Iver Heron

Safety and immunogenicity of a new Neisseria meningitidis serogroup C-tetanus toxoid conjugate vaccine in healthy adults.

UNLABELLED We evaluated the safety and immunogenicity of a single dose of a new serogroup C O-deacetylated meningococcal polysaccharide-tetanus toxoid conjugate vaccine in 30 healthy adult volunteers. The vaccine was well tolerated with no serious adverse events and minimal local reactions and systemic symptoms. All subjects developed a fourfold or greater increase in serum bactericidal antibody (SBA) to serogroup C meningococcus. SBA geometric mean titre increased from 11 to 3649 (p<0.001). Serogroup C-specific IgG levels increased postvaccination from 0.65 to 17.02 microg/ml (p<0.001). Bactericidal titres pre- and postimmunisation showed significant correlation with serogroup C-specific IgG (r(2)=0.693). Antibody levels fell by 6 months postvaccination, however, meningococcal C IgG avidity increased indicating the successful induction of a T-cell-dependent antibody response. CONCLUSION meningococcal C-tetanus toxoid conjugate vaccine is immunogenic and well tolerated in healthy adults.

Immunity against Diphtheria and Tetanus in the Age Group 30–70 Years

In Denmark, childhood primary vaccination against diphtheria and tetanus has been recommended since 1950. No routine revaccinations or general vaccination of adults have been offered. In most other western countries revaccinations are recommended later in childhood. However, death still occurs from diphtheria and tetanus in countries with such vaccination programs although it is generally accepted that protective immunity can be obtained by vaccination. On this basis the immunity against diphtheria and tetanus was assessed in a random sample of 351 subjects in the age range 30-70 years. Diphtheria antitoxin titres were determined by in vitro neutralization technique using VERO cells. 26% had diphtheria antitoxin titres below protective level (0.01 international antitoxin units/ml serum). The highest number of unprotected against diphtheria was found among 30-39 year old women (68%) and 60-69 year old subjects (36%). Tetanus titres were determined by a combination of ELISA technique and an in vivo neutralization assay. 51% were unprotected against tetanus (less than 0.01 international antitoxin units/ml serum). The highest number of unprotected against tetanus was found among 60-69 year old subjects (68%) and especially among females in this age group (77%). To avoid epidemics of diphtheria in the future the immunity among adults must be raised within the coming years. Thus, revaccination must be recommended and high attendance ensured. One revaccination is sufficient only when complete primary vaccination is documented.

Intranasal booster vaccination against diphtheria and tetanus in man

The booster responses of three different formulations of intranasal (i.n.) diphtheria-tetanus (D-T) vaccines were determined in military recruits and compared with a conventional subcutaneous D-T vaccine. The vaccines for mucosal delivery were sprayed into one nostril and contained D and T toxoids in an enhancer mixture of polysorbate and caprylic/capric glycerides. All of the vaccines gave rise mainly to a systemic IgG response. Among 51 persons with anti-D antibody concentrations in serum below a protective level of 0.01 international units (IU ml-1) before vaccination, all except two attained protective antibody concentrations 4 weeks after vaccination. The median increase in anti-D antibody concentration was 113-fold with the most efficient i.n. formulation. The median increase in anti-T antibody level was 2.4-fold, however, the pre-vaccination levels for this antigen were very high. Within the examined levels, the booster response depended mainly on the dose of the antigen in the vaccine rather than on the concentration of the vehicle mixture. Compared with the parenteral D-T vaccine containing aluminium hydroxide as an adjuvant, all of the tested i.n. formulations showed somewhat lower immunogenicity in man as well as in pre-clinical guinea-pig studies. Among 215 persons immunized i.n., 61% preferred this route of administration rather than a parenteral injection, although the formulations were all associated with varying local symptoms, frequently stinging and pronounced, nasal secretion.

Booster vaccination against diphtheria and tetanus in man. Comparison of three different vaccine formulations—III

Adverse reactions and antibody levels were compared following a booster vaccination of 177 Danish military recruits with a plain, an aluminium hydroxide (0.5 mg Al per human dose, HD) and a calcium phosphate (0.25 mg Ca per HD) adsorbed diphtheria-tetanus (D-T) vaccine. The calcium phosphate adsorbed vaccine was given in a HD of 3 Lf of D and T toxoids and proved to be of equal efficacy as the aluminium hydroxide adsorbed vaccine which was injected in a dose containing twice the antigen amount. The calcium phosphate vaccine caused fewer adverse reactions than the one adsorbed to aluminium hydroxide. The plain vaccine (6 Lf per HD of D and T toxoid) had the highest efficacy with a similar low occurrence of adverse reactions as the calcium phosphate adsorbed vaccine. Potency assays in mice were in accordance with these immunogenicity results in man if a two dose immunization schedule was followed, but not if the vaccines were compared after a single immunization as requested by the procedure for potency testing according to current WHO and European Pharmacopoeia requirements. Both of the adsorbed vaccines primed mice for specific IgE antibody formation. This could be detected after a second immunization with either of the adsorbed vaccines or with the plain D-T vaccine. Also in humans, immunization with the plain vaccine boosted specific IgE formation to a detectable level. This may be ascribed to adjuvant priming during the primary vaccination series some 20 years previously.

Multivalent pneumococcal capsular polysaccharide conjugate vaccines employing genetically detoxified pneumolysin as a carrier protein

A genetically detoxified pneumolysin, pneumolysoid (PLD), was investigated as a carrier protein for pneumococcal capsular polysaccharide (CPS). Such a CPS-PLD conjugate might provide additional protection against pneumococcal infections and resultant tissue damage. A single point mutant of pneumolysin was selected, which lacked measurable haemolytic activity, but exhibited the overall structural and immunological properties of the wild type. PLD conjugates were prepared from CPS serotypes 6B, 14, 19F, and 23F by reductive amination. The structural features of free PLD, as well as the corresponding CPS-PLD, as assessed by circular dichroism spectroscopy, were virtually indistinguishable from the wild type counterpart. Each of the CPS monovalent and tetravalent conjugate formulations were examined for immunogenicity in mice at both 0.5 and 2.0 micrograms CPS per dose. Tetanus toxoid (TT) conjugates were similarly created and used for comparison. The resultant conjugate vaccines elicited high levels of CPS-specific IgG that was opsonophagocytic for all serotypes tested. Opsonophagocytic titres, expressed as reciprocal dilutions resulting in 50% killing using HL-60 cells, ranged from 100 to 30,000, depending on the serotype and formulation. In general, the lower dose and tetravalent formulations yielded the best responses for all serotypes (i.e., either equivalent or better than the higher dose and monovalent formulations). The PLD conjugates were also generally equivalent to or better in CPS-specific responses than the TT conjugates. In particular, both the PLD conjugate and the tetravalent formulations induced responses for type 23F CPS that were approximately an order of magnitude greater than that of the corresponding TT conjugate and monovalent formulations. In addition, all the PLD conjugates elicited high levels of pneumolysin-specific IgG which were shown to neutralize pneumolysin-induced haemolytic activity in vitro. As a result of these findings, PLD appears to provide an advantageous alternative to conventional carrier proteins for pneumococcal multivalent CPS conjugate vaccines.

Evaluation of vaccination requirements to secure continuous antitoxin immunity to tetanus

To investigate minimal requirements for tetanus revaccination to secure continuous protection, recently recommended by WHO, 637 subjects with documented vaccination history were studied. Antitoxin concentration in serum relative to time corresponded to a steep decline in the first years after vaccination continuing exponentially. By multiple regression analyses duration of immunity after three-dose primary vaccination was calculated to be 5 years (upper 95% confidence limit of estimated risk of serum antitoxin concentration below 0.01 IU ml-1 still less than 0.1%). Serum antitoxin concentration relative to time after revaccination depended upon age at revaccination and interval from primary vaccination. When given in childhood 5 years after primary vaccination revaccination was calculated to offer protection for approximately equal to 21 years, but protection was considerably shorter when given to the elderly. Cross-sectional and longitudinal analyses were compared and statistical approaches were introduced which may be generally applicable for evaluation of vaccination programmes. It was concluded that a vaccination programme consisting of primary vaccination in infancy and one revaccination 5 years later will secure continuous protection to about the age of 25 years. This is considerably simpler than programmes recommended in many countries, in which risk of hyperimmunization is apparent.

Booster vaccination against diphtheria and tetanus in man. Comparison of calcium phosphate and aluminium hydroxide as adjuvants--II.

Diphtheria and tetanus antibody levels were measured before and four weeks after booster vaccination of 313 Danish military recruits participating in a clinical trial to compare aluminium hydroxide and calcium phosphate as adjuvants in diphtheria-tetanus vaccines (DT). Twenty-eight percent of the men had a diphtheria pre-vaccination content below a protective level of 0.01 IU ml-1. The calcium phosphate adsorbed vaccine showed the highest efficacy for both antigens. Adverse reactions were rare but more frequent in the calcium group than in the aluminium group. No correlation was found between pre- or post-vaccination levels and adverse reactions and both vaccines gave rise to specific IgE formation. The results show that calcium phosphate is more effective but not a safer alternative to aluminium hydroxide when compared in vaccines containing 1.0 mg ml-1 of Ca or of Al.

Adjuvanticity of aluminium hydroxide and calcium phosphate in diphtheria-tetanus vaccines—I

The potencies of two diphtheria-tetanus vaccines (DT) adsorbed to either aluminium hydroxide or calcium phosphate were compared in mice and guinea pigs. The vaccines were made from the same batches of purified toxoids and contained the same amounts of antigens. Immunizations were done once or twice with different doses of vaccine injected undiluted, diluted in saline or diluted in the corresponding adjuvant. The various potency assays showed that the adjuvanticity of calcium phosphate was lower than or equal to aluminium hydroxide. Despite the range of potency assays done, none of the methods reflected the efficacy of these vaccines in revaccination of humans. A simplified potency assay is suggested for release of final vaccine formulations to reduce the number of animals in quality control.

Preclinical studies on a recombinant group B meningococcal porin as a carrier for a novel Haemophilus influenzae type b conjugate vaccine

In anticipation of future combination vaccines, a recombinant class 3 porin (rPorB) of group B meningococci was evaluated as an alternative carrier protein for a Haemophilus influenzae type b (Hib) polyribosylribotol phosphate (PRP) conjugate vaccine. The use of rPorB may avoid undesirable immunologic interactions among vaccine components, including epitopic suppression from conventional carriers (e.g. tetanus toxoid [TT]), as well as provide desirable immunomodulatory effects. Rats were found to be more reliable and consistent than mice or guinea pigs for studying antibody responses to the Hib conjugates. Different Hib conjugates, Hib-TT and Hib-rPorB, consisting of PRP conjugated by reductive amination to TT or rPorB, were compared in rats. Commercially available, licensed vaccines, HbOC (HibTITER) and PRP-T (OmniHib), were used as reference controls. Maximum geometric mean ELISA IgG titers were obtained in rats after only two doses, showing booster effects for all. However, Hib-rPorB immunization consistently resulted in responses that were 1-2 orders of magnitude greater than those for the other conjugates, including the licensed control vaccines. A maximum 4600-fold rise was observed for Hib-rPorB after two doses, and, unlike the other conjugates, a 100% response rate was always achieved without adjuvant. These results warrant further investigation of Hib-rPorB in combination with DTaP.

Improvement of a Vero cell assay to determine diphtheria antitoxin content in sera

Diphtheria antitoxin content in sera were determined automatically in Vero cell assay by spectrophotometric determination of the equivalence point between toxin and antitoxin followed by computer analysis of absorption values. The method was more accurate than visual reading and made handling of many samples easy.