Dorothy K.L. Xing

Physicochemical and immunological studies on the stability of free and microsphere-encapsulated tetanus toxoid in vitro

The stability of tetanus toxoid released from experimental, slow-release microsphere vaccines was compared with that of free toxoid under different conditions over a 3 month period. The amount of antigenicity remaining, as measured by ELISA, correlated well with loss of toxoid structure, as determined by circular dichroism and fluorescence spectroscopy. At 37 degrees C and low pH, pH 2.5 for free toxoid, or under the acidic conditions generated by the hydrolysis of fast-release microspheres, a gradual unfolding of the polypeptide chain was observed within the first few weeks with more rapid denaturation beyond 30 days.

Toxicity and potency evaluation of pertussis vaccines

Current methods for determining the potency and toxicity of pertussis vaccines are outdated and require improvement. The intracerebral challenge test is effective for determining the potency of whole-cell vaccines but is objectionable on animal welfare and technical grounds. The same applies to its modification for assaying acellular pertussis vaccines. Respiratory challenge methods offer an interim solution pending establishment of validated in vitro correlates of protection, for example nitric oxide induction. Their evaluation is being promoted by the World Health Organization through the Pertussis Vaccines Working Group. Current toxicity assays based on weight gain and histamine sensitization of mice are imprecise and need replacement. Limits need to be established for specific toxin content of both acellular and whole-cell vaccines and should be supported by specific assays. More precise methods based on determination of ribosyltransferase activity in tandem with receptor-binding assays are under evaluation. Genome sequence data and the use of gene microarrays to screen responses triggered by vaccine components may also provide leads to improved methods for assessing both toxicity and immunogenicity.

Investigation of cellular and humoral immune responses to whole cell and acellular pertussis vaccines

New generation acellular pertussis vaccines were compared with the established whole cell pertussis vaccine for the induction of humoral and cellular immune-responses in mice. At the same time, the in vivo protective effect of these two types of vaccine was also compared in both intracerebral (ic) and aerosol challenge models. In general, whole cell vaccine induced lower antibody titres to pertussis toxin, filamentous haemagglutinin and pertactin than the acellular vaccine. Nitric oxide concentration in macrophage cultures was used as a marker for macrophage activation. The nitric oxide concentrations in the macrophage cultures from mice following immunisation with the whole cell vaccine were higher than those from mice immunised with the acellular vaccine, which indicated that the whole cell vaccine was more effective than the acellular vaccine in activating macrophages. This was associated with better protection in vivo after challenge. After ic challenge of mice following immunisation with whole cell or acellular vaccine, 90% of the whole cell vaccine group survived compared with 40% of the acellular vaccine group at the vaccine dose selected. Following aerosol challenge, mice in the whole cell vaccine group showed faster clearance of bacteria from the lungs than those in the acellular vaccine group. Our findings suggest that the different types of pertussis vaccines may achieve protection in different ways and that CMI may play an important role in eliminating bacteria which escape humoral defence mechanisms.

An aerosol challenge model of Bordetella pertussis infection as a potential bioassay for acellular pertussis vaccines

Whole cell and five different types of acellular pertussis vaccine were assayed using a mouse aerosol challenge model which permitted delivery of a controlled, consistent dose of Bordetella pertussis to the lower respiratory tract. Using this system, the viable counts in the lungs of vaccinated mice were immunisation dose-dependent and allowed the protective capacity of different vaccine preparations to be distinguished. This model may thus provide the basis for a protection assay for pertussis vaccines. Comparison of acellular vaccines with a whole cell pertussis vaccine showed that the latter gave better active protection in mice but with a different dose-response relationship. Thus the two types of vaccine are not directly comparable in the same assay and require different reference standards. A pentavalent type acellular vaccine is suggested as a possible candidate standard for the acellular vaccine potency test. The results suggest that this aerosol challenge model has potential for use as a potency test for acellular pertussis vaccines.

International Bordetella pertussis assay standardization and harmonization meeting report. Centers for Disease Control and Prevention, Atlanta, Georgia, United States, 19–20 July 2007

n Abstractn n An international meeting on Bordetella pertussis assay standardization and harmonization was held at the Centers for Disease Control and Prevention (CDC), Atlanta, GA, 19–20 July 2007. The goal of the meeting was to harmonize the immunoassays used for pertussis diagnostics and vaccine evaluation, as agreed upon by academic and government researchers, regulatory authorities, vaccine manufacturers, and the World Health Organization (WHO).n The primary objectives were (1) to provide epidemiologic, laboratory, and statistical background for support of global harmonization; (2) to overview the current status of global epidemiology, pathogenesis and immunology of pertussis; (3) to develop a consensus opinion on existing gaps in understanding standardization of pertussis assays used for serodiagnosis and vaccine evaluation; and (4) to search for a multicenter process for addressing these priority gaps. Presentations and discussions by content experts addressed these objectives. A prioritized list of action items to improve standardization and harmonization of pertussis assays was identified during a group discussion at the end of the meeting. The major items included: (1) to identify a group that will organize, prepare, maintain, and distribute proficiency panels and key reagents such as reference and control sera; (2) to encourage the development and identification of one or more reference laboratories that can serve as an anchor and resource for other laboratories; (3) to define a performance-based assay method that can serve as a reference point for evaluating laboratory differences; (4) to develop guidance on quality of other reagents, e.g., pertussis toxin and other antigens, and methods to demonstrate their suitability; (5) to establish an international working group to harmonize the criteria to evaluate the results obtained on reference and proficiency panel sera; (6) to create an inventory to determine the amount of appropriate and well-characterized sera that are available globally to be used as bridging reagents for vaccine licensure; and (7) to seek specific guidance from regulatory authorities regarding the expectations and requirements for the licensure of new multicomponent pertussis vaccines.n n

Bordetella pertussis isolates in Finland: Serotype and fimbrial expression

BackgroundBordetella pertussis causes whooping cough or pertussis in humans. It produces several virulence factors, of which the fimbriae are considered adhesins and elicit immune responses in the host. B. pertussis has three distinct serotypes Fim2, Fim3 or Fim2,3. Generally, B. pertussis Fim2 strains predominate in unvaccinated populations, whereas Fim3 strains are often isolated in vaccinated populations. In Finland, pertussis vaccination was introduced in 1952. The whole-cell vaccine contained two strains, 18530 (Fim3) since 1962 and strain 1772 (Fim2,3) added in 1976. After that the vaccine has remained the same until 2005 when the whole-cell vaccine was replaced by the acellular vaccine containing pertussis toxin and filamentous hemagglutinin. Our aims were to study serotypes of Finnish B. pertussis isolates from 1974 to 2006 in a population with > 90% vaccination coverage and fimbrial expression of the isolates during infection. Serotyping was done by agglutination and serotype-specific antibody responses were determined by blocking ELISA.ResultsAltogether, 1,109 isolates were serotyped. Before 1976, serotype distributions of Fim2, Fim3 and Fim2,3 were 67%, 19% and 10%, respectively. From 1976 to 1998, 94% of the isolates were Fim2 serotype. Since 1999, the frequency of Fim3 strains started to increase and reached 83% during a nationwide epidemic in 2003. A significant increase in level of serum IgG antibodies against purified fimbriae was observed between paired sera of 37 patients. The patients infected by Fim3 strains had antibodies which blocked the binding of monoclonal antibodies to Fim3 but not to Fim2. Moreover, about one third of the Fim2 strain infected patients developed antibodies capable of blocking of binding of both anti-Fim2 and Fim3 monoclonal antibodies.ConclusionDespite extensive vaccinations in Finland, B. pertussis Fim2 strains were the most common serotype. Emergence of Fim3 strains started in 1999 and coincided with nationwide epidemics. Results of serotype-specific antibody responses suggest that Fim2 strains could express Fim3 during infection, showing a difference in fimbrial expression between in vivo and in vitro.

Effect of pertussis toxin on the induction of nitric oxide synthesis in murine macrophages and on protection in vivo

Macrophages from mice immunised with whole cell pertussis vaccine (WCV) responded in vitro to selected antigens by nitric oxide (NO) synthesis. This process was closely associated with macrophage activation. Because of the postulated role of traces of pertussis toxin (PT) in the protective effects of WCV, native PT and a genetically detoxified PT (g-PT) in combination with either a heat-treated whole cell pertussis vaccine (dWCV) or a three component acellular vaccine (ACV), were examined for their effects on NO induction in murine macrophages. The protective effects of these two forms of PT were examined in parallel using the intracerebral (ic) and aerosol challenge routes. Cultures of macrophages from mice immunised with dWCV and ACV, PT or g-PT produced less NO than comparable cultures from mice vaccinated with WCV. However, vaccination with either dWCV or ACV in combination with PT but not with g-PT, induced a significant increase (126-157%) in NO production by cultured cells and was associated with increased protection against challenge by both the ic and aerosol routes. These data indicate that a low concentration of PT acting as a co-factor in combination with other Bordetella pertussis antigens, can potentiate the activation of macrophages and that this process plays a key role in protection against infection.

Comparison of the bioactivity of reference preparations for assaying Bordetella pertussis toxin activity in vaccines by the histamine sensitisation and Chinese hamster ovary-cell tests: assessment of validity of expression of activity in terms of protein concentration.

Pertussis toxin (PT) in its detoxified form is an important antigenic component of both acellular and whole cell pertussis vaccines. Limits on the content of active PT in acellular vaccines are set in official monographs (EP, WHO, USP) and evidence of compliance is therefore, required by regulatory authorities. The two assay methods which are currently used by most manufacturers and official national control laboratories to monitor residual PT activity in acellular pertussis vaccines (and also in whole cell vaccines) are histamine sensitising (HIST) assays and Chinese hamster ovary (CHO) cell assays. Currently, different reference preparations of PT are used by individual laboratories for these tests. We therefore organised an international collaborative study to examine, by these two assay methods, two freeze-dried purified preparations of PT, one preparation in ampoules coded JNIH-5 and one preparation in ampoules coded 90/518, together with in-house reference (IHR) preparations in current use. Data from this study confirm that both JNIH-5 and 90/518 show biological activity both in HIST assays and in CHO-cell assays. Both HSD50 and ED50 values obtained in this study differ significantly between laboratories and thus show that biological activity is not determined by the nominal masses of preparations. Estimates of relative potency of 90/518 in terms of JNIH-5 per ampoule for the HIST assays do not differ significantly between laboratories. The overall mean estimates of relative potency of 90/518 in terms of JNIH-5 do not differ significantly between the two methods. Data from this study further indicate that the biological activity of different preparations was not directly related to their stated protein content. The use of protein content to indicate the level of PT activity in different preparations would give misleading results. Thus, use of a common standard is shown to greatly improve between laboratory agreement of estimates.

Nitric oxide induction in murine macrophages and spleen cells by whole-cell Bordetella pertussis vaccine.

Nitric oxide (NO) induction was studied in the peritoneal macrophages and spleen cells of female NIH mice immunised with whole cell Bordetella pertussis vaccines of moderate and high potency, respectively. Compared with controls receiving diluent only, the macrophages and spleen cells of the vaccinated mice developed high levels of reactive nitrogen intermediates from the third day after injection. The nitrite concentrations achieved maximum values at the 10th day, but significant levels persisted until the 25th day. Heat-killed B. pertussis cells were the most effective inducer of NO synthesis, followed by lipopolysaccharide and agglutinogens Fim 2 and 3. Pertussis toxoid, filamentous haemagglutinin and pertactin were poor inducers of NO synthesis. The specific nitric oxide synthase inhibitor, aminoguanidine, and anti-IFN-gamma antibody blocked formation of nitrite by the macrophages and spleen cells. The production of NO in response to in vitro culture with bacterial antigen was clearly associated with protective immunity in vivo as determined by i.c. challenge. These results suggest that reactive nitrogen intermediates play a role in the immune response induced by whole cell pertussis vaccines.

Modifications of the catalytic and binding subunits of pertussis toxin by formaldehyde: effects on toxicity and immunogenicity.

A panel of pertussis toxin (PT) preparations with varying levels of residual toxicity was prepared by treatment of native PT with formaldehyde (0-1.00% (w/v)) with the purpose of investigating the effects of residual toxicity on immunogenicity. The catalytically inactive mutant PT (PT-9K/129G) was used for comparison. Results from in vitro ADP-ribosyl transferase and Chinese hamster ovary (CHO)-cell toxicity assays demonstrated a formaldehyde-dependent reduction in PT toxicity, and implied that both A and B domain functions of PT were modified. The in vivo histamine sensitisation and leukocyte proliferation tests suggested that the formaldehyde-treated native PT preparations were subject to reversion to toxicity. Reversion was confirmed by in vitro toxicity assays, which demonstrated recovery of A and B domain functions. The presence of high molecular weight aggregated and cross-linked species of PT in these preparations did not appear to be detrimental to the production of a neutralising antibody response. IgG responses to native and non-catalytic mutant PT suggested that low levels of residual activity in the native PT enhanced the antibody response, while higher levels of activity inhibited the response. Using the non-catalytic mutant PT showed that formaldehyde-induced changes were not detrimental to the magnitude of the PT-specific antibody response but did reduce the PT-specific neutralising activity. In conclusion, the residual toxicity of PT preparations following formaldehyde treatment may play an important role in the immune response to pertussis vaccine, potentially altering the quality, class and magnitude of the antibodies produced to PT.