Culture phenotypes and molecular characterization of Mycobacterium avium subsp. paratuberculosis isolates from small ruminants.

In this study the suitability of different solid media was investigated for the isolation of Mycobacterium avium subsp. paratuberculosis (Map) in order to identify the optimum single or combination of media to permit the isolation of all strain types from small ruminants. A subset of these Map strains was then further characterized by molecular typing methods to assess the genetic diversity of Map strains in the study area (Northern Greece). Map strains were isolated from tissues and faeces of infected goats (n=52) and sheep (n=8) and were analysed for polymorphisms in IS1311 to classify the strain type as Type C or S. The study found that M7H11 supplemented with mycobactin j, OADC and new born calf serum (M7H11+Mj) is the best single choice of medium for the primary isolation of Map of both Type C and S from small ruminants. The combination of M7H11+Mj and Herrolds egg yolk medium supplemented with mycobactin j and sodium pyruvate allowed the detection of all Map isolates in this study. Nineteen Map isolates were characterised by pulsed-field gel electrophoresis and the isolates demonstrated significant genetic diversity. Twelve different SnaBI and 16 distinct SpeI profiles were detected of which 25 have not been described previously and are new profiles. The combination of both enzyme profiles gave 13 different multiplex profiles. Ten different multiplex profiles were detected in goats and three in sheep. One ovine isolate gave the same multiplex profile as a caprine isolate and two different profiles were found within a single goat herd.

Nucleophosmin is a novel Bax chaperone that regulates apoptotic cell death.

The proapoptotic B-cell lymphoma-2 family protein Bax is a key regulatory point in the intrinsic apoptotic pathway. However, the factors controlling the process of Bax activation and translocation to mitochondria have yet to be fully identified and characterized. We performed affinity chromatography using peptides corresponding to the mitochondrial-targeting region of Bax, which is normally sequestered within the inactive structure. The molecular chaperone nucleophosmin was identified as a novel Bax-binding protein by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Reciprocal co-immunoprecipitation and proximity assays confirmed the Bax-nucleophosmin protein-protein interaction and verified that nucleophosmin only bound to activated conformationally altered Bax. Confocal microscopy in a cell-based apoptosis model, demonstrated that nucleophosmin translocation from nucleolus to cytosol preceded Bax movement. Specific knockdown of nucleophosmin expression using RNAi attenuated apoptosis as measured by mitochondrial cytochrome c release and activation of the caspase cascade. In a mouse model of ischaemic stroke, subcellular fractionation studies verified that nucleophosmin translocation occurred within 3 h, at a time before Bax translocation but after Bax conformational changes have occurred. Thus, we have elucidated a novel molecular mechanism whereby Bax becomes activated and translocates to the mitochondria to orchestrate mitochondrial dysfunction and apoptotic cell death, which opens new avenues for therapeutic intervention.

[Immunogenicity and safety of Di-Te-Ki-Pol vaccine "SSI" in Danish infants]. / Immunogenicitet og sikkerhed af Di-Te-Ki-Pol vaccine "SSI" hos danske spaedbørn.

This study compared a tetravalent DTaP-IPV vaccine (Di-Te-Ki-Pol vaccine "SSI") with the vaccination regimen used in Denmark at that time, DT-IPV plus wholecell pertussis vaccine. Two hundred and seventy children were included at their five-week routine examination. The children were allocated to one of the two vaccination regimens. No hypotonic-hyporesponsive episodes or other vaccine-related serious adverse events were seen. Local reactions, febrile and crying episodes following the investigational vaccine were similar to the reactions seen after Di-Te-Pol vaccine. All children achieved protective antibody levels to polio, diphtheria and tetanus after completing the vaccination schedule. A significantly better response to pertussis toxin was seen after the investigational vaccine. We conclude that the Di-Te-Ki-Pol vaccine is safe and immunogenic when used according to the schedule tested.

DTaP vaccines from north american vaccine (NAVA): composition and critical parameters.

NAVA's acellular pertussis vaccine is based on highly purified pertussis toxin (PT) inactivated with H(2)O(2). PT was analysed using advanced biochemical methodology including mass spectroscopy (LC/MS), yielding mass and peptide mapping information on the subunits. Pertactin, adenylate cyclase, and Fim 1, 2 were below detection levels and only trace amounts of filamentous haemagglutinin (FHA) have been identified as a minor impurity. The vaccine does not induce anti-FHA antibodies during the course of a 3-dose primary immunization series in infants. B and T cell epitopes are preserved to a higher extent after H(2)O(2)detoxification when compared with chemical inactivation with formaldehyde, thus providing new information explaining why vaccines employing formaldehyde detoxified PT may need additional pertussis components added to induce high levels of protection. Anti-PT antibodies generated by NAVA diphtheria, tetanus, and acellular pertussis vaccine (DTaP) showed a positive correlation with protection against WHO-defined pertussis. The safety profiles for these vaccines showed low reactogenicity with no serious adverse events due to the vaccines.

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.

Bordetella pertussis and chronic cough in adults.

To evaluate Bordetella pertussis as a cause of persistent cough in adults, we examined 201 patients who had a cough for 2-12 weeks and no pulmonary disease. We obtained the following at presentation: medical history, chest radiograph, respiratory function measurement, nasopharyngeal aspirate for polymerase chain reaction (PCR), nasopharyngeal swab specimen for culture, and a blood sample (acute serum). Four weeks later a second blood sample (convalescent serum) was obtained. Control sera were obtained from 164 age-matched healthy blood donors with no history of cough during the previous 12 weeks. Four patients were B. pertussis culture-positive; 11 (including the culture-positive patients) were B. pertussis PCR-positive; and 33, including 10 of the 11 PCR-positive patients, had serological evidence of recent B. pertussis infection. Pertussis-positive and -negative patients could not be discriminated by a history of cough. We conclude that B. pertussis infection is a common cause of persistent cough in adults. This is of concern, because these patients may be B. pertussis reservoirs from which transmission may occur to infants, in whom the disease can be devastating.

Immunogenicity and safety of a tetravalent diphtheria-tetanus-acellular pertussis-inactivated poliovirus vaccine.

The objective of this study was to investigate whether a tetravalent vaccine containing diphtheria, tetanus, monocomponent acellular pertussis and inactivated poliovirus (DTaP-IPV) was immunogenic and safe compared with the vaccination regime used in Denmark at the time of the study. The study was performed as an open controlled study in which 270 Danish children were enrolled at their 5 weeks' routine examination. The children were allocated to receive either (i) DTaP-IPV (12.5 Lf, 7 Lf, 40 microg, 40, 8, 32 DU) at 3, 5 and 12 months of age (n = 186) or (ii) DT-IPV (50 Lf, 12.5 Lf, 40, 8, 32 DU) at 5, 6 and 15 months of age plus whole-cell pertussis vaccine (> or = 4 IU) at 5 and 9 weeks and at 10 months of age (n = 84). No hypotonic hyporesponsive episodes or other vaccine-related serious adverse events were seen. Local reactions, febrile and crying episodes with the investigational vaccine (DTaP-IPV) were similar to the reactions seen with the existing DT-IPV vaccine. One month after completing the vaccination schedule, all children had antibodies above the defined protective antibody concentrations to polio, tetanus and diphtheria. For pertussis toxin, there was a significantly better response in the investigational vaccine group. We therefore conclude that, when used according to the schedule tested, the tetravalent DTaP-IPV vaccine is safe and immunogenic. In addition, the number of visits and the number of injections necessary are reduced with this vaccine and vaccination schedule.

Mutual interactions between DTaP-IPV and Haemophilus influenzae type b (Hib)-conjugated vaccines in laboratory animal models.

Potency and/or immunogenicity of three different Haemophilus influenzae type b-conjugated vaccines (Hib) and a DTaP-IPV vaccine alone, and their mutual interactions in DTaP-IPV-Hib combination was tested. In a mouse model, only combination of Act-Hib, in which tetanus toxoid (TT) was as active as non-conjugated TT, significantly increased the immunogenicity and potency of TT component of DTaP-IPV vaccine. Also, only combination of Hib-TITER, in which CRM197 was used as the carrier with DTaP-IPV, increased the potency of diphtheria toxoid (DT) component of DTaP-IPV vaccine significantly. It shows that the additive effect of tested Hib vaccines on immunogenicity and/or potency of TT and DT was mostly due to the existence of TT and CRM197, respectively, as the carrier in the mentioned Hib vaccines. No difference was shown in inoculation of DTaP-IPV and Hib conjugated vaccines in the same syringe or at separate sites. DTaP-IPV had dual effects on anti-Hib capsular polysaccharide (HibCP) responses to Hib vaccines in the mouse model. This duality was probably related to the carrier B-cell epitopes activity of Hib conjugated vaccines. The immunogenicity of TT component of Act-Hib and Amvax Hib-TT in the guinea pig model was shown and combination of mentioned Hib vaccines with DTaP-IPV, remarkably increased anti-TT antibody responses to the TT component of DTaP-IPV vaccine. These confirmed our results in the mouse model. Using two different protocols to evaluate the guinea pig model for induction of anti-HibCP immunity showed that a "long interval" protocol does not have any advantage over the "short interval" protocol. Also, combination of DTaP-IPV with Hib vaccines did not have any noticeable effect on anti-HibCP antibodies in the guinea pig model. Taken together, our observations in laboratory animal models may facilitate a better understanding of the mutual interactions between the different antigen components of a combined vaccine such as DTaP-IPV-Hib vaccine.

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.

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.

Intranasal vaccination: pharmaceutical evaluation of the vaccine delivery system and immunokinetic characteristics of the immune responses.

The purpose of this study was to analyze the effect of some pharmaceutical excipients when used for mucosal vaccine formulations and to characterize the achieved immune response. After conducting various pharmaceutical evaluations of the formulations, immunokinetic studies were performed in mice, guinea pigs, and rabbits. The kinetics and the characteristics (antibody isotypes, etc.) of the immune response were studied, as well as the induced level of toxin neutralizing IgG antibodies, which are usually used as the only measures of the potency of vaccines. Results in mice show that intranasal vaccination results in a potent and rapid immune response, similar to that seen after subcutaneous immunization. In guinea pigs and rabbits, however, the subcutaneous immunization produced significantly stronger response than did intranasal vaccination. The most promising excipients were found to be either Polysorbate 20 or Cremophor EL in an aqueous mixture together with caprylic/capric glyceride. The results indicate that nontoxic and pharmaceutically acceptable excipients can be used for mucosal vaccination, providing an interesting alternative to parenteral vaccination.

The effect of conjugation on immunogenicity and potency of protein carriers of polyribosylribitol phosphate (PRP) conjugated vaccines in the mouse model.

Immunogenicity and vaccine potency of carrier proteins of two different PRP-tetanus toxoid (PRP-T) conjugated vaccines, produced using different size PRP (Act-Hib & Amvax Hib-T), and one PRP-CRM197 (Hib-TITER) were studied. The immunogenicity and the vaccine potency of the carrier component of the tested PRP-conjugated vaccines, and their influences on the potency of the tetanus toxoid (T) and of the diphtheria toxoid (D) component of diphtheria toxoid-tetanus toxoid-acellular pertussis-inactivated polio vaccine (DTaP-IPV) were variable. The T component of Act-Hib (large size PRP) was as immunogenic and potent as the T component of the DTaP-IPV vaccine, and a combination of Act-Hib and DTaP-IPV resulted in a more than five-fold increase in the potency of the T However, Amvax Hib-T (small size PRP) did not show any anti-T response on its own, and a combination of Amvax Hib-T and DTaP-IPV did not affect the T potency of the DTaP-IPV vaccine. In immunogenicity studies with multiple shots, Hib-TITER (small size PRP) produced significantly less anti-D antibodies than non-conjugated D. Hib-TITER did not show any D potency on its own, while a combination of a Hib-TITER and DTaP-IPV increased the potency of the D component of DTaP-IPV vaccine significantly. Thus, in the case of a combination of T-and D-containing vaccines with a PRP-conjugated vaccine in which either T or CRMI 97 has been used as the carrier, the influence of these carriers on basic immunogenicity and vaccine potency of T and D, respectively, should be considered carefully. We propose the techniques employed in this study for the quality control of combined vaccines consisting of diphtheria, tetanus, and PRP-conjugated vaccines.

Improved ELISA for determination of anti-diphtheria and/or anti-tetanus antitoxin antibodies in sera.

Double-antigen ELISAs for detection and quantification of anti-tetanus or anti-diphtheria antibodies in serum have been developed. The assays showed good correlations with established toxin neutralizing assays and were functionally specific for IgG antibodies. The double-antigen set-up allows specific antibodies to bind to antigen-coated microtitre wells with one arm and the free arm to bind to biotin-labelled antigen. The amount of antibodies able to bind labelled antigen was assessed by adding enzyme-conjugated streptavidin and colour substrate followed by measurement of the colour using an ELISA reader. The double-antigen principle makes it possible to compare samples of different species on the same plate, permitting the direct use of existing international references of animal or human origin. The double-antigen ELISAs showed a detection limit of 0.00002 IU/ml for both antibodies and were suitable for quantifying antibodies in blood samples collected on filter paper as well as in serum. The assays required no special equipment compared to traditional ELISA.

In vivo cell-mediated immunity and vaccination response following prolonged, intense exercise.

Epidemiological and experimental studies have shown increased frequency and severity of infections after intense, long-term exercise. This study examines whether an in vivo impairment of the cell-mediated immunity and antibody production can be demonstrated after intense, long-term exercise. Twenty-two male triathletes performed one-half an ironman (group A). Vaccinations with tetanus and diphtheritis toxoid and purified pneumococcal polysaccharide were given after the exercise. Furthermore, a skin test with seven different antigens was applied on the forearm. Antibody titers were measured before and 2 wk after the exercise. The skin test was read 48 h after the application. Eleven non-exercising triathletes (group B) and 22 moderately trained men (group C) were used as control groups. Group A revealed a significantly lower skin test response to the tetanus antigen than both groups B and C. In group A, a smaller cumulative response (sum of the diameters of indurations and number of positive skin test spots) was found than in both groups B and C. No differences in antibody titers were found among the three groups. Thus, the in vivo cell-mediated immunity was impaired in the first days after prolonged, high intensity exercise, whereas there was no impairment of the in vivo antibody production measured 2 wk after vaccination.

Immunodominant domains present on the Bordetella pertussis vaccine component filamentous hemagglutinin.

To identify immunologically important domains on filamentous hemagglutinin (FHA), a Bordetella pertussis protein included in new acellular pertussis vaccines (ACPVs), a series of monoclonal antibodies, sera from infants vaccinated with ACPVs or whole cell pertussis vaccine (WCPV), and sera from patients with pertussis were analyzed by immunoblots containing FHA fragments and recombinant FHA proteins. Immunodominant domains located at the COOH-terminus of FHA (type I domain) and near the NH2-terminus (type II domain) were defined by the reactivity with monoclonal antibodies. The sera from patients with pertussis and sera from infants vaccinated with WCPV or with 6 different investigational ACPVs specifically recognized well-defined regions within the type I and type II domains. Identification of these prominent immunologic epitopes on FHA should be useful for the construction of more well-defined pertussis vaccines and for the interpretation of human serologic responses, which may correlate with efficacy of pertussis vaccines.

Immunity to tetanus and diphtheria in rural Africa.

To assess the effect of the Expanded Program on Immunization (EPI) in rural Africa, blood samples were collected in two Kenyan sublocations. Serum antibodies against tetanus toxoid were measured in 155 individuals 1-70 years of age. Titers greater than the protective level of 0.01 IU/ml were found in 47% of the population. Protection was significantly higher in children born after the launching of the EPI (68%) and in women who had been at childbearing age since then (69%). Significantly lower protection was demonstrated in other age and sex-groups. The level of protection in children was equal in the two populations, whereas protection in fertile women was significantly lower in the population living a long distance from a health center. Diphtheria anti-toxin was measured in the samples from one sublocation, and 70 of 84 individuals (83%) had antibody levels greater than the protective level. No age or sex difference could be found, and there was no correlation between response levels to diphtheria and tetanus. This implicates natural infections as an important source of diphtheria antibodies. Our findings demonstrate a need for better coverage of the adult population against tetanus. Furthermore, diphtheria transmission still appears to take place, underscoring the importance of diphtheria vaccination of travelers to rural Africa.

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.

Fall-off in immunity following diphtheria revaccination--an 8 year follow-up study.

Diphtheria may occur even among previously vaccinated persons and knowledge of the duration of immunity is of crucial importance when designing effective vaccination programmes. In a follow-up study of 42 representative probands revaccinated 8 years previously, a continuous fall-off in antitoxic immunity was demonstrated. 98% were still protected (antitoxin concentration > 0.01 IU/ml). From the distribution of titres in the group the individual risk of susceptibility 8 years after revaccination was calculated to be 0.8/1000 (0.2-2.9/1000, 95% confidence limits). Thus, repeated revaccinations are required to secure continuous protection. The fall-off pattern for diphtheria antitoxin was approximately the same as for tetanus antitoxin. Peak values following revaccination are decisive for the duration of immunity. As peak values following vaccination depend on naturally acquired immunity and consequently decrease as indigenous diphtheria in a population disappears, highly potent vaccines are required to secure long-term immunity following diphtheria revaccination. The effects of dose and adjuvant are discussed.

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.