Pneumococcal Surface Protein A does not affect the immune responses to a combined diphtheria tetanus and pertussis vaccine in mice.

The Pneumococcal Surface Protein A (PspA) is a promising candidate for the composition of a protein vaccine against Streptococcus pneumoniae. We have previously shown that the whole cell Bordetella pertussis vaccine (wP) is a good adjuvant to PspA, inducing protective responses against pneumococcal infection in mice. In Brazil, wP is administered to children, formulated with diphtheria and tetanus toxoids (DTPw) and aluminum hydroxide (alum) as adjuvant. A single subcutaneous dose of PspA5-DTPlow (a formulation containing PspA from clade 5 and a new generation DTPw, containing low levels of B. pertussis LPS and Alum) induced high levels of systemic anti-PspA5 antibodies in mice and conferred protection against respiratory lethal challenges with two different pneumococcal strains. Here we evaluate the mucosal immune responses against PspA5 as well as the immune responses against the DTP antigens in mice vaccinated with PspA5-DTPlow. Subcutaneous immunization of mice with PspA5-DTPlow induced high levels of anti-PspA5 IgG in the airways but no IgA. In addition, no differences in the influx of cells to the respiratory mucosa, after the challenge, were observed in vaccinated mice, when compared with control mice. The levels of circulating anti-pertussis, -tetanus and -diphtheria antibodies were equivalent in mice vaccinated with DTPlow or PspA5-DTPlow. Antibodies induced by DTPlow or PspA5-DTPlow showed similar ability to neutralize the cytotoxic effects of the diphtheria toxin on Vero cells. Furthermore, combination with PspA5 did not affect protection against B. pertussis and tetanus toxin challenges in mice. Our results support the proposal for a combined PspA-DTP vaccine.

Immunogenicity and reactogenicity of a combined fully liquid DTPw-HepB-Hib pentavalent vaccine in healthy infants: no clinically relevant impact of a birth dose of hepatitis B vaccine.

OBJECTIVES: In this open-label, non-randomized phase II study, the safety and immunogenicity of a fully liquid diphtheria-tetanus-whole cell pertussis-hepatitis B-Haemophilus influenzae type b (DTPw-HepB-Hib) combination vaccine (Quinvaxem(®)) were assessed in infants who had or had not received a birth dose of hepatitis B (HepB) vaccine. STUDY DESIGN: Two groups of infants, 'HepB at birth' (n=110) and 'no HepB at birth' (n=108), were enrolled and received a primary vaccination course using a 2-4-6 months schedule. RESULTS: Seroprotection/seroconversion rates of >95% were achieved against all antigens included in the combination vaccine for both study groups. Although significantly higher anti-hepatitis B virus (p<0.001) and anti-tetanus (p=0.031) antibody titers were achieved in group 'HepB at birth' when compared with group 'no HepB at birth', the proportion of 'no HepB at birth' subjects achieving protective titers was non-inferior to the proportion of subjects in group 'HepB at birth'. The birth dose of HepB vaccine did not seem to influence the safety pattern of the DTPw-HepB-Hib combination vaccine. CONCLUSIONS: The present study demonstrated that the fully liquid DTPw-HepB-Hib vaccine was safe and immunogenic when administered using a 2-4-6 months immunization schedule, regardless of whether or not infants had received a dose of HepB vaccine at birth.

Carrier potential properties of Bacillus thuringiensis Cry1A toxins for a diphtheria toxin epitope.

The N-terminal half or toxic fragment of Bacillus thuringiensis Cry proteins is comprised of three structural domains. In a previous paper, we showed that this region plays an important role in the immunogenicity of the B. thuringiensis Cry proteins. Due to this ability and along with their stability it is worthy of investigating whether this region has carrier potential. To approach this, an eight amino acid hydrophobic motif in alpha-helix 7 of wild-type (WT) Cry1A toxins was exchanged for a diphtheria toxin epitope (DTB). The resultant recombinant toxins were tested for their ability to induce specific anti-Cry and anti-diphtheria toxin antibodies in mice after intraperitoneal and nasal immunization. We found that recombinant Cry1A toxins retained their ability to induce serum and mucosal anti-Cry Ab as well as IgG subclasses, although with a varied magnitude. By the systemic route, the effect of the amino acid substitution in the ratio of the IgG1/IgG2a Ab, leading in some sites toward IgG1 or IgG2a is more evident. Interestingly, mice produced specific anti-DTB IgG, and IgA after intranasal immunization. Together, our results support and show the immunogenic properties of the WT Cry1A toxins as well as its carrier potential for a DTB.

Induction of neutralizing antibodies against diphtheria toxin by priming with recombinant Mycobacterium bovis BCG expressing CRM(197), a mutant diphtheria toxin.

BCG, the attenuated strain of Mycobacterium bovis, has been widely used as a vaccine against tuberculosis and is thus an important candidate as a live carrier for multiple antigens. With the aim of developing a recombinant BCG (rBCG) vaccine against diphtheria, pertussis, and tetanus (DPT), we analyzed the potential of CRM(197), a mutated nontoxic derivative of diphtheria toxin, as the recombinant antigen for a BCG-based vaccine against diphtheria. Expression of CRM(197) in rBCG was achieved using Escherichia coli-mycobacterium shuttle vectors under the control of pBlaF*, an upregulated beta-lactamase promoter from Mycobacterium fortuitum. Immunization of mice with rBCG-CRM(197) elicited an anti-diphtheria toxoid antibody response, but the sera of immunized mice were not able to neutralize diphtheria toxin (DTx) activity. On the other hand, a subimmunizing dose of the conventional diphtheria-tetanus vaccine, administered in order to mimic an infection, showed that rBCG-CRM(197) was able to prime the induction of a humoral response within shorter periods. Interestingly, the antibodies produced showed neutralizing activity only when the vaccines had been given as a mixture in combination with rBCG expressing tetanus toxin fragment C (FC), suggesting an adjuvant effect of rBCG-FC on the immune response induced by rBCG-CRM(197). Isotype analysis of the anti-diphtheria toxoid antibodies induced by the combined vaccines, but not rBCG-CRM(197) alone, showed an immunoglobulin G1-dominant profile, as did the conventional vaccine. Our results show that rBCG expressing CRM(197) can elicit a neutralizing humoral response and encourage further studies on the development of a DPT vaccine with rBCG.

Clinical acceptability and immunogenicity of a pentavalent parenteral combination vaccine containing diphtheria, tetanus, acellular pertussis, inactivated poliomyelitis and Haemophilus influenzae type b conjugate antigens in two-, four- and six-month-old Chilean infants.

BACKGROUND: In recent years additional parenteral vaccines have been recommended for routine immunization of infants in the US and elsewhere. The ability to administer multiple vaccines as a single injection without unacceptably increasing reactogenicity or decreasing immunogenicity of any component would offer many practical advantages. METHODS: A randomized, open, controlled trial was conducted to assess the tolerance profile and immunogenicity, as well as to identify potential antigenic interferences, resulting from administration of a parenteral combination vaccine for infants. The vaccine contains diphtheria and tetanus toxoids, acellular pertussis antigens (DTaP), enhanced inactivated poliovirus (eIPV) and Haemophilus influenzae type b-tetanus toxoid conjugate (PRP-T). Infants (n=711) were randomly assigned to receive 1 of 5 regimens as the primary series at 2, 4 and 6 months of age, by group: (1) DTaP plus oral polio vaccine (OPV); (2) DTaP plus eIPV (separate injections); (3) DTaP-eIPV combined as a single injection; (4) DTaP-eIPV combined, plus a separate injection of PRP-T; or (5) DTaP-eIPV combined and reconstituting PRP-T, as a single injection. At 3, 5 and 7 months Groups 1, 2 and 3 received PRP-T. At 12 months all infants received a booster dose of DTaP reconstituting PRP-T as a single injection, plus a separate injection of measles, mumps and rubella vaccine. Groups 2, 3, 4 and 5 received OPV at 7 months, and all infants received OPV at 13 months. Serum immune responses were measured to the primary series at 2 and 7 months and to the booster dose at 12 and 13 months. RESULTS: Reaction rates were similar among groups. In the primary series combining eIPV with DTaP decreased geometric mean titers (GMTs) to diphtheria, tetanus and pertussis. In addition concomitant PRP-T (either simultaneous or combined) with DTaP-eIPV lowered anti-PRP and further decreased tetanus GMTs. Nonetheless in 100% of infants protective titers were achieved against diphtheria and tetanus (>0.01 IU/ml each) and against the poliovirus types 1, 2 and 3 after eIPV (Groups 2 to 5); 99% of infants (Groups 4 and 5) had protective titers against PRP (> or = 0.15 microg/ml). After boosting with DTaP/PRP-T decreased GMTs to diphtheria and PRP antigens were observed in the groups that received DTaP and eIPV combined. Nonetheless protective titers to diphtheria, tetanus and PRP occurred consistently. In contrast concomitant PRP-T with DTaP-eIPV enhanced the pertussis GMTs. CONCLUSIONS: We conclude that combined DTaP, eIPV and PRP-T in a single injection is well-tolerated and elicits an acceptable immune response to each component.

Potency assay of diphtheria antitoxin in Vero cell microcultures.

Laboratory conditions were established for the titration of diphtheria toxin and antitoxin in Vero Cell Cultures (CCT) and a comparison was made with the intradermal test (IDT) as currently used throughout the world. Working dilutions and cut off values were established by reading both the change in color of phenol red used as pH indicator and changes in cell viability in the microscope. CCT shows high reproducibility and higher detectability than IDT in the titration of both WHO Reference Standard and high titer horse antisera. In sera of guinea pigs immunized for potency testing of diphtheria toxoid the titre was approximately 10 times lower than in the IDT. The explanation is a subject of speculation. The Vero cell titration might be adopted as such for titration of diphtheria antitoxin. In the case of the toxoid potency test it could be used if the limit for the titration is adjusted to 0.2 IU considering the equivalence obtained between the two tests, by taking into account a ratio of 1:10 between CCT and IDT.