Prospects and problems in synthetic vaccine development: what did we learn from the cholera toxin system?
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Adjuvants, Immunologic↗
Biomedical subjects
C O Jacob
Publications and source records attributed to C O Jacob.
Molecular characterization of MHC class II antigens (beta 1 domain) in the BB diabetes-prone and -resistant rat.
The BB or BB/Worcester (BB/W) rat is widely recognized as a model for human insulin-dependent diabetes mellitus (IDDM). Of at least three genes implicated in genetic susceptibility to IDDM in this strain, one is clearly linked to the major histocompatibility complex (MHC). In an attempt to define the diabetogenic gene(s) linked to the MHC of the BB rat, cDNA clones encoding the class II MHC gene products of the BB diabetes-prone and diabetes-resistant sublines have been isolated and sequenced. For comparison, the beta 1 domain of class II genes of the Lewis rat (RT1L) were sequenced. Analysis of the sequence data reveals that the first domain of RT1.D beta and RT1.B beta chain of the BB rat are different from other rat or mouse class II sequences. However, these sequences were identical in both the BB diabetes-prone and BB diabetes-resistant sublines. The significance of these findings is discussed in relation to MHC class II sequence data in IDDM patients and in the nonobese diabetic (NOD) mouse strain.
Amino Acid Sequence↗
A molecular basis for MHC class II--associated autoimmunity.
Class II major histocompatibility (MHC) molecules have an immunoregulatory role. These cell-surface glycoproteins present fragments of protein antigens (or peptides) to thymus-derived lymphocytes (T cells). Nucleotide sequence polymorphism in the genes that encode the class II MHC products determines the specificity of the immune response and is correlated with the development of autoimmune diseases. This study identifies certain class II polymorphic amino acid residues that are strongly associated with susceptibility to insulin-dependent diabetes mellitus, rheumatoid arthritis, and pemphigus vulgaris. These findings implicate particular class II MHC isotypes in susceptibility to each disease and suggest new prophylactic and therapeutic strategies.
Amino Acid Sequence↗
Tumour necrosis factor-alpha in murine autoimmune 'lupus' nephritis.
The (NZB x NZW)F1 hybrid mouse develops a severe autoimmune disease similar to systemic lupus erythematosus in humans. Both the human and murine form of the disease show strong associations with alleles of the major histocompatibility complex (MHC) gene products. The severe form of the disease found in F1 mice is due, in part, to dominant NZW gene(s) mapping with the H-2 complex (the murine MHC). Here we present evidence that the tumour necrosis factor (TNF-alpha) gene, which is located within the H-2 complex (the murine major histocompatibility complex), could be involved in the pathogenesis of lupus nephritis in F1 mice. Thus, a restriction fragment length polymorphism in the TNF-alpha gene correlates with the reduced levels of TNF-alpha produced by NZW mice. Furthermore, replacement therapy with recombinant TNF-alpha induces a significant delay in the development of the nephritis.
Animals↗
In vivo treatment of (NZB X NZW)F1 lupus-like nephritis with monoclonal antibody to gamma interferon.
The (NZB X NZW)F1 mouse is recognized as an important animal model of the human disease systemic lupus erythematosus (SLE). Groups of NZB/W F1 mice were treated either with IFN-gamma or with PBS. The results demonstrate that IFN-treated animals have accelerated development of fatal immune complex glomerulonephritis relative to age-matched controls. On the other hand, administration of mAbs specific for IFN-gamma to such mice from 4 to 7 mo of age resulted in significant remission. Development of both proteinuria and anti-DNA antibodies were delayed and survival at 11 mo was increased from less than 20% to 95% in treated mice relative to controls (p less than or equal to 0.001). These findings may have therapeutic implications for the treatment of SLE.
Animals↗
Anti-cholera response elicited by a completely synthetic antigen with built-in adjuvanticity administered in aqueous solution.
Some synthetic peptides derived from the B subunit of cholera toxin, conjugated to tetanus toxoid have been shown by us to be efficient when administered in complete Freund's adjuvant (CFA), in induction of anticholera toxin response with neutralizing capacity. When the peptide CTP3, corresponding to residues 50-64 within the sequence of the B subunit, was attached to the hardly immunogenic A-L (multichain poly-DL-alanine) the resulting conjugate did not lead to a significant anti-peptide response even when given in CFA. On the other hand, when CTP3 was coupled to the immunogenic (T,G)-A-L, the resulting CTP3-(T,G)-A-L led, in CFA, to protective anti-cholera antibodies. Moreover, covalent attachment of the synthetic adjuvant N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) to the latter immunogen led to MDP-CTP3-(T,G)-A-L which, when administered in aqueous solution, elicited in rabbits antibodies with neutralizing capacity.
Acetylmuramyl-Alanyl-Isoglutamine↗
Synthetic peptides with antigenic specificity for bacterial toxins.
The attachment of a diphtheria toxin-specific synthetic antigenic determinant and a synthetic adjuvant to a synthetic polymeric carrier led to production of a totally synthetic macromolecule which provoked protective antibodies against diphtheria when administered in aqueous solution. When peptides related to the B subunit of cholera toxin were synthesized and attached to tetanus toxoid, antibodies produced against the conjugate reacted in some but not all cases with intact cholera toxin and (especially with peptide CTP 3, residues 50-64) neutralized toxin reactivity, as tested by permeability in rabbit skin, fluid accumulation in ligated small intestinal loops and adenylate cyclase activation. Polymerization of the peptide without any external carrier, or conjugation with the dipalmityl lysine group, had as good an effect in enhancing the immune response as its attachment to tetanus toxoid. Prior exposure to the carrier suppressed the immune response to the epitope attached to it, whereas prior exposure to the synthetic peptide had a good priming effect when the intact toxin was given; when two different peptides were attached to the same carrier, both were expressed. Antisera against peptide CTP 3 were highly cross-reactive with the heat-labile toxin of Escherichia coli and neutralized it to the same extent as cholera toxin, which is not surprising in view of the great homology between the two proteins. A synthetic oligonucleotide coding for CTP 3 has been used to express the peptide in a form suitable for immunization. It led to a priming effect against the intact cholera toxin.
Adjuvants, Immunologic↗
Priming immune response to cholera toxin induced by synthetic peptides.
A systematic study has been conducted of the priming effect in the immunization against cholera toxin (CT). We demonstrate that a priming phenomenon can be achieved by synthetic peptides of the CT B subunit, leading (after a subsequent booster with a subimmunizing dose of the intact toxin) to an efficient anti-CT neutralizing antibody response. This effect is obtained even upon a single administration of a peptide conjugate and even by peptides that as such are not able to induce CT cross-reactive antibodies whatsoever. This effect is specific and dose dependent. A macromolecular carrier as well as an adjuvant are essential for the induction of anti-toxin response. In this respect, a totally synthetic priming agent, CTP3-poly(DL-alanyl)--poly(L-lysine), was adequate for an effective priming response. The specificity of the antibodies formed after the booster was mainly towards the whole CT molecule and only a small fraction of them were specific towards the peptide used for priming. The ability of synthetic peptides to prime the immune system towards a secondary stimulus with whole organism or native protein might be of general practical value, especially in endemic areas where the population is probably constantly exposed to a low level of a particular infectious agent. This exposure, which has no influence on the unprimed immune system, could serve as a booster in the case of individuals primed with an appropriate peptide, leading to a secondary immune response.
Adjuvants, Immunologic↗
Antibodies to cholera toxin synthetic peptides of increasing size and their reactivity with related toxins.
The role of the size of synthetic peptides in their immunological cross-reactivity with the parent native protein, was investigated using the cholera toxin system. The results show that elongation of a peptide may affect the reactivity of the antibodies it elicits in either positive or negative directions. Thus, elongation by six amino acid residues of each of two peptides of cholera toxin that were incapable of inducing cross-reactive antibodies with the parent protein (CTP2a and CTP5) converted them into peptides with cross-reacting capacity (CTP2b and CTP4, respectively). On the other hand, a similar elongation of another peptide (CTP3) which as such leads to cross-reactive antibodies resulted in a peptide (CTP3a) which was less cross-reactive with a series of proteins related immunologically to the parent protein.
Amino Acid Sequence↗
Immunity to heat-labile enterotoxins of porcine and human Escherichia coli strains achieved with synthetic cholera toxin peptides.
Antiserum to a synthetic peptide, CTP3, consisting of residues 50 to 64 of the B subunit of cholera enterotoxin (CT), a sequence which is conserved in heat-labile enterotoxins from Escherichia coli strains of human (H-LT) or porcine (P-LT) origin, cross-reacted with and neutralized all three enterotoxins. It also cross-reacted with genetically engineered chimeric B subunit proteins in which H-LT amino acids had been substituted for the corresponding residues in P-LT. Antisera against another CT peptide, CTP1, consisting of residues 8 to 20, were weakly cross-reactive with H-LT but not with P-LT, whereas antisera against four other CT peptides were not cross-reactive with the heterologous enterotoxins. A single dose of CTP3-linked tetanus toxoid, by itself nonimmunogenic, primed rabbits to a vigorous immune response to subsequent administration of single, small, subimmunizing doses of any of the three enterotoxins.
Animals↗
Induction of rat secretory IgA antibodies against cholera toxin by a synthetic peptide.
There is accumulating evidence concerning the possible importance of secretory IgA antibodies in defence mechanisms against infections of the gastrointestinal tract, including cholera. Intestinal IgA antibodies are also thought to play a major role in protection against the diarrhoeogenic effects of cholera toxin. We therefore attempted to induce secretory IgA antibodies towards a reactive synthetic peptide from the cholera toxin B subunit sequence. We report that rat biliary secretory IgA antibodies against the CTP3 peptide (residues 50-64 of the B subunit) were obtained by three intra-Peyer's patch immunizations, at 2-week intervals, with CTP3 conjugated to tetanus toxoid in complete Freund's adjuvant. Purified secretory IgA fractions from bile of such immunized rats reacted with the carrier toxoid, but also with the CTP3 peptide, and with the native cholera toxin, they also partially neutralized its biological activity, as assayed by inhibition of in vitro cholera toxin-induced cAMP production in mouse thymocytes.
Animals↗
Priming immunization against cholera toxin and E. coli heat-labile toxin by a cholera toxin short peptide-beta-galactosidase hybrid synthesized in E. coli.
A synthetic oligodeoxynucleotide encoding for a small peptide was employed for the expression of this peptide in a form suitable for immunization. The encoded peptide, namely, the region 50-64 of the B subunit of cholera toxin (CTP3), had previously been identified as a relevant epitope of cholera toxin. Thus, multiple immunizations with its conjugate to a protein carrier led to an efficient neutralizing response against native cholera toxin. Immunization with the resulting fusion protein of CTP3 and beta-galactosidase, followed by a booster injection of a sub-immunizing amount (1 microgram) of cholera toxin, led to a substantial level of neutralizing antibodies against both cholera toxin and the heat-labile toxin of Escherichia coli.
Animals↗
Effect of carrier on the immunogenic capacity of synthetic cholera vaccine.
In the design of the synthetic antigens and synthetic vaccines, primary consideration should be given to the choice of the carrier. Since small peptides which are being used as the relevant antigenic determinants are likely to be poor immunogens as such, the augmentation of their immunogenic capacity by the carrier or any other means is crucial for the induction of immunity. In the present study, we explored several approaches for the enhancement of the immune response towards synthetic peptides derived from the B-subunit of cholera toxin. The results indicate that the use of tetanus toxoid as a macromolecular carrier, polymerization of the peptide without any external carrier and the conjugation of dipalmityl side chain had comparable effects in enhancing the immune response to several synthetic peptides. This effect was manifested both at the level of antibodies produced and in their capacity to neutralize the biological activity of the cholera toxin. Prior exposure to the carrier resulted in a dose-dependent suppression against the synthetic epitope attached to it.
Animals↗
Neutralization of heat-labile toxin of E. coli by antibodies to synthetic peptides derived from the B subunit of cholera toxin.
Antibodies elicited by six synthetic peptides corresponding to various fragments of B subunit of cholera toxin (CT) were evaluated for their cross-reactivity with heat-labile toxin (LT) of Escherichia coli. The antiserum directed towards the peptide CTP3 (residues 50-64) was found highly cross-reactive with the LT, in radioimmunoassay and immunoblotting. This peptide was also the most cross-reactive with intact CT. The antiserum against CTP1 (residues 8-20) was also cross-reactive with the two toxins, although to a much lower extent. Antisera to both CTP1 and CTP3, which are inhibitory towards CT, were found equally effective in neutralizing the biological activity of the E. coli LT. This was manifested by inhibition of both adenylate cyclase activity and fluid secretion into ligated ileal loops of rats. These results might indicate the potential of such synthetic peptides as the basis for a general vaccine against several types of infectious diarrhea.
Adenylyl Cyclases↗
[Acute radiation syndrome due to whole body exposure to ionizing radiation].
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Humans↗
Both cholera toxin-induced adenylate cyclase activation and cholera toxin biological activity are inhibited by antibodies against related synthetic peptides.
The immune response against six synthetic peptides corresponding to various segments of the B subunit of cholera toxin was evaluated. Conjugates in which the peptides were covalently linked to tetanus toxoid served for immunization of rabbits. As previously reported, four of these conjugates elicited antibodies cross-reactive with intact cholera toxin. We report here that antisera against two of these synthetic peptides inhibit the entire spectrum of activities of the intact cholera toxin. This is manifested both on the biochemical level (adenylate cyclase induction) and on the biological effect (intestinal fluid secretion). These results indicate that these peptides may serve as suitable candidates for preparation of a synthetic anticholera vaccine.
Adenylyl Cyclases↗
Effect of dexamethasone on the survival of whole-body irradiated mice.
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Animals↗