A genetically inactivated two-component acellular pertussis vaccine, alone or combined with tetanus and reduced-dose diphtheria vaccines, in adolescents: a phase 2/3, randomised controlled non-inferiority trial.

BACKGROUND: Increasing evidence shows that protection induced by acellular pertussis vaccines is short-lived, requiring repeated booster vaccination to control pertussis disease. We aimed to assess the safety and immunogenicity of a recombinant acellular pertussis vaccine containing genetically inactivated pertussis toxin and filamentous haemagglutinin, as either a monovalent vaccine (aP[PTgen/FHA]) or in combination with tetanus and reduced-dose diphtheria vaccines (TdaP[PTgen/FHA]), versus a licensed tetanus and reduced-dose diphtheria and acellular pertussis combination vaccine (Tdap). METHODS: We did this phase 2/3, randomised controlled non-inferiority trial at two sites in Bangkok, Thailand. Healthy adolescents (aged 12-17 years) were randomly assigned (1:1:1), via a computer-generated randomisation list with block sizes of three, to receive one dose (0·5 mL) of aP(PTgen/FHA), TdaP(PTgen/FHA), or Tdap (comparator). Clinical research staff responsible for participant randomisation, vaccine preparation and administration, and accountability were aware of group allocation. However, allocation was concealed from all other site study staff, data management personnel, statisticians, laboratory staff, and study participants. The primary outcome was non-inferior immunogenicity of TdaP(PTgen/FHA) to Tdap based on seroconversion rates (a four-fold increase or more) for pertussis toxin and filamentous haemagglutinin IgG antibodies 28 days after vaccination, with a predefined 10% margin of equivalence. We did analysis by per protocol. This study is registered with the Thai Clinical Trial Registry, number TCTR20150703002. FINDINGS: Between July 6 and Aug 20, 2015, we allocated 450 participants to receive one dose of TdaP(PTgen/FHA) (n=150), aP(PTgen/FHA) (n=150), or comparator Tdap (n=150). 28 days after vaccination, seroconversion rates for anti-pertussis toxin IgG were 96·6% (95% CI 93·8-99·5; n=144) in the TdaP(PTgen/FHA) group and 55·0% (47·1-63·0; n=82) in the comparator Tdap group (difference 41·6%, 95% CI 33·1-50·1; p<0·0001). Seroconversion rates for anti-filamentous haemagglutinin were 82·6% (95% CI 76·5-88·6; n=123) in the TdaP(PTgen/FHA) group and 54·4% (46·4-62·4; n=81) in the comparator group (difference 28·2%, 95% CI 18·1-38·2 p<0·0001). 28 days after vaccination, seroconversion rates in the aP(PTgen/FHA) group were 96·0% (95% CI 92·8-99·1; n=142) for anti-pertussis toxin IgG and 93·2% (89·2-97·3; n=138) for anti-filamentous haemagglutinin IgG. These findings support the non-inferior immunogenicity of TdaP(PTgen/FHA) over comparator Tdap. Reactogenicity and incidence of adverse events were similar between groups. INTERPRETATION: The new TdaP(PTgen/FHA) vaccine is safe and induces higher pertussis responses 28 days after vaccination than does the available licensed Tdap booster vaccine. Results of our trial led to the licensure of new acellular pertussis vaccines containing genetically inactivated pertussis toxin in Thailand. The availability of recombinant monovalent pertussis vaccines that induce high antibody responses provides the medical community and consumers with the opportunity to vaccinate against pertussis when immunisation against diphtheria and tetanus is not required or not desired. Studies are underway to pave the way for licensure studies of this acellular pertussis vaccine in other countries. FUNDING: BioNet-Asia.

Pertussis Antibody Transfer to Preterm Neonates After Second- Versus Third-Trimester Maternal Immunization.

Preterm infants are most vulnerable to pertussis. Whether they might benefit from maternal immunization is unknown. Extending our previous results in term neonates, this observational study demonstrates that second- rather than third-trimester maternal vaccination results in higher birth anti-pertussis toxin titers in preterm neonates.

Molecular Cloning, Structural Modeling and the Production of Soluble Triple-Mutated Diphtheria Toxoid (K51E/G52E/E148K) Co-expressed with Molecular Chaperones in Recombinant Escherichia coli.

CRM197 is a diphtheria toxin (DT) mutant (G52E) which has been used as a carrier protein for conjugate vaccines. However, it still possesses cytotoxicity toward mammalian cells. The goal of this project was to produce a non-toxic and soluble CRM197EK through introduction of triple amino acid substitutions (K51E/G52E/E148K) in Escherichia coli. The expression of CRM197EKTrxHis was optimized and co-expressed with different molecular chaperones. The soluble CRM197EKTrxHis was produced at a high concentration (97.33 ± 17.47 µg/ml) under the optimal condition (induction with 0.1 mM IPTG at 20 °C for 24 h). Cells containing pG-Tf2, expressing trigger factor and GroEL-GroES, accumulated the highest amount of soluble CRM197EKTrxHis at 111.24 ± 10.40 µg/ml after induction for 24 h at 20 °C. The soluble CRM197EKTrxHis still possesses nuclease activity and completely digest λDNA at 25 and 37 °C with 8- and 4-h incubation, respectively. Molecular modeling of diphtheria toxin, CRM197 and CRM197EK indicated that substitutions of two amino acids (K51E/E148K) may cause poor NAD binding, consistent with the lack of toxicity. Therefore, CRM197EK might be used as a new potential carrier protein. However, further in vivo study is required to confirm its roles as functional carrier protein in conjugate vaccines.

Safety and immunogenicity of a combined Tetanus, Diphtheria, recombinant acellular Pertussis vaccine (TdaP) in healthy Thai adults.

BACKGROUND: An acellular Pertussis (aP) vaccine containing recombinant genetically detoxified Pertussis Toxin (PTgen), Filamentous Hemagglutinin (FHA) and Pertactin (PRN) has been developed by BioNet-Asia (BioNet). We present here the results of the first clinical study of this recombinant aP vaccine formulated alone or in combination with tetanus and diphtheria toxoids (TdaP). METHODS: A phase I/II, observer-blind, randomized controlled trial was conducted at Mahidol University in Bangkok, Thailand in healthy adult volunteers aged 18-35 y. The eligible volunteers were randomized to receive one dose of either BioNet's aP or Tetanus toxoid-reduced Diphtheria toxoid-acellular Pertussis (TdaP) vaccine, or the Tdap Adacel® vaccine in a 1:1:1 ratio. Safety follow-up was performed for one month. Immunogenicity was assessed at baseline, at 7 and 28 d after vaccination. Anti-PT, anti-FHA, anti-PRN, anti-tetanus and anti-diphtheria IgG antibodies were assessed by ELISA. Anti-PT neutralizing antibodies were assessed also by CHO cell assay. RESULTS: A total of 60 subjects (20 per each vaccine group) were enrolled and included in the safety analysis. Safety laboratory parameters, incidence of local and systemic post-immunization reactions during 7 d after vaccination and incidence of adverse events during one month after vaccination were similar in the 3 vaccine groups. One month after vaccination, seroresponse rates of anti-PT, anti-FHA and anti-PRN IgG antibodies exceeded 78% in all vaccine groups. The anti-PT IgG, anti-FHA IgG, and anti-PT neutralizing antibody geometric mean titers (GMTs) were significantly higher following immunization with BioNet's aP and BioNet's TdaP than Adacel® (P< 0.05). The anti-PRN IgG, anti-tetanus and anti-diphtheria GMTs at one month after immunization were comparable in all vaccine groups. All subjects had seroprotective titers of anti-tetanus and anti-diphtheria antibodies at baseline. CONCLUSION: In this first clinical study, PTgen-based BioNet's aP and TdaP vaccines showed a similar tolerability and safety profile to Adacel® and elicited significantly higher immune responses to PT and FHA.

Maternal Immunization Earlier in Pregnancy Maximizes Antibody Transfer and Expected Infant Seropositivity Against Pertussis.

BACKGROUND: Maternal immunization against pertussis is currently recommended after the 26th gestational week (GW). Data on the optimal timing of maternal immunization are inconsistent. METHODS: We conducted a prospective observational noninferiority study comparing the influence of second-trimester (GW 13-25) vs third-trimester (≥GW 26) tetanus-diphtheria-acellular pertussis (Tdap) immunization in pregnant women who delivered at term. Geometric mean concentrations (GMCs) of cord blood antibodies to recombinant pertussis toxin (PT) and filamentous hemagglutinin (FHA) were assessed by enzyme-linked immunosorbent assay. The primary endpoint were GMCs and expected infant seropositivity rates, defined by birth anti-PT >30 enzyme-linked immunosorbent assay units (EU)/mL to confer seropositivity until 3 months of age. RESULTS: We included 335 women (mean age, 31.0 ± 5.1 years; mean gestational age, 39.3 ± 1.3 GW) previously immunized with Tdap in the second (n = 122) or third (n = 213) trimester. Anti-PT and anti-FHA GMCs were higher following second- vs third-trimester immunization (PT: 57.1 EU/mL [95% confidence interval {CI}, 47.8-68.2] vs 31.1 EU/mL [95% CI, 25.7-37.7], P < .001; FHA: 284.4 EU/mL [95% CI, 241.3-335.2] vs 140.2 EU/mL [95% CI, 115.3-170.3], P < .001). The adjusted GMC ratios after second- vs third-trimester immunization differed significantly (PT: 1.9 [95% CI, 1.4-2.5]; FHA: 2.2 [95% CI, 1.7-3.0], P < .001). Expected infant seropositivity rates reached 80% vs 55% following second- vs third-trimester immunization (adjusted odds ratio, 3.7 [95% CI, 2.1-6.5], P < .001). CONCLUSIONS: Early second-trimester maternal Tdap immunization significantly increased neonatal antibodies. Recommending immunization from the second trimester onward would widen the immunization opportunity window and could improve seroprotection.

Needle-free and adjuvant-free epicutaneous boosting of pertussis immunity: Preclinical proof of concept.

The limited durability of pertussis vaccine-induced protection requires novel approaches to reactivate immunity and limit pertussis resurgence in older children and adults. We propose that periodic boosters could be delivered using a novel epicutaneous delivery system (Viaskin) to deliver optimized pertussis antigens such as genetically-detoxified pertussis toxin (rPT). To best mimic the human situation in which vaccine-induced memory cells persist, whereas antibodies wane, we developed a novel adoptive transfer murine model of pertussis immunity. This allowed demonstrating that a single application of Viaskin delivering rPT and/or pertactin and filamentous hemagglutinin effectively reactivates vaccine-induced pertussis immunity and protects against Bordetella pertussis challenge. Recalling pertussis immunity without needles nor adjuvant may considerably facilitate the acceptance and application of periodic boosters.

Construction of Bordetella pertussis strains with enhanced production of genetically-inactivated Pertussis Toxin and Pertactin by unmarked allelic exchange.

BACKGROUND: Acellular Pertussis vaccines against whooping cough caused by Bordetella pertussis present a much-improved safety profile compared to the original vaccine of killed whole cells. The principal antigen of acellular Pertussis vaccine, Pertussis Toxin (PT), must be chemically inactivated to obtain the corresponding toxoid (PTd). This process, however, results in extensive denaturation of the antigen. The development of acellular Pertussis vaccines containing PTd or recombinant PT (rPT) with inactivated S1, Filamentous Hemagglutinin (FHA), and Pertactin (PRN) has shown that the yield of PRN was limiting, whereas FHA was overproduced. To improve antigen yields and process economics, we have constructed strains of Bordetella pertussis that produce enhanced levels of both rPT and PRN. RESULTS: Three recombinant strains of Bordetella pertussis were obtained by homologous recombination using an allelic exchange vector, pSS4245. In the first construct, the segment encoding PT subunit S1 was replaced by two mutations (R9K and E129G) that removed PT toxicity and Bp-WWC strain was obtained. In the second construct, a second copy of the whole cluster of PT structural genes containing the above mutations was inserted elsewhere into the chromosome of Bp-WWC and the Bp-WWD strain was obtained. This strain generated increased amounts of rPT (3.77 ± 0.53 µg/mL) compared to Bp-WWC (2.61 ± 0.16 µg/mL) and wild type strain (2.2 µg/mL). In the third construct, a second copy of the prn gene was inserted into the chromosome of Bp-WWD to obtain Bp-WWE. Strain Bp-WWE produced PRN at 4.18 ± 1.02 µg/mL in the cell extract which was about two-fold higher than Bp-WWC (2.48 ± 0.10 µg/mL) and Bp-WWD (2.31 ± 0.17 µg/mL). Purified PTd from Bp-WWD at 0.8-1.6 µg/well did not show any toxicity against Chinese hamster ovary (CHO) cell whereas purified PT from WT demonstrated a cell clustering endpoint at 2.6 pg/well. CONCLUSIONS: We have constructed Bordetella pertussis strains expressing increased amounts of the antigens, rPT or rPT and PRN. Expression of the third antigen, FHA was unchanged (always in excess). These strains will be useful for the manufacture of affordable acellular Pertussis vaccines.

Experimental design to optimize an Haemophilus influenzae type b conjugate vaccine made with hydrazide-derivatized tetanus toxoid.

The introduction of type b Haemophilus influenzae conjugate vaccines into routine vaccination schedules has significantly reduced the burden of this disease; however, widespread use in developing countries is constrained by vaccine costs, and there is a need for a simple and high-yielding manufacturing process. The vaccine is composed of purified capsular polysaccharide conjugated to an immunogenic carrier protein. To improve the yield and rate of the reductive amination conjugation reaction used to make this vaccine, some of the carboxyl groups of the carrier protein, tetanus toxoid, were modified to hydrazides, which are more reactive than the ε -amine of lysine. Other reaction parameters, including the ratio of the reactants, the size of the polysaccharide, the temperature and the salt concentration, were also investigated. Experimental design was used to minimize the number of experiments required to optimize all these parameters to obtain conjugate in high yield with target characteristics. It was found that increasing the reactant ratio and decreasing the size of the polysaccharide increased the polysaccharide:protein mass ratio in the product. Temperature and salt concentration did not improve this ratio. These results are consistent with a diffusion controlled rate limiting step in the conjugation reaction. Excessive modification of tetanus toxoid with hydrazide was correlated with reduced yield and lower free polysaccharide. This was attributed to a greater tendency for precipitation, possibly due to changes in the isoelectric point. Experimental design and multiple regression helped identify key parameters to control and thereby optimize this conjugation reaction.