Comparative seroepidemiology of diphtheria in six European countries and Israel.

Serological surveys for diphtheria were conducted in six European countries including Czech Republic, Hungary, Ireland, Latvia, Luxembourg, Slovakia and one country outside Europe, Israel. For each country, a nationally representative population sample was collected across the entire age range and was tested for antibodies to diphtheria toxin. Although each national laboratory used its preferred assay, the results were all standardized to those of the in vitro neutralization test and expressed in international units (IU) which allowed comparative analyses to be performed. The results showed that increasing age is related to a gradual increase in seronegative subjects (<0·01 IU/ml of diphtheria antitoxin antibodies). This may reflect waning immunity following childhood vaccination without repeated booster vaccinations in adults. Differences in seronegativity were also found according to gender. In subjects aged 1-19 years, geometric mean titres of antitoxin are clearly related to the different vaccination schedules used in the participating countries. Although clinical disease remains rare, the susceptibility to diphtheria observed in these serosurveys highlights the importance of strengthened surveillance.

Evaluation of two serological methods for potency testing of whole cell pertussis vaccines.

The European Pharmacopoeia (Ph. Eur.) and the World Health Organization (WHO) require the performance of extensive quality control testing including a potency test before a vaccine batch is released for human use. Whole cell pertussis (wP) vaccine potency is assessed by a mouse protection test (MPT) based on the Kendrick test. This test compares the vaccine dose necessary to protect 50% of mice against the effect of a lethal intracerebral dose of Bordetella pertussis and the dose of a suitable reference vaccine needed to give the same protection level. Due to the large variability in the results of this test and the severe distress which is inflicted on the many animals involved, its replacement by an alternative method is highly desirable. At the initiative of the European Directorate for the Quality of Medicines and HealthCare (EDQM) of the Council of Europe, in collaboration with the WHO and the In-vitro toxicology Unit/European Centre for the Validation of Alternative Methods (ECVAM) of the European Commission (EC) Joint Research Centre-Institute for Health and Consumer Protection (JRC-IHCP), wP vaccine specialists from all over the world were invited to present an overview of candidate alternatives at a symposium organised in Geneva (Switzerland) in March 2005. Although no alternative method was found suitable for immediate implementation of batch potency control, the Pertussis Serological Potency Test (PSPT), initially developed in mice and recently transferred to guinea pigs (gps), was identified as a model of interest. Using the PSPT in gps to test several components of combined vaccines such as Diphtheria-Tetanus-wP vaccines in the same animal series would allow further implementation of the European 3Rs policy to batch potency control, by additional method refinement and reduction of animal use. The present study evaluated 2 features of the serological response to wP vaccination: 1) the overall antibody response as measured by a "whole cell" ELISA (PSPT-wC-ELISA) which uses the B. pertussis 18323 challenge strain prescribed for the MPT to coat the assay plates and 2) the functional neutralising antibodies to pertussis toxin (PT, one of the main virulence factors of B. pertussis), as measured by the Chinese Hamster Ovary (CHO) cell assay. The results showed that 1) the gp model can be used for wP vaccine potency testing; 2) despite good repeatability and precision, the CHO cell assay did not generate results comparable to the MPT. Moreover, the CHO cell assay showed significant differences in the ability of wP vaccines to induce neutralising anti-PT antibodies, which did not correlate to the overall antibody response evaluated by PSPT-wC-ELISA; 3) comparable potencies were obtained in the MPT and the PSPT-wC-ELISA. This study, supported by the previous ones correlating the PSPT-wC-ELISA in mice with the MPT, confirms that PSPT-wC-ELISA in gps is a promising approach for batch release potency testing of wP vaccines for which consistency in production has already been demonstrated by the MPT. However, a large scale validation study is required prior to the adoption of PSPT-wC-ELISA as a compendial reference method for wP vaccines batch release control.

Collaborative study on saccharide quantification of the Haemophilus influenzae type b component in liquid vaccine presentations.

Before release onto the market, it must be demonstrated that the total and free polysaccharide (poly ribosyl-ribitol-phosphate, PRP) content of Haemophilus influenzae type b (Hib) vaccine complies with requirements. However, manufacturers use different methods to assay PRP content: a national control laboratory must establish and validate the relevant manufacturer methodology before using it to determine PRP content. An international study was organised by the World Health Organization (WHO), in collaboration with the Biological Standardisation Programme (BSP) of the Council of Europe/European Directorate for the Quality of Medicines & HealthCare (EDQM) and of the European Union Commission, to verify the suitability of a single method for determining PRP content in liquid pentavalent vaccines (DTwP-HepB-Hib) containing a whole-cell pertussis component. It consists of HCl hydrolysis followed by chromatographic separation and quantification of ribitol on a CarboPac MA1 column using high-performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD). The unconjugated, free, PRP is separated from the total PRP using C4 solid-phase extraction cartridges (SPE C4). Ten quality control laboratories performed two independent analyses applying the proposed analytical test protocol to five vaccine samples, including a vaccine lot with sub-potent PRP content and very high free PRP content. Both WHO PRP standard and ribitol reference standard were included as calibrating standards. A significant bias between WHO PRP standard and ribitol reference standard was observed. Study results showed that the proposed analytical method is, in principle, suitable for the intended use provided that a validation is performed as usually expected from quality control laboratories.

Adherence of glucan-positive and glucan-negative strains of Streptococcus bovis to human epithelial cells.

Adherence to buccal epithelial cells (BEC) and the role played in the binding by lipoteichoic acid (LTA) and other superficial components have been studied in reference and clinical strains of Streptococcus bovis either glucan-positive biotype I or glucan-negative biotype II. To avoid the synthesis of glucan by biotype I strains, adherence was studied in bacteria grown in Todd-Hewitt broth, a sucrose deficient medium. Both biotypes were shown to bind to BEC and clinical isolates, irrespective of biotype attached to the same degree but in greater numbers than reference strains. Inhibition studies suggest that at least two mechanisms,--LTA and protein-mediated--are responsible for the adherence of both glucan-positive and negative strains of S. bovis. Moreover, in glucan-positive strains capsular polysaccharides may be also involved.

Protective activity of a murine monoclonal antibody against acute and chronic experimental infection with type IV group B streptococcus.

A murine IgM monoclonal antibody (MAb H11) was developed against the type polysaccharide capsular antigen of group B streptococcus (GBS), serotype IV, after intraperitoneal immunisation of BALB/c mice with heat-killed bacteria. MAb H11 reacted in immunodiffusion with the purified polysaccharide in both its sialylated and desialylated form, giving a line of identity, and opsonised type IV GBS strains in an in vitro assay. When administered at the time of intraperitoneal lethal challenge with homologous GBS, or 4 h earlier, MAb H11 protected 90% of the mice. Protection was still observed when MAb H11 was given 4 h after the challenge. This MAb was strongly effective in preventing septic arthritis induced by type IV GBS.

Role of group B streptococcal capsular polysaccharides in the induction of septic arthritis.

The ability of different serotypes of group B streptococci (GBS) to induce septic arthritis in mice was compared. Types II, III, IV, V, VI and VII GBS were investigated. A highly capsulate strain of type III GBS, COH1, and its mutants, COH1-11 (lacking capsular sialic acid) and COH1-13 (non-capsulate), obtained by transposon insertional mutagenesis, were used to assess the role of type-specific polysaccharide on the induction of arthritis. At an intravenous dose of 10(7) cfu/mouse, reference strains of types II, III, IV, VI and VII and type III strain COH1 induced arthritis with an incidence ranging from 70 to 90%. For type V and strain COH1-11, 10(8) cfu/mouse was required to obtain a 50% incidence of arthritis; lesions were not evident with strain COH1-13. The presence of the capsule played a major role in the induction of GBS septic arthritis. The presence and amount of sialic acid in capsular polysaccharide influenced the incidence of articular lesions. The bacterial dose affected the manifestations of arthritis; the less virulent strains of GBS also induced articular lesions when an adequate number of micro-organisms reached the joints.

Antibody-independent protection in mice against type Ia group B streptococcus lethal infection.

There is ample evidence that protection against group B streptococcal (GBS) disease, both in experimental animals and in humans, is related to the presence of specific antibodies and complement. However, until now the possibility of increasing resistance to GBS infection by potentiating natural cell-mediated immunity in the host, has not been explored. In this study we examine the effect of administering in vivo MVE-2 (a polymer fraction of 1,2-co-polymer of divinyl ether and maleic anhydride) and inactivated Candida albicans (CA) cells on mouse resistance to the reference strain type Ia 090 GBS (GBS-090) lethal infection. MVE-2 and CA, respectively a synthetic and a microbial biological response modifier (BRM), are strong inducers and activators of natural resistance effectors, such as natural killer (NK) cells, macrophages and polymorphonuclear cells (PMN). The results showed that MVE-2 protected 100% CD-1 mice from a systemic lethal challenge with GBS-090 (5 x 10(3) microorganisms/mouse) when administered 3 days before infection at dose of 50 mg kg-1. CA treatment, in five doses (CA-5d) over 14 days protected 100% mice when administered at 2 x 10(7) cells/mouse and when the last CA injection was given 1 day before the GBS-090 challenge. Instead, when the GBS-090 challenge was performed by intraperitoneal route, protection was obtained with CA-5d treatment but not with MVE-2. The possibility that MVE-2 or CA stimulated a rapid production of specific antibodies against GBS-090 infection was excluded by the ELISA assay. Evidence exists that NK cells do not play a primary role as effectors in the MVE-2 and CA conferred protection since the strong reduction in NK activity, due to in vivo administration of anti-asialo GM1 antibodies before GBS-090 infection, did not influence the BRM-induced protection. Besides, high NK activity levels, induced by in vivo rhIL-2 administration, did not protect the mice against GBS-090 infection. Both studies on in vivo clearance and in vitro microbicidal activity, showed that, after 1 h, immunopotentiated effectors were unable to kill GBS-090, but were highly effective against GBS type VI. These results seem to indicate that intracellular GBS-090 killing is a slow process requiring more than 1 h. This study demonstrates that it is possible to increase resistance to GBS-090 lethal infection by BRMs, by potentiating the antibody-independent microbicidal activity of the phagocytes.

Determination of the molecular size distribution of Haemophilus influenzae type b-tetanus toxoid conjugate vaccines by size-exclusion chromatography.

The molecular size of Haemophilus influenzae (Hib) type b polysaccharide-protein conjugate vaccines is an important physico-chemical parameter which correlates with immunogenicity. This paper describes the experimental conditions for high-performance size-exclusion chromatography on a PL Aquagel-OH 60 column to determine the size distribution of Hib high-molecular-weight conjugate vaccines.

Structural elucidation of the novel type VII group B Streptococcus capsular polysaccharide by high resolution NMR spectroscopy.

The type VII capsular polysaccharide isolated from the newly discovered group B Streptococcus (GBS) strain contains D-glucose, D-galactose, N-acetyl-D-glucosamine and N-acetylneuraminic acid in the molar ratio 2:2:1:1. High-resolution one- and two-dimensional (1D and 2D) 1H and 13C NMR spectroscopy of the native and desialylated polysaccharides showed the type VII GBS capsular polysaccharide to contain the following branched hexasaccharide repeating unit: [formula: see text] Despite extensive structural similarity with the previously described GBS polysaccharides, the type VII polysaccharide showed no cross-reaction with the heterologous antisera.

Antibody classes & subclasses induced by mucosal immunization of mice with Streptococcus pyogenes M6 protein & oligodeoxynucleotides containing CpG motifs.

BACKGROUND & OBJECTIVES: Type-specific antibodies against M protein are critical for human protection as they enhance phagocytosis and are protective. An ideal vaccine for the protection against Streptococcus pyogenes would warrant mucosal immunity, but mucosally administered M-protein has been shown to be poorly immunogenic in animals. We used a recombinant M type 6 protein to immunize mice in the presence of synthetic oligodeoxynucleotides containing CpG motifs (immunostimulatory sequences: ISS) or cholera toxin (CT) to explore its possible usage in a mucosal vaccine. METHODS: Mice were immunized by intranasal (in) or intradermal (id) administration with four doses at weekly intervals of M6-protein (10 microg/mouse) with or without adjuvant (ISS, 10 microg/mouse or CT, 0,5 microg/mouse). M6 specific antibodies were measured by enzyme linked immunosorbent assay using class and subclass specific monoclonal antibodies. RESULTS: The use of ISS induced an impressive anti M-protein serum IgG response but when id administered was not detectable in the absence of adjuvant. When used in, M-protein in the presence of both ISS and CT induced anti M-protein IgA in the bronchoalveolar lavage, as well as specific IgG in the serum. IgG were able to react with serotype M6 strains of S. pyogenes. The level of antibodies obtained by immunizing mice in with M-protein and CT was higher in comparison to M-protein and ISS. The analysis of anti-M protein specific IgG subclasses showed high levels of IgG1, IgG2a and IgG2b, and low levels of IgG3 when ISS were used as adjuvant. Thus, in the presence of ISS, the ratio IgG2a/IgG1 and (IgG2a+IgG3)/IgG1 >1 indicated a type 1-like response obtained both in mucosally or systemically vaccinated mice. INTERPRETATION & CONCLUSION: Our study offers a reproducible model of anti-M protein vaccination that could be applied to test new antigenic formulations to induce an anti-group A Streptococcus (GAS) vaccination suitable for protection against the different diseases caused by this bacterium.

Surveillance of systemic invasive disease caused by group A Streptococcus in Italy 1994-1996.

In late May 1994, following reports of an outbreak of group A beta-haemolytic streptococcus (GAS) in the United Kingdom, the Istituto Superiore di Sanita (ISS) set up a surveillance system for systemic invasive GAS disease. The surveillance system was als

Studies of the antimicrobial activity of ovotransferrin.

The antibacterial activity of ovotransferrin (conalbumin) against different bacterial species was studied in vitro. The most sensitive species were Pseudomonas sp., E. coli, S. mutans; and the most resistant ones S. aureus, Proteus sp., Klebsiella. The bacteriostatic activity of conalbumin in various conditions was also tested. The presence of bicarbonate ions always increased the activity of conalbumin, while an antagonistic effect of citrate was observed in bacteria with a receptor for the iron-citrate complex. Experiments with conalbumin covalently linked to Sepharose 4B indicated that its antibacterial activity may not be due simply to the removal of iron from the medium, but probably involves other metals and an interaction with the bacterial surface. The in vitro studies carried out with conalbumin and lactoferrin demonstrated that the two proteins produced a similar inhibition of growth of E. coli and S. mutans. The in vivo studies showed that the protective effect of conalbumin in newborn guinea pigs with E. coli by gastrointestinal route was similar to that naturally provided by the lactoferrin present in milk.

Screening for Corynebacterium diphtheriae and Corynebacterium ulcerans in patients with upper respiratory tract infections 2007-2008: a multicentre European study.

Diphtheria is now rare in most European countries but, when cases do arise, the case fatality rate is high (5-10%). Because few countries continue to routinely screen for the causative organisms of diphtheria, the extent to which they are circulating amongst different European populations is largely unknown. During 2007-2008, ten European countries each screened between 968 and 8551 throat swabs from patients with upper respiratory tract infections. Six toxigenic strains of Corynebacterium diphtheriae were identified: two from symptomatic patients in Latvia (the country with the highest reported incidence of diphtheria in the European Union) and four from Lithuania (two cases, two carriers); the last reported case of diphtheria in Lithuania was in 2002. Carriage rates of non-toxigenic organisms ranged from 0 (Bulgaria, Finland, Greece, Ireland, Italy) to 4.0 per 1000 (95% CI 2.0-7.1) in Turkey. A total of 28 non-toxigenic strains were identified during the study (26 C. diphtheriae, one Corynebacterium ulcerans, one Corynebacterium pseudotuberculosis). The non-toxigenic C. ulcerans strain was isolated from the UK, the country with the highest reported incidence of cases due to C. ulcerans. Of the eleven ribotypes detected, Cluj was seen most frequently in the non-toxigenic isolates and, amongst toxigenic isolates, the major epidemic clone, Sankt-Petersburg, is still in circulation. Isolation of toxigenic C. diphtheriae and non-toxigenic C. diphtheriae and C. ulcerans in highly-vaccinated populations highlights the need to maintain microbiological surveillance, laboratory expertise and an awareness of these organisms amongst public health specialists, microbiologists and clinicians.

Penicillin tolerance amongst non-toxigenic Corynebacterium diphtheriae isolated from cases of pharyngitis.

Twenty-four strains of non-toxigenic Corynebacterium diphtheriae biotype gravis from the throats of patients with pharyngitis/tonsillitis were assayed for susceptibility to penicillin and erythromycin using determination of MIC, MBC and time-kill curves. There were no differences between the MICs of penicillin for susceptible and tolerant strains. All but one strain had penicillin MBCs > or = 2 mg/L. Seventy-one per cent (17/24) of the strains were tolerant to penicillin. In contrast, all strains were susceptible to erythromycin (MIC < or = 0.016 mg/L). These aspects should be considered when choosing the therapy for treating non-toxigenic C. diphtheriae pharyngitis/tonsillitis.