Broadly protective semi-synthetic glycoconjugate vaccine against pathogens capable of producing poly-ß-(1→6)-N-acetyl-d-glucosamine exopolysaccharide.

Poly-ß-(1→6)-N-acetylglucosamine (PNAG) was first discovered as a major component of biofilms formed by Staphylococcus aureus and some other staphylococci but later this exopolysaccharide was also found to be produced by pathogens of various nature. This common antigen is considered as a promising target for construction of a broadly protective vaccine. Extensive studies of PNAG, its de-N-acetylated derivative (dPNAG, containing around 15% of residual N-acetates) and their conjugates with Tetanus Toxoid (TT) revealed the crucial role of de-N-acetylated glucosamine units for the induction of protective immunity. Conjugates of synthetic penta- (5GlcNH2) and nona-ß-(1→6)-d-glucosamines (9GlcNH2) were tested in vitro and in different animal models and proved to be effective in passive and active protection against different microbial pathogens. Presently conjugate 5GlcNH2-TT is being produced under GMP conditions and undergoes safety and effectiveness evaluation in humans and economically important animals. Current review summarizes all stages of this long-termed study.

Brucellosis: Improved Diagnostics and Vaccine Insights from Synthetic Glycans.

Brucellosis is a serious zoonotic bacterial disease that is ranked by the World Health Organization among the top seven "neglected zoonoses" that threaten human health and cause poverty. It is a costly, highly contagious disease that affects ruminants, cattle, sheep, goats, and other productive animals such as pigs. Symptoms include abortions, infertility, decreased milk production, weight loss, and lameness. Brucellosis is also the most common bacterial disease that is transmitted from animals to humans, with approximately 500 000 new human cases each year. Detection and slaughter of infected animals is required to eradicate the disease, as vaccination alone is currently insufficient. However, as the most protective vaccines compromise serodiagnosis, this creates policy dilemmas, and these often result in the failure of eradication and control programs. Detection of antibodies to the Brucella bacterial cell wall O-polysaccharide (OPS) component of smooth lipopolysaccharide is used in diagnosis of this disease, and the same molecule contributes important protective efficacy to currently deployed veterinary whole-cell vaccines. This has set up a long-standing paradox that while Brucella OPS confers protective efficacy to vaccines, its presence results in similar antibody profiles in infected and vaccinated animals. Consequently, differentiation of infected from vaccinated animals (DIVA) is not possible, and this limits efforts to combat the disease. Recent clarification of the chemical structure of Brucella OPS as a block copolymer of two oligosaccharide sequences has provided an opportunity to utilize unique oligosaccharides only available via chemical synthesis in serodiagnostic tests for the disease. These oligosaccharides show excellent sensitivity and specificity compared with the native polymer used in current commercial tests and have the added advantage of assisting discrimination between brucellosis and infections caused by several bacteria with OPS that share some structural features with those of Brucella. During synthesis and immunochemical evaluation of these synthetic antigens, it became apparent that an opportunity existed to create a polysaccharide-protein conjugate vaccine that would not create antibodies that give false positive results in diagnostic tests for infection. This objective was reduced to practice, and immunization of mice showed that antibodies to the Brucella A antigen could be developed without reacting in a diagnostic test based on the M antigen. A conjugate vaccine of this type could readily be developed for use in humans and animals. However, as chemical methods advance and modern methods of bacterial engineering mature, it is expected that the principles elucidated by these studies could be applied to the development of an inexpensive and cost-effective vaccine to combat endemic brucellosis in animals.

Synthesis of antifungal vaccines by conjugation of ß-1,2 trimannosides with T-cell peptides and covalent anchoring of neoglycopeptide to tetanus toxoid.

Selective strategies for the construction of novel three component glycoconjugate vaccines presenting Candida albicans cell wall glycan (ß-1,2 mannoside) and polypeptide fragments on a tetanus toxoid carrier are described. The first of two conjugation strategies employed peptides bearing an N-terminal thiopropionyl residue for conjugation to a trisaccharide equipped with an acrylate linker and a C-terminal S-acetyl thioglycolyl moiety for subsequent linking of neoglycopeptide to bromoacetylated tetanus toxoid. Michael addition of acrylate trisaccharides to peptide thiol under mildly basic conditions gave a mixture of N- and C- terminal glyco-peptide thioethers. An adaptation of this strategy coordinated S-acyl protection with anticipated thioester exchange equilibria. This furnished a single chemically defined fully synthetic neoglycopeptide conjugate that could be anchored to a tetanus toxoid carrier and avoids the introduction of exogenous antigenic groups. The second strategy retained the N-terminal thiopropionyl residue but replaced the C-terminal S-acetate functionality with an azido group that allowed efficient, selective formation of neoglycopeptide thioethers and subsequent conjugation of these with propargylated tetanus toxoid, but introduced potentially antigenic triazole linkages.

Synthesis and immunological activity of an oligosaccharide-conjugate as a vaccine candidate against Group A Streptococcus.

The synthesis and immunogenicity of a tetanus toxoid (TT)-conjugate of the hexasaccharide portion of the cell-wall polysaccharide (CWPS) of the Group A Streptococcus (GAS) is described. The synthesis relies on the reaction of an allyl glycoside of the hexasaccharide with cysteamine, followed by the reaction of the resultant amine with diethyl squarate to give the monoethyl squarate adduct. Subsequent reaction with the lysine ε-amino groups on TT gives the glycoconjugate containing 30 hexasaccharide haptens per TT molecule. The immunogenicity in mice is similar to that obtained with a native CWPS-TT conjugate, validating the glycoconjugate as a vaccine candidate against GAS infections.

Ex vivo characterization and isolation of rare memory B cells with antigen tetramers.

Studying human antigen-specific memory B cells has been challenging because of low frequencies in peripheral blood, slow proliferation, and lack of antibody secretion. Therefore, most studies have relied on conversion of memory B cells into antibody-secreting cells by in vitro culture. To facilitate direct ex vivo isolation, we generated fluorescent antigen tetramers for characterization of memory B cells by using tetanus toxoid as a model antigen. Brightly labeled memory B cells were identified even 4 years after last immunization, despite low frequencies ranging from 0.01% to 0.11% of class-switched memory B cells. A direct comparison of monomeric to tetrameric antigen labeling demonstrated that a substantial fraction of the B-cell repertoire can be missed when monomeric antigens are used. The specificity of the method was confirmed by antibody reconstruction from single-cell sorted tetramer(+) B cells with single-cell RT-PCR of the B-cell receptor. All antibodies bound to tetanus antigen with high affinity, ranging from 0.23 to 2.2 nM. Furthermore, sequence analysis identified related memory B cell and plasmablast clones isolated more than a year apart. Therefore, antigen tetramers enable specific and sensitive ex vivo characterization of rare memory B cells as well as the production of fully human antibodies.

Synthesis and immunogenicity of a glycopolymer conjugate.

A protective ß-mannan trisaccharide epitope from the Candida albicans cell wall phosphomannan has been synthesized and activated for copolymerization with acrylamide. The resulting glycopolymer displayed 33 trisaccharide haptens and was derivatized for conjugation to the immunogenic carrier protein, chicken serum albumin. The resulting conjugate achieves a high degree of oligosaccharide substitution while limiting the sites of substitution on the protein. The murine immune response against this conjugate was compared with the response to a trisaccharide-tetanus toxoid conjugate vaccine. The glycopolymer was shown to induce a more robust immune response with higher trisaccharide-specific antibody titers and with a significantly larger proportion of responding mice developing antibodies that bound the target, native cell wall antigen of C. albicans.

Structural and immunogenicity analysis of chimeric B-cell epitope constructs derived from the gp46 and gp21 subunits of the envelope glycoproteins of HTLV-1.

B-cell epitopes were selected from the gp21 and gp46 subunits of the envelope glycoprotein of human T-cell lymphotropic virus type 1 (HTLV-1) by computer-aided analyses of protein antigenicity. Molecular modeling was used to design and synthesize the epitopes as chimeric constructs with promiscuous T-helper epitopes derived either from the tetanus toxoid (amino acids 947-967) or measles virus fusion protein (amino acids 288-302). Circular dichroism measurements revealed that the peptides had a secondary structure that correlated well with the crystal structure data or predicted structure. The chimeric peptides were then evaluated for their immunogenicity in rabbits or mice. Antibodies against one of the epitopes derived from the gp21 subunit were found to be neutralizing in its ability to inhibit the formation of virus-induced syncytia. These studies underscore the importance of the gp21 transmembrane region for the development of vaccine candidates. The applicability of a chimeric approach is discussed in the context of recent findings regarding the role of gp21 transmembrane region in the viral fusion process.

Serological response to immunization with tetanus toxoid.

A total of two hundred women were immunized with tetanus toxoid vaccine. Two batches of toxoid prepared at the Institute of Public Health (IPH), Dhaka and one batch of imported vaccines, were being used by the EPI in Bangladesh for immunization. Each hundred women were immunized by IPH and imported vaccine. Two human doses were given in one month interval. Blood samples from all the study subjects were collected on the day of 1st dose and one month after second dose. Both the preimmunized sera and the sera after vaccination were tested to determine the antibody titre against tetanus toxoid by the haemagglutination method. The preimmunized sera showed the presence of protective antibody in 50(25%) subjects who had the history of previous immunization. Including these initial antitoxin positive cases the seroconversions found among 95% and 96% of the study population respectively after immunization with IPH and imported toxoids, which were 93.05% and 94.87% when these 50 subjects were excluded. No significant difference (p = 1.0) was observed between the immunity of the subjects after receiving IPH and imported vaccine. Antibody titre of initial tetanus-antitoxin positive cases raised eight folds after getting more doses. The result gave fair indication of the antigenicity of all the toxoids used in the study.

Exogenously provided peptides fail to complex with intracellular class II molecules for presentation by antigen-presenting cells.

Exogenously supplied antigenic peptides can bind to and be presented by cell surface class II molecules of APCs without prior processing. However, it has been unclear whether peptide Ags exogenously supplied to APCs can also form complexes with nascent intracellular class II molecules that contribute to Ag presentation. We found that exogenously provided peptide Ags, unlike whole protein Ags, are presented as efficiently by fixed as by unfixed B lymphoblastoid APCs, suggesting that intracellular processes do not contribute to the presentation of exogenously supplied peptides by unfixed APCs. Consistent with this finding, exogenously provided peptides do not bind detectably to nascent intracellular class II molecules. We studied the basis for this failure. First, as compared with whole proteins, exogenously supplied peptides accumulate very poorly intracellularly. Second, peptides are more rapidly exocytosed. The limited ability of APCs to accumulate exogenously supplied peptides intracellularly provides a likely explanation for the failure of these peptides to associate with nascent intracellular class II molecules. Exogenously supplied peptides probably never reach the intracellular vesicles in which peptide loading of class II molecules occurs. These findings have implications for the use of peptides therapeutically to block presentation of autoantigens in autoimmune disease.

The synthesis of Streptococcus pneumoniae polysaccharide-tetanus toxoid conjugates and the effect of chain length on immunogenicity.

To study the relationship between length of pneumococcal polysaccharide and immunologic performance in rabbits we took well defined fragments of the capsular polysaccharides of S. pneumoniae types 3, 6A, 18C, 19F and 23F and pneumococcal C-polysaccharide and linked them terminally by reductive amination to tetanus toxoid. Contrary to other reports we found little variation in antibody titers with increasing length. In general the opsonophagocytic titers determined using activated HL60 cells and rabbit peritoneal cells correlated well with the antibody titers except for that of type 3, which despite the presence of high polysaccharide antibody titers gave unexpectedly low opsonophagocytic titers. The C-polysaccharide-conjugate was also immunogenic when injected in both rabbits and mice but gave low opsonophagocytic titers. It was demonstrated that opsonophagocytosis was solely dependent on the presence of phosphoryl choline-specific antibody and that the induction of these antibodies was species dependent.

Synthesis, characterization, and immunological properties in mice of conjugates composed of detoxified lipopolysaccharide of Salmonella paratyphi A bound to tetanus toxoid with emphasis on the role of O acetyls.

Salmonella paratyphi A, the second most common cause of enteric fever in Southeast Asia, is a habitant of and a pathogen for humans only. Lipopolysaccharides (LPS) are both essential virulence factors and protective antigens for systemic infections caused by groups A, B, C, and D nontyphoidal salmonellae. The O-specific polysaccharide of S. paratyphi A is composed of a trisaccharide, -->2-alpha-D)-Manp-(1-->4)-alpha-L-Rhap-(1-->3)-alpha-D-Galp -(1-->, with a branch of D-paratose from the C-3 of alpha-D-mannose, and the C-3 of beta-L-rhamnose is partially O acetylated (C. G. Hellerqvist, B. Lindberg, K. Samuelsson, and A. A. Lindberg, Acta Chem. Scand. 25:955-961, 1971). On the basis of data from our investigational vaccines for enteric bacterial pathogens, including group B salmonellae (D. C. Watson, J. B. Robbins, and S. C. Szu, Infect. Immun. 60:4679-4686, 1992), conjugates composed of the detoxified LPS of S. paratyphi A bound to tetanus toxoid (TT) were prepared by several schemes. LPS was detoxified with acetic acid or with hydrazine; the latter removed O acetyls from the O-specific polysaccharide. The detoxified polysaccharides were activated with cyanogen bromide (CNBr) or with 1-cyano-4-dimethylaminopyridinium tetratfluoroborate (CDAP) and bound to TT with or without a spacer. Solutions of 2.5 microgram of saccharide, alone or as a conjugate, were injected subcutaneously into young mice, and LPS and TT antibodies were measured by enzyme-linked immunosorbent assaying. A conjugate synthesized with higher-molecular-weight O-SP elicited the highest anti-LPS levels. Only conjugates with O acetyls elicited serum immunoglobulin G anti-LPS with bactericidal activity. There were no statistically significant differences between LPS antibody levels elicited by conjugates synthesized with or without a spacer. The conjugate with O-specific polysaccharide activated by CDAP and bound to TT without a spacer elicited the highest level of TT antibodies. Clinical evaluation (if S. paratyphi A conjugates is planned.

[Detoxification of tetanus toxin]. / Untersuchungen zur Entgiftung von Tetanustoxin.

Reported in this paper are experiments conducted to compare formaldehyde, glyoxal, malondialdehyde, succindialdehyde, glutardialdehyde, adipindialdehyde, beta-propiolacton, and pyrocarbonic acid diethylester for their toxifying properties. Tetanus toxin, 1.5 million dlm/ml or 120 binding units, was used. Glutardialdehyde proved to be the most favourable compound and was followed by formaldehyde against the background of the experimental arrangement used, that is determination of concentrations for inactivation and antigenic properties of the toxoids. Glyoxal, malondialdehyde, succindialdehyde, and beta-propiolactone followed in some distance, whereas no inactivation at all was obtained from adipindialdehyde and pyrocarbonic acid diethylester.