Molecular and cellular signatures underlying superior immunity against Bordetella pertussis upon pulmonary vaccination.

This corrects the article DOI: 10.1038/mi.2017.81.

Molecular and cellular signatures underlying superior immunity against Bordetella pertussis upon pulmonary vaccination.

Mucosal immunity is often required for protection against respiratory pathogens but the underlying cellular and molecular mechanisms of induction remain poorly understood. Here, systems vaccinology was used to identify immune signatures after pulmonary or subcutaneous immunization of mice with pertussis outer membrane vesicles. Pulmonary immunization led to improved protection, exclusively induced mucosal immunoglobulin A (IgA) and T helper type 17 (Th17) responses, and in addition evoked elevated systemic immunoglobulin G (IgG) antibody levels, IgG-producing plasma cells, memory B cells, and Th17 cells. These adaptive responses were preceded by unique local expression of genes of the innate immune response related to Th17 (e.g., Rorc) and IgA responses (e.g., Pigr) in addition to local and systemic secretion of Th1/Th17-promoting cytokines. This comprehensive systems approach identifies the effect of the administration route on the development of mucosal immunity, its importance in protection against Bordetella pertussis, and reveals potential molecular correlates of vaccine immunity to this reemerging pathogen.

Toxicity and immunogenicity of pertussis whole cell vaccine in one animal model.

The required in vivo assays for the release of Whole Cell Pertussis Vaccine are the Mouse Weight Gain test (MWGT) for assessment of specific toxicity and the Mouse Protection test (MPT) to estimate the potency. Despite the fact that both assays are criticised for the use of large number of animals, poor precision or insensitivity, more precise alternatives--such as the Leukocytosis Promotion test (LP-test) and Pertussis Serological Potency Test (PSPT)--are still not fully accepted. During the optimisation of the production process, the need for more accurate parameters to determine toxicity and potency became essential. To reduce substantially the number of animals, we have combined the MWGT with the LP-test and PSPT in one model, named the Mouse Toxicity and Immunogenicity test (MTI-test). Animals are inoculated with half a human dose and are weighed individually pre-immunisation and 16 hours (parameter for endotoxin), three and seven days post immunisation. Additionally, the number of leukocytes (parameter for PT) is determined on day 7 and after 28 days the animals are bled individually for immunogenicity testing (parameter for potency). A lyophilised reference vaccine is included as a positive control to standardise the assay and to determine its reproducibility. The advantage of this modified procedure is that the data on toxicity and immunogenicity are obtained from the individual mouse, which enables statistical analysis to be made. The leucocytosis data can be used to check whether mice were vaccinated correctly, allowing for the elimination of incorrectly vaccinated mice from the assay. Furthermore, this assay can be used to determine the consistency of production, the influence of adjuvant on toxicity, as well as the composition and stability of vaccines.

The Pertussis Serological Potency Test. Collaborative study to evaluatereplacement of the Mouse Protection Test.

The Pertussis Serological Potency Test (PSPT)--based on in vitro assessment of the humoral immune response against Bordetella pertussis--was developed as an alternative for the Mouse Protection Test (MPT). A small-scale collaborative study was carried out in five laboratories to evaluate the relevance and reliability of the PSPT. The study has been divided into three separate phases, each with its own objective. A pilot-phase study of the antibody detection assay, the 18323-whole cell ELISA (WCE), was included for training purposes. Significant differences in absorbance and antibody concentrations between the laboratories were found. In the Phase I study, the intra-assay, inter-assay and inter-laboratory precisions of the 18323-WCE were assessed. Although a precision of less than 20% was not always established and significant differences in antibody concentrations were found at random throughout the Phase I study, the ranking of the antibody concentrations corresponded well between the laboratories and should warrant a reliable potency estimation of whole cell vaccines (WCV's) in the PSPT. Phase II was a comparative study of the PSPT and the MPT to evaluate the implementation of the PSPT, to demonstrate correlation and to compare the reproducibility and reliability of both tests. The mean antibody concentrations per vaccine dose in the PSPT and the survival of mice in the MPT differed significantly within and between the laboratories. Nevertheless, the potencies of the vaccines under test estimated in both test models did not differ significantly (P>0.05). The PSPT and MPT correlated well in chi2-test of homogeneity within and between the laboratories. The potencies were similar (overall ratio=0.877), but the PSPT is more reproducible and reduces the chance of re-testing due to the smaller 95% confidence intervals. We have demonstrated that the PSPT is a valid model to estimate the potencies of pertussis WCV's from different manufacturers. Moreover, the 18323-WCE is easy to carry out and the intra-assay precision and antibody ranking warrants a reliable potency testing of pertussis WCV's in the PSPT.

Production, characterization and control of a Neisseria meningitidis hexavalent class 1 outer membrane protein containing vesicle vaccine.

An experimental serogroup B meningococcal vaccine was prepared from two genetically engineered strains; each expressing three different class 1 outer membrane proteins (OMPs) (PorA). The two strains expressed the subtypes P1.7,16;P1.5,2;P1.19,15 and P1.5c,10;P1.12,13;P1.7h,4, respectively. Outer membrane vesicles (OMV) were prepared from these strains by deoxycholate extraction, mixed with aluminiumphosphate as adjuvant and formulated to final vaccines. The class 1 OMPs represent ca 90% of the protein in the vaccine. The vaccine was found safe for human use and induced a bactericidal immune response in mice against five of the six wild type strains, which served as donors for the various por A genes.

Pertussis serological potency test as an alternatively to the intracerebral mouse protection test.

The current potency test for pertussis vaccines, the intracerebral protection test (MPT), is still the only mandatory laboratory model available. This test, however, is a valid, but inhumane and imprecise test and therefore a good candidate for replacement. Recently we have developed the Pertussis Serological Potency Test (PSPT) as an alternative for the MPT. The PSPT is based on in vitro assessment of the humoral immune response against the whole range of surface -antigens of B. pertussis in mice after immunisation with Whole Cell Vaccine (WCV). We have demonstrated a relationship between the mean pertussis antibody concentration at the day of challenge and the proportion of surviving mice at each vaccine dose in the MPT (R = 0.91). The PSPT is a model in which mice (20-24 g) are immunised i.p. with graded doses of vaccine and bled after four weeks. Sera are titrated in a whole cell ELISA and potency based on the vaccine dose-dependent antibody response is estimated by means of a parallel line analysis. In an in-house validation study 13 WCVs were tested in the PSPT and MPT. Homogeneity of both tests was proven by means of the chi-square test; potencies were significantly similar (p = 0.95). Compared to the MPT, the PSPT is more reproducible as is indicated by its smaller 95% confidence intervals. Moreover, by using the PSPT the animal distress can be reduced to an acceptable level and the PSPT also results in a reduction of more than 25% in use of mice. Additional experiments showed that estimation of WCV-potency in the PSPT based on specific antibody responses against protective antigens (PT, FHA, 69- and 92-kDa OMPS) was not possible or did not correlate with protection in MPT. Sera obtained from the PSPT showed a correlation between pertussis antibody levels and complement-mediated killing by pertussis antibodies in in vitro assays. In conclusion, the PSPT is a promising substitute for the MPT though further validation and additional studies on functional validity should finally warrant replacement of the MPT by this serological model.

Development of pertussis serological potency test. Serological assessment of antibody response induced by whole cell vaccine as an alternative to mouse protection in an intracerebral challenge model.

The current potency test for pertussis vaccines, the mouse protection test (MPT), has many disadvantages. However, no alternative is yet available. The purpose of this study is to develop a serological alternative for the MPT based on in vitro assessment of the humoral immune response against pertussis in mice. After immunization with pertussis whole cell vaccine, the MPT shows a normal primary and secondary antibody response. Moreover, the i.c. challenge has a distinct booster effect on the pertussis IgG response. The relationship between the concentration of IgG antibodies against the surface-antigens of pertussis bacteria and the survival of mice after the i.c. challenge was demonstrated in a modified MPT (R = 0.91). To this end a protecting antibody level of > or = 45 EU/ml was selected as a level at which concentration most of the mice survived. Survival of mice in the MPT could be predicted, based on the antibody concentration at the day of challenge. Potencies estimated with the predicted and actual survival corresponded well (P = 0.990). This confirmed the essential role of vaccine induced pertussis antibodies in the protection against a lethal i.c. challenge and offered a possibility to develop a pertussis potency test based on serology. We developed a model in which mice (20-24 g) are immunized (i.p.) with graded doses of vaccine and bled after four weeks. Sera are titrated in Bordetella pertussis whole cell ELISA and potency based on vaccine dose dependent antibody response is estimated by means of a parallel line analysis. The potency of vaccines tested in the Pertussis Serological Potency Test (PSPT) and MPT are significantly similar, a P-value of 0.92 was found by means of the chi 2 test. Compared to the MPT, the PSPT is more reproducible as is indicated by its smaller 95% confidence intervals. Moreover, by using the PSPT the animal distress can be reduced to an acceptable level and the PSPT also results in a reduction of more than 25% in use of mice.

Sudden unexpected death due to Taxus poisoning. A report of five cases, with review of the literature.

The toxicity of yew (Taxus) has been known since antiquity. However, in the past 31 years, to our knowledge only six cases of Taxus poisoning have been reported in the literature. In the present paper we add five cases. From a forensic point of view, intoxication with Taxus has three important aspects: (i) the clinical presentation, which among other causes should suggest Taxus intoxication; (ii) the fact that the diagnosis may often be easily made by examination of the contents of stomach, duodenum and small bowel and (iii) the widespread availability in the near future of Taxol, an anti-neoplastic drug which is an alkaloid extracted from Taxus. The clinical and autopsy findings are summarized, the diagnostic aspects are discussed and the literature concerning Taxus is reviewed.

CD59 expressed by human endothelial cells functions as a protective molecule against complement-mediated lysis.

CD59 is a 18-20-kDa membrane glycoprotein that inhibits formation of the membrane attack complex of complement (C) on homologous cells. In the present study we analyzed the expression and function of CD59 on human endothelial cells. Immunohistochemical analysis of renal cortex demonstrated a predominant expression of CD59 on peritubular capillary endothelial cells and glomerular endothelial cells. Flow cytometry analysis showed that human umbilical vein endothelial cells (HUVEC) expressed CD59 and the fluorescence intensity was approximately four times that of peripheral blood lymphocytes. CD59 is detected on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single 20-kDa molecule in 2% deoxycholate extracts of HUVEC. CD59 was released from the surface of HUVEC by phosphatidylinositol-specific phospholipase C, demonstrating that it is attached to the cell membrane by means of a glycolipid anchor. The functional activity of CD59 expressed on HUVEC was studied. Blocking of CD59 antigen with F(ab')2 fragments of polyclonal anti-CD59 enhanced markedly the susceptibility of HUVEC to C-mediated lysis. This effect was dependent on the amount of blocking antibodies added. Northern blot analysis revealed the presence of three species of mRNA expressed in HUVEC, which hybridized to a cDNA probe specific for CD59, with sizes of about 800, 1400 and 2000 bp. These findings suggest that CD59 may be important in protection of endothelial cells against C-mediated damage at local sites of inflammation, thereby maintaining the vascular integrity in vivo.

Interleukin 2 mediates stimulation of complement C3 biosynthesis in human proximal tubular epithelial cells.

Previous reports have suggested the production of complement components C4, C2, and factor B by renal tissue. However, the cells involved in production of complement have not been identified. In this study metabolic labeling experiments demonstrated that human proximal tubular epithelial cells (PTEC) synthesize a 180-kD precursor of C3 that is secreted after proteolytic cleavage into a disulphide linked two-chain molecule as found in plasma. C3 present in culture supernatants of PTEC was functionally active, however, during the culture period there was a partial inactivation of the C3 molecule as assessed by hemolytic titration. Recombinant IL-2 enhances the rate of C3 synthesis in a dose-dependent manner reaching maximal stimulation at doses of 200-400 U/ml IL-2. Northern blot analysis demonstrated a 5.2-kb C3 mRNA species present in PTEC that was increased within 24 h of IL-2 treatment. IL-2-induced enhancement of C3 production by PTEC could be neutralized with specific antibodies to IL-2. This study demonstrates that C3 synthesis in PTEC is upregulated by IL-2, the major cytokine produced by activated T cells.

Differential regulation of complement factor H and C3 production in human umbilical vein endothelial cells by IFN-gamma and IL-1.

Human umbilical vein endothelial cells (HUVEC) synthesize and secrete C component factors H and C3. Addition of T cell growth factor to HUVEC enhanced factor H production, and caused a profound increase in C3 production. In the present study, investigations were initiated to characterize the effects of purified rIL-1 and rIFN-gamma on the production of factor H and C3 at the protein and mRNA level. IFN-gamma enhanced factor H production in a dose-dependent fashion and a two-fold increase was observed with an optimal dose of 200 U/ml, whereas IFN-gamma had no effect on C3 production. IL-1 inhibited factor H secretion, but the production of C3 was increased 10-fold at an optimal dose of 500 pg/ml of IL-1. Kinetic experiments demonstrated that addition of IL-1 to HUVEC resulted in an induction of C3 production after more than 24 h, whereas IFN-gamma already had a significant effect on factor H production after 8 h of culture. IL-1 in combination with IFN-gamma had a synergistic effect on C3 production. The effects of IL-1 and IFN-gamma on factor H and C3 production by HUVEC could be blocked by using neutralizing amounts of antibodies specific for these cytokines. Northern blot analysis showed that factor H mRNA expression was enhanced in IFN-gamma-treated HUVEC and C3 mRNA was induced in IL-1-treated HUVEC, indicating that the observed increase of factor H and C3 probably is controlled by enhancement of transcription or stability of the transcript.

Elevated production of polymeric and monomeric IgA1 by the bone marrow in IgA nephropathy.

In bone marrow cultures of 15 patients with primary IgA nephropathy we found significantly (P = 0.02) increased synthesis of both monomeric and polymeric IgA1 compared to 23 controls, by using high performance liquid chromatography (HPLC) fractionation of culture supernatants. The relative contribution of polymeric to total IgA1 produced was not different for the two groups. Two-color immunofluorescence studies of the percentage of bone marrow IgA1 plasma cells able to bind secretory component in vitro showed no difference between patients and controls. In the sera of patients with primary IgA nephropathy the relative contribution of IgA1 polymers to total IgA1 was also similar to controls. These results indicate that in IgA nephropathy, the increased IgA production in the bone marrow is restricted to the IgA1 subclass. The production of both monomeric and polymeric IgA1 is increased in patients during a quiescent phase of the disease.

Biosynthesis of complement factor H by human umbilical vein endothelial cells. Regulation by T cell growth factor and IFN-gamma.

The present studies were initiated to characterize a 150-kDa molecule with inhibitory activity for C3bBb formation, which is present in human umbilical vein endothelial cells (HUVEC). Therefore, human endothelial culture supernatants (HECS) were analyzed for the presence of human complement factor H by ELISA. It was found that H was present in HECS. An immunoblot analysis of affinity purified H from HECS showed that the size of HUVEC H was identical to that of plasma H. The mean production of H by HUVEC of first passage cultures was 40 ng/10(6) cells/day. The synthesis of HUVEC H was fully inhibitable by the addition of cycloheximide to the cultures, suggesting that H is de novo synthesized. Additional evidence for de novo synthesis was obtained by using biosynthetic labeling with [35S] methionine, immunoprecipitation, and SDS-PAGE. It was demonstrated that, indeed, HUVEC produce and secrete factor H. Two forms of the protein were identified, the 150-kDa form and also a 45-kDa form, both forms have been identified in plasma. The functional activity of HUVEC H is identical to that of plasma H. IFN-gamma induced enhanced synthesis of H by HUVEC, whereas it had no effect on C3 synthesis. Supernatant from stimulated PBMC, T cell growth factor, enhanced synthesis of both H and C3. The present studies indicate that H is produced by HU-VEC and that H may function as an inhibitor of complement activation at the endothelial cell level and, thereby, together with molecules like decay accelerating factor and membrane cofactor protein, may influence resistance of endothelial cells to complement mediated damage.

Serum levels and in vitro production of IgA subclasses in patients with primary IgA nephropathy.

Patients with primary IgA nephropathy have deposits of IgA1 in their kidneys, and increased plasma levels of macromolecular IgA1. Total serum IgA concentrations are frequently elevated, but studies on the subclass distribution have been few and conflicting. Several investigators found that production of IgA by peripheral blood lymphocytes in culture is increased. However, the distribution of the IgA subclasses produced has not been studied previously. We studied the serum IgA subclasses in 14 patients with IgA nephropathy, and found a significant (P less than 0.001) increase in IgA1 (3.71 +/- 1.34 mg/ml, mean +/- s.d.) compared with controls (1.77 +/- 1.10 mg/ml). Serum IgA2 was not different in patients and controls. The ratio of serum IgA1 to total IgA was also significantly (P less than 0.001) higher in patients (92.2 +/- 4.9%) than in controls (80.2 +/- 6.6%). Studies of immunoglobulin production by peripheral blood mononuclear cells showed a significant increase in IgA1 synthesis, expressed as a fraction of total IgA synthesis in unstimulated cultures (P less than 0.05) and in PWM stimulated cultures (P less than 0.01). Polymeric IgA and polymeric IgA1 production were not higher in patients than in controls. IgM production in unstimulated cultures was significantly (P less than 0.05) higher in patients than in controls. Together with the observed deposition of exclusively IgA1 in the mesangium, our results indicate that patients with IgA nephropathy preferentially produce antibodies of the IgA1 subclass.

Immunoglobulin synthesis by peripheral blood lymphocytes in patients with ankylosing spondylitis.

Immunoglobulin synthesis by peripheral blood lymphocytes and serum IgA subclasses were investigated in patients with ankylosing spondylitis (AS) with and without accompanying microscopic hematuria, and HLA-B27 positive and negative healthy controls. An increase in serum IgA, restricted to subclass IgA1, was found in patients with AS, especially in those with microscopic hematuria. IgA synthesis was not increased, but a significant shift to subclass IgA1 was found. Our results resemble abnormalities previously noted in primary IgA nephropathy, and further support the pathogenetic role of IgA in AS.

The bone marrow as production site of the IgA deposited in the kidneys of patients with IgA nephropathy.

Patients with primary IgA nephropathy have increased plasma levels of polymeric IgA1 and deposits of IgA1 in their kidneys. The origin of this material is unknown. The production of IgA and its subclasses was investigated in the bone marrow of 14 patients and 19 controls using two colour immunofluorescence and tissue culture. Patients had an increase in the percentage of plasma cells containing IgA (45.8 +/- 7.2 mean +/- s.d.) compared to controls (40.1 +/- 10.5) (P = 0.08). IgA plasma cells containing subclass IgA1 were significantly (P less than 0.01) increased in patients (89.9 +/- 2.7%) compared to controls (84.1 +/- 6.7%). Correspondingly IgA plasma cells containing subclass IgA2 were significantly decreased (P less than 0.01) in patients (7.4 +/- 3.0%) compared to controls (13.5 +/- 5.9%). Production of IgA in bone marrow culture in patients was increased (1,684 +/- 1,151 ng/culture) compared to controls (1,087 +/- 937), but this difference was not significant (P = 0.2). However, in patients the IgA1 subclass contributed significantly (P less than 0.01) more to the IgA synthesis in culture (ratio of IgA1 over IgA: 0.96 +/- 0.02) than in controls (ratio 0.90 +/- 0.06). These findings suggest that the bone marrow may well be the site of long-term overproduction of the IgA1 found in the circulation and mesangial deposits in IgA nephropathy.

Weakly basic impurities in illicit amphetamine.

In this paper the isolation and identification of two pyrimidines, five pyridines, and one pyridone as impurities in illicit amphetamines prepared by the Leuckart synthesis are reported. Isolation was achieved by repeated thin-layer chromatography with various solvent mixtures, while identification was done by both high and low resolution mass spectrometry and 1H and 13C NMR spectroscopy. Some chromatographic data are reported and a quantitative analysis of a reaction mixture and an illicit amphetamine is given.