Efficacy of combined porcine reproductive and respiratory syndrome virus and Mycoplasma hyopneumoniae vaccination in piglets.

Three vaccination challenge studies were performed to evaluate the impact on vaccine efficacy of combining porcine reproductive and respiratory syndrome virus (PRRSV) and Mycoplasma hyopneumoniae vaccines. Piglets were vaccinated with either a M hyopneumoniae bacterin, a modified live PRRSV vaccine based on a European-type PRRSV strain, or a combination of both vaccines, followed by experimental infection with either M hyopneumoniae or PRRSV. Vaccine efficacy was evaluated by assessing lung lesion scores for M hyopneumoniae and measuring viraemia for PRRSV. There were no significant differences between the protective efficacy of the combined vaccine protocol and the protective efficacy of the two single vaccines, indicating that PRRSV vaccination did not interfere with M hyopneumoniae vaccine efficacy and vice versa.

Interaction of pertussis toxin with human T lymphocytes.

The binding of pertussis toxin (PT) to the human T-cell line Jurkat was examined by using flow cytometry. Fluorescein isothiocyanate (FITC)-labeled PT bound rapidly to the cells in a specific manner as determined by blocking experiments with unlabeled toxin, B oligomer, and the S2-S4 and S3-S4 dimers. Monoclonal antibodies against the S3 subunit of the toxin also significantly inhibited the binding of FITC-PT. Sialidase treatment of the cells resulted in decreased binding of FITC-PT, indicating that sialic acid residues are involved in the binding process. In addition, we studied the effect of PT binding on the expression of cell surface molecules. On binding of PT to the cell surface, a rapid down-regulation of the T-cell receptor (TCR)-CD3 complex was observed. The modulation of the TCR-CD3 complex was independent of the toxin's enzymatic activity, as the B oligomer and a nonenzymatic toxin mutant induced modulation comparable to that caused by the native holotoxin. Isolated dimers did not cause down-regulation. Stimulation of the TCR-CD3 complex, leading to reduced cell surface expression of this complex, provides a possible explanation for the second messenger production associated with the interaction of PT or B oligomer with T lymphocytes. We therefore conclude that PT activates T cells by divalent binding to the TCR-CD3 complex itself or by binding a structure closely associated with it.

Mechanism of pertussis toxin B oligomer-mediated protection against Bordetella pertussis respiratory infection.

Immunization with the B oligomer of pertussis toxin protected neonatal mice from a lethal respiratory challenge with Bordetella pertussis. All mice immunized with 8 micrograms of B oligomer survived aerosol challenge and had peripheral leukocyte counts and weight gains similar to those of mice immunized with pertussis toxoid before challenge and to those of control mice that were not challenged. Unprotected mice challenged with an aerosol of B. pertussis had an increase in peripheral leukocyte count, failed to gain weight, and died within 21 days of challenge. Protection appeared to be dose dependent, since a dose of 1 microgram of B oligomer per mouse prevented death in 100% of the mice challenged with B. pertussis, whereas 0.4 micrograms of B oligomer protected 50% of the challenged mice. Mice immunized with the B oligomer had increases in immunoglobulin G (IgG) anti-B oligomer in sera and in IgG and IgA anti-B oligomer in bronchoalveolar lavage fluids 1 to 3 weeks after respiratory challenge. Specific anti-B oligomer antibodies could not be detected in unimmunized, infected mice at the same time after challenge. Intravenous administration of the monoclonal antibody 170C4, which binds to the S3 subunit of the B oligomer, protected neonatal mice from B. pertussis respiratory challenge, while administration of an IgG1 anti-tetanus toxin monoclonal antibody, 18.1.7, was not protective. We conclude that anti-B-oligomer-mediated neutralization of pertussis toxin is one mechanism of protection in the mouse model of B. pertussis aerosol challenge.

Binding of pertussis toxin to eucaryotic cells and glycoproteins.

The binding of pertussis toxin and its subunits to cell surface receptors and purified glycoproteins was examined. The interaction of pertussis toxin with components of two variant Chinese hamster ovary (CHO) cell lines was studied. These cell lines are deficient in either sialic acid residues (LEC 2) or sialic acid and galactose residues (LEC 8) on cell surface macromolecules. The binding of pertussis toxin to components of these cells differed from the binding of the toxin to wild-type components. Although the toxin bound to a 165,000-dalton glycoprotein found in N-octylglucoside extracts of wild-type cells, it did not bind to components found in extracts of LEC 2 cells. In contrast, the toxin bound to components found in extracts of LEC 8 cells, which are variant cells that contain increased amounts of terminal N-acetylglucosamine residues on cell surface macromolecules. These results suggest that the receptor for pertussis toxin on CHO cells contains terminal acetamido-containing sugars. The cytopathic effect of the toxin on both types of variant cells was much reduced compared with its effects on wild-type cells. Thus, optimal functional binding of pertussis toxin appears to require a complete sialyllactosamine (NeuAc----Gal beta 4GlcNAc) sequence on surface macromolecules. In addition to studying the nature of the eucaryotic receptor for pertussis toxin, we examined corresponding binding sites for glycoproteins on the toxin molecule. Binding of both S2-S4 and S3-S4 dimers of the toxin to cellular components and purified glycoproteins was observed. The two dimers bound to a number of glycoproteins containing N-linked oligosaccharides but not O-linked oligosaccharides, and differences in the binding of the two dimers to some glycoproteins was noted. These data indicate that the holotoxin molecule contains at least two glycoprotein-binding sites which may have slightly different specificities for glycoproteins.

Biochemical properties of pertussis toxin.

Pertussis toxin is an exotoxin produced by the organism Bordetella pertussis. The toxin binds to receptors on the eukaryotic cell surface. After introduction into the eukaryotic cell, the toxin is activated by ATP and subsequently ADP-ribosylates a family of GTP-binding regulatory proteins interrupting signal transduction within the cell. We have examined the location of several critical sites on the toxin molecule. These sites include the receptor binding site and the ATP binding site. The B oligomer of the toxin was found to contain at least two sites capable of binding glycoproteins suggesting that the B oligomer may have more than one eukaryotic cell receptor binding site. ATP was also shown to bind to a site on the B oligomer. These results indicate that the B oligomer contains several sites necessary for toxin action.

O-polysaccharide-protein conjugates induce high levels of specific antibodies to Pseudomonas aeruginosa immunotype 3 lipopolysaccharide.

A semi-synthetic vaccine against Pseudomonas aeruginosa immunotype 3 was prepared by chemical coupling of P. aeruginosa immunotype 3 O-polysaccharide to tetanus toxoid. The O-polysaccharide was obtained by mild acid hydrolysis of immunotype 3 lipopolysaccharide, and purified by gel permeation chromatography. The purification was evaluated by high performance liquid chromatography. Additional analyses revealed a high grade of purity of the O-polysaccharide, and an at least 1000-fold reduction of endotoxic activity as compared to homologous lipopolysaccharide. O-Polysaccharide was conjugated to tetanus toxoid, using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide as coupling reagent. Antigenic determinants of both O-polysaccharide and tetanus toxoid were retained after conjugation, as tested in a sandwich enzyme-linked immunosorbent assay. Immunization of mice revealed that O-polysaccharide was nonimmunogenic in mice, while the O-specific part of the conjugate was able to induce high levels of IgG antibodies reacting with immunotype 3 lipopolysaccharide in an enzyme-linked immunosorbent assay. By immunoblotting it was shown that the antibodies were directed to high molecular weight lipopolysaccharide only, demonstrating specificity for its O-polysaccharide moiety.

Comparison of meningococcal outer membrane protein vaccines solubilized with detergent or C polysaccharide.

Outer membrane proteins (OMPs) were isolated from meningococcal strain H44/76 (B:15:P1.16) by detergent extraction of bacteria. A final product containing class 1 (P1.16), 3(15), 4 OMPs and 5% (w/w) lipooligosaccharide was obtained. Two experimental vaccines were prepared: OMP-detergent and OMP-C polysaccharide. The OMP-detergent vaccine tended to show a better bactericidal: ELISA ratio for the antibodies induced as compared to the OMP-C polysaccharide vaccine. The vaccine induced bactericidal antibodies appeared for the greater part to be directed against the class 1 OMP (P1.16). By comparison of cultures grown in Mueller Hinton Broth with and without 0.25% (w/v) glucose, it was found that monoclonal antibodies against the serotype OMP (class 2 or 3) were not bactericidal against meningococci grown in MHB without glucose. Antibodies against class 1 OMP and lipooligosaccharide were not influenced by this. A new major outer membrane protein (appr. 40 kd) is described that may function as a cation-specific porin.

Class 1/3 outer membrane protein vaccine against group B, type 15, subtype 16 meningococci.

Neisseria meningitidis capsular polysaccharides and outer membrane proteins have been incorporated in vaccines and the potential of these vaccines has been evaluated in man. Polysaccharides are the most attractive candidates for a vaccine against group A and C meningococci whereas outer membrane proteins may have a potential for a vaccine against group B meningococci. This paper describes the characteristics of the five classes of outer membrane proteins of group B meningococci and the protective (bactericidal) activity of monoclonal antibodies against class 1 and 2 or 3 outer membrane proteins. Monoclonal antibodies against class 1 outer membrane proteins were bactericidal irrespective of the growth conditions of the bacterium. On the other hand, these conditions influenced the bactericidal activity of monoclonal antibodies against class 2 or 3 outer membrane proteins. These data indicate that class 1 outer membrane protein is an attractive component of a vaccine. The Blake and Gotschlich procedure for the isolation of gonococcal outer membrane protein II (1) was adapted for the isolation of a combination of class 1 and 3 outer membrane proteins from group B, type 15 meningococci. The combination of both outer membrane proteins was adsorbed to ALPO4 in the presence of the detergent Zwittergent 3-14. The vaccine was injected into mice. The antibodies were strongly bactericidal and Western blot analysis indicated that both outer membrane proteins induced antibodies. The vaccine may have a potential to combat an epidemic caused by group B, type 15 meningococci. Such an epidemic was observed in some N.W. - European countries.