RÉSUMÉ
The aim of this study was to identify the potential vaccine antigens in Corynebacterium diphtheriae strains by in silico analysis of the amino acid variation in the 67-72p surface protein that is involved in the colonization and induction of epithelial cell apoptosis in the early stages of infection. The analysis of pili structural proteins involved in bacterial adherence to host cells and related to various types of infections was also performed. A polymerase chain reaction (PCR) was carried out to amplify the genes encoding the 67-72p protein and three pili structural proteins (SpaC, SpaI, SapD) and the products obtained were sequenced. The nucleotide sequences of the particular genes were translated into amino acid sequences, which were then matched among all the tested strains using bioinformatics tools. In the last step, the affinity of the tested proteins to major histocompatibility complex (MHC) classes I and II, and linear B-cell epitopes was analyzed. The variations in the nucleotide sequence of the 67-72p protein and pili structural proteins among C. diphtheriae strains isolated from various infections were noted. A transposition of the insertion sequence within the gene encoding the SpaC pili structural proteins was also detected. In addition, the bioinformatics analyses enabled the identification of epitopes for B-cells and T-cells in the conserved regions of the proteins, thus, demonstrating that these proteins could be used as antigens in the potential vaccine development. The results identified the most conserved regions in all tested proteins that are exposed on the surface of C. diphtheriae cells.The aim of this study was to identify the potential vaccine antigens in Corynebacterium diphtheriae strains by in silico analysis of the amino acid variation in the 6772p surface protein that is involved in the colonization and induction of epithelial cell apoptosis in the early stages of infection. The analysis of pili structural proteins involved in bacterial adherence to host cells and related to various types of infections was also performed. A polymerase chain reaction (PCR) was carried out to amplify the genes encoding the 6772p protein and three pili structural proteins (SpaC, SpaI, SapD) and the products obtained were sequenced. The nucleotide sequences of the particular genes were translated into amino acid sequences, which were then matched among all the tested strains using bioinformatics tools. In the last step, the affinity of the tested proteins to major histocompatibility complex (MHC) classes I and II, and linear B-cell epitopes was analyzed. The variations in the nucleotide sequence of the 6772p protein and pili structural proteins among C. diphtheriae strains isolated from various infections were noted. A transposition of the insertion sequence within the gene encoding the SpaC pili structural proteins was also detected. In addition, the bioinformatics analyses enabled the identification of epitopes for B-cells and T-cells in the conserved regions of the proteins, thus, demonstrating that these proteins could be used as antigens in the potential vaccine development. The results identified the most conserved regions in all tested proteins that are exposed on the surface of C. diphtheriae cells.
Sujet(s)
Adhésines bactériennes/génétique , Antigènes bactériens/génétique , Corynebacterium diphtheriae/génétique , Anatoxine diphtérique/génétique , Diphtérie/prévention et contrôle , Variation génétique , Protéines membranaires/génétique , Adhésines bactériennes/immunologie , Antigènes bactériens/immunologie , Biologie informatique , Séquence conservée , Corynebacterium diphtheriae/immunologie , Anatoxine diphtérique/immunologie , Déterminants antigéniques des lymphocytes B/génétique , Déterminants antigéniques des lymphocytes B/immunologie , Déterminants antigéniques des lymphocytes T/génétique , Déterminants antigéniques des lymphocytes T/immunologie , Antigènes d'histocompatibilité de classe I/immunologie , Antigènes d'histocompatibilité de classe II/immunologie , Protéines membranaires/immunologie , Réaction de polymérisation en chaîne , Liaison aux protéines , Analyse de séquence d'ADNRÉSUMÉ
The majority of conjugate vaccines focus on inducing an antibody response to the polysaccharide antigen and the carrier protein is present primarily to induce a T-cell dependent response. In this study conjugates consisting of poly(ribosylribitolphosphate) (PRP) purified from Haemophilus influenzae Type b bound to Hepatitis B virus surface antigen (HBsAg) virus like particles were prepared with the aim of inducing an antibody response to not only the PRP but also the HBsAg. A conjugate consisting of PRP bound to HBsAg via an adipic acid dihydrazide (ADH) spacer induced strong IgG antibodies to both the PRP and HBsAg. When conjugation was performed without the ADH spacer the induction of an anti-PRP response was equivalent to that seen by conjugate with the ADH spacer, however, a negligible anti-HBsAg response was induced. For comparison, PRP was conjugated to diphtheria toxoid (DT) and Vi polysaccharide purified from Salmonella Typhi conjugated to HBsAg both using an ADH spacer. The PRPAH-DT conjugate induced strong anti-PRP and anti-DT responses, the Vi-AHHBsAg conjugate induced a good anti-HBsAg response but not as strong as that induced by the PRPAH-HBsAg conjugate. This study demonstrated that in mice it was possible to induce robust antibody responses to both polysaccharide and carrier protein provided the conjugate has certain physico-chemical properties. A PRPAH-HBsAg conjugate with the capacity to induce anti-PRP and anti-HBsAg responses could be incorporated into a multivalent pediatric vaccine and simplify formulation of such a vaccine.
Sujet(s)
Vaccins anti-Haemophilus/immunologie , Haemophilus influenzae type B/immunologie , Antigènes de surface du virus de l'hépatite B/génétique , Antigènes de surface du virus de l'hépatite B/immunologie , Vaccins anti-hépatite B/immunologie , Polyosides/immunologie , Vaccins conjugués/immunologie , Animaux , Anticorps antibactériens/immunologie , Anatoxine diphtérique/génétique , Anatoxine diphtérique/immunologie , Vaccin diphtérie-tétanos-coqueluche/administration et posologie , Vaccin diphtérie-tétanos-coqueluche/immunologie , Haemophilus influenzae type B/composition chimique , Haemophilus influenzae type B/génétique , Antigènes de surface du virus de l'hépatite B/composition chimique , Immunoglobuline G/immunologie , Souris , Polyosides/génétique , Polyosides/isolement et purification , Salmonella typhi/génétique , Salmonella typhi/immunologie , Vaccins conjugués/administration et posologie , Vaccins à pseudo-particules virales/immunologieRÉSUMÉ
BACKGROUND: Diphtheria is a highly communicable disease caused by toxin-producing strains of Corynebacterium diphtheriae. OBJECTIVES: To evaluate the efficacy of A and B subunits of diphtheria toxin (DT-A, DT-B) as potential vaccines against C. diphtheriae. A culture of C. diphtheriae (strain PW 8) was grown on Loeffler plates while Lingood medium was used for production of diphtheria toxin (DT). MATERIALS AND METHODS: DT was purified and digested to obtain pure DT-A and DT-B and detoxified to obtain diphtheria toxin. Four groups of mice were immunised with different antigens (Ag) of C. diphtheriae. RESULTS: The antibody (Ab) titres were significantly increased with immunised groups subsequent to three injections. On the other hand, Ab titres were estimated after the three immunisations and the levels of different Ab isotypes were comparatively measured. The levels of various isotypes immune responses showed variation between immunised groups where the IgG subclasses were significantly increased mainly with DPT immunised group. The IgM and IgA were significantly increased with DT-A more than others. Additionally, the evaluation of the cellular immune responses demonstrated that spleen cells from DPT and DT-A groups gave highly significant proliferative response with production of high levels of IL-2 and IFN-γ (Th1/Th2). Separation and purification of DT gene were performed using polymerase chain reaction (PCR) and sub-cloned in pGEM-T vector, for further studying of recombinant vaccine. CONCLUSION: Our results showed the possibility to prepare a potent recombinant vaccine containing whole DT gene or DT-A against C. diphtheriae or could be used in treatment of cancer as it give high levels of IL-2 and IFN-γ.
Sujet(s)
Toxine diphtérique/immunologie , Anatoxine diphtérique/immunologie , Animaux , Anticorps antibactériens/sang , Prolifération cellulaire , Corynebacterium diphtheriae , Toxine diphtérique/génétique , Anatoxine diphtérique/administration et posologie , Anatoxine diphtérique/génétique , Femelle , Immunoglobuline A/sang , Immunoglobuline G/sang , Immunoglobuline M/sang , Interféron gamma/métabolisme , Interleukine-2/métabolisme , Agranulocytes/immunologie , Souris , Souris de lignée BALB C , Sous-unités de protéines/génétique , Sous-unités de protéines/immunologie , Rate/immunologie , Vaccins synthétiques/administration et posologie , Vaccins synthétiques/génétique , Vaccins synthétiques/immunologieSujet(s)
Infections à Corynebacterium/prévention et contrôle , Corynebacterium , Antitoxine diphtérique/usage thérapeutique , Anatoxine diphtérique , Corynebacterium/effets des médicaments et des substances chimiques , Corynebacterium/génétique , Infections à Corynebacterium/thérapie , Antitoxine diphtérique/pharmacologie , Anatoxine diphtérique/génétique , Humains , Réaction de polymérisation en chaîneRÉSUMÉ
We immunized mice with four different combinations of diphtheria toxoid or tetanus toxoid with aluminum phosphate or Freund's adjuvant and studied the resulting gene expression profiles in spleen lymphocytes. Genes, which are unique for each combination or shared in several combinations, were found activated, with functions in immune response but also in other cellular processes like apoptosis or signal transduction. Using bioinformatic tools we show, that some of the genes may serve as indicators for adverse reactions, while other genes may be new immune response markers. The results also suggest that adjuvant participates in the formation of an immunological memory.
Sujet(s)
Anatoxine diphtérique/génétique , Anatoxine diphtérique/immunologie , Régulation de l'expression des gènes/immunologie , Lymphocytes/immunologie , Rate/immunologie , Anatoxine tétanique/génétique , Anatoxine tétanique/immunologie , Adjuvants immunologiques , Animaux , Séquence nucléotidique , Amorces ADN , ADN complémentaire/génétique , Souris , Modèles statistiques , ARN messager/génétique , RT-PCR , Vaccins combinés/immunologieRÉSUMÉ
Dog zona pellucida glycoprotein 2 (dZP2), excluding the N-terminal signal sequence and the C-terminal transmembrane-like domain, was cloned and expressed as a polyhistidine fusion protein in Escherichia coli to evaluate the immunocontraceptive efficacy of ZP glycoproteins. The recombinant dZP2 (rec-dZP2) revealed a 70 kDa band corresponding to the full length transcript, as well as several low molecular mass fragments in western blot analysis. In addition to rec-dZP2, E. coli expressed recombinant dog ZP glycoprotein 3 (rec-dZP3), which has also been evaluated for its efficacy to block fertility in a homologous system. Three groups of female dogs (n = 4 per group) were immunized with rec-dZP2 conjugated to diphtheria toxoid (rec-dZP2-DT), rec-dZP3 conjugated to DT (rec-dZP3-DT) and DT alone. Immunization of female dogs with rec-dZP2-DT and rec-dZP3-DT led to generation of antibodies against the respective ZP proteins as well as to DT. Subsequent to mating, the four female dogs immunized with rec-dZP2-DT all conceived, which is indicative of failure of the anti-rec-dZP2 antibodies to block fertility. In the group of dogs immunized with rec-dZP3-DT, three of four animals did not conceive when mated with males of proven fertility. The block in fertility was associated with anti-dZP3 antibody titres. Ovarian histopathology revealed that the block in fertility in the group immunized with rec-dZP3-DT is probably manifested by inhibition in the development of follicles and is due to atretic changes in the zona pellucida. These results, although preliminary, indicate that immunization with dZP3 may be a feasible proposition to control dog populations provided that adequate antibody titres are achieved.
Sujet(s)
Immunocontraception/médecine vétérinaire , Anatoxine diphtérique/génétique , Protéines d'oeuf/génétique , Vecteurs génétiques/administration et posologie , Glycoprotéines membranaires/génétique , Récepteurs de surface cellulaire , Animaux , Chiens , Escherichia coli/métabolisme , Femelle , Modèles animaux , Protéines de fusion recombinantes/administration et posologie , Glycoprotéines de la zone pellucideRÉSUMÉ
Deleting an important active-site residue of diphtheria toxin, glutamic acid-148, reduces the toxin's ADP-ribosyltransferase activity by a factor of greater than 10(4). We considered using this mutation to construct a recombinant toxoid for expression by live attenuated vaccines and explored second-site mutations that might cause reversion. Activity was partially restored by substituting glutamic acid for valine-147 or by extending the deletion by five residues toward the NH2 terminus, thereby placing glutamic acid-142 immediately adjacent to tyrosine-149. In both mutants the indicated glutamic acid may occupy a spatial locus similar to that of glutamic acid-148 in the unmutated protein. Simply deleting a crucial residue does not, therefore, provide confidence that a second-site mutation could not readily restore activity to a toxoid.
Sujet(s)
Toxine diphtérique/génétique , Séquence d'acides aminés , Sites de fixation , Toxine diphtérique/toxicité , Anatoxine diphtérique/génétique , Données de séquences moléculaires , Mutagenèse dirigée , Mutation , Poly(ADP-ribose) polymerases/génétique , Poly(ADP-ribose) polymerases/métabolisme , Conformation des protéines , Protéines recombinantes , Relation structure-activité , Vaccins synthétiques/génétiqueRÉSUMÉ
Monoclonal antibodies (Mab) were raised against CRM197, a non-toxic mutant of diphtheria toxin (DT). The ability of four Mabs to bind DT and the six functional mutants CRM197, CRM176, CRM228, CRM1001, CRM45 and CRM30 was assessed by immunoblotting and by a radioimmunoassay in which the protein antigen in solution competes with labeled CRM197 for the Mab binding site. The results show that the peptides recognized by Mab11.3, Mab53 and Mab23 are accessible in the mutant molecules in solution but not when they are part of the native DT structure, which could therefore be described for this purpose as 'closed' in contrast with an 'open' conformation of CRM197, CRM176 and CRM228. In particular, the behaviour of Mab53 indicates that the single amino acid substitutions in the A fragments of CRM197 and CRM176 also affect the conformation of their B fragments.