid and trehalose-based lipooligosaccharide antigens which do not cross react with antisera raised against other mycobacteria. The phenolic glycolipid and an uncharacterised polar glycolipid, with the properties of a lipooligosaccharide, from Mycobacterium marinum are also shown to be specific antigens. An acylated trehalose glycolipid antigen from Mycobacterium tuberculosis H37Rv reacts strongly with antisera raised against the same strain and sera from eight out of ten tuberculosis patients. The phenolic glycolipid antigen, isolated only from Mycobacterium tuberculosis "Canetti" variants, did not react with antisera raised against the type strain, Mycobacterium tuberculosis H37Rv, although it had been shown previously to react with sera from tuberculosis patients. It is apparent that there are populations of the tubercle bacillus which differ in the lipid antigens expressed on their cell surface.

Characterization of mycobacterial immunoprecipitates by selective staining of enzymes.

Immune precipitation patterns of Mycobacterium intracellulare, M. phlei and M. smegmatis were analysed by selective enzyme staining procedures in order to characterize individual mycobacterial antigens. Enzyme activity was shown in eight precipitinogens of M. intracellulare, seven of M. phlei, and six of M. smegmatis. The identification of mycobacterial precipitinogens as enzymes is important since only a few mycobacterial antigens have been functionally characterized.

Experimental studies on cholera immunization. II. Evidence for protective antitoxic immunity mediated by serum antibodies as well as local antibodies.

By use of the ileal loop technique, the resistance to challenge with cholera enterotoxin was compared between unimmunized rabbits and rabbits immunized with toxin or toxoids. It was shown that subcutaneous as well as intraintestinal immunization induced protective immunity, the toxin being a better immunogen than Formalin-induced toxoid and much better than heat-induced toxoid. The relation between protection and serum antitoxin titer was poor, e.g., protection was seen in the absence of demonstrable serum antibodies. However, intravenous administration of antitoxic antiserum conferred some protection, suggesting that local as well as serum-mediated antitoxic immunity is operating in the host defence against cholera.