A chimeric glycoprotein formulated with a combination adjuvant induces protective immunity against both human respiratory syncytial virus and parainfluenza virus type 3.

Human respiratory syncytial virus (RSV) and parainfluenza virus type 3 (PIV3) are major causes of serious lower respiratory tract disease in infants. Currently there is no licensed vaccine against RSV or PIV3. To make an effective bivalent subunit vaccine, a chimeric truncated FRHN protein containing the N-terminal ectodomain of the RSV fusion (F) protein linked to the C-terminal ectodomain of the PIV3 haemagglutinin-neuraminidase (HN) protein was produced in HEK293T cells. Mice, cotton rats and hamsters were immunized intramuscularly (IM) with both RSV F and PIV3 HN (FR+HN) or FRHN, formulated with TriAdj, which consists of poly(I:C), innate defense regulator peptide and poly[di(sodium carboxylatoethylphenoxy)]-phosphazene. Both formulations were immunogenic and elicited full protection from RSV; however, animals vaccinated with FRHN/TriAdj were significantly better protected from PIV3 than animals vaccinated with FR+HN/TriAdj. To develop a potentially more effective subunit vaccine, a chimeric glycoprotein (FRipScHN), encoding the RSV F ectodomain stabilized in the pre-fusion form linked to PIV3 HN was generated. Intramuscular vaccination with FRipScHN/TriAdj induced virus neutralizing antibodies followed by complete protection from RSV and PIV3 replication in the lungs of challenged cotton rats. Furthermore, intranasal vaccination with FRipScHN/TriAdj significantly reduced both RSV and PIV3 replication in cotton rats. Mucosal immunization with FRipScHN/TriAdj also elicited strong antigen-specific mucosal and systemic immune responses in a lamb model. In conclusion, the chimeric FRipScHN protein combined with TriAdj has potential for development of a safe, effective, bivalent vaccine against both RSV and PIV3.

Index coronary angiography use in Manitoba, Canada: a population-level descriptive analysis of First Nations and non-First Nations recipients.

OBJECTIVES: To investigate recipient characteristics and rates of index angiography among First Nations (FN) and non-FN populations in Manitoba, Canada. SETTING: Population-based, secondary analysis of provincial administrative health data. PARTICIPANTS: All adults 18 years or older who received an index angiogram between 2000/2001 and 2008/2009. PRIMARY AND SECONDARY OUTCOME MEASURES: (1) Descriptive statistics for age, sex, income quintile by rural and urban residency and Charlson Comorbidity Index for FN and non-FN recipients. (2) Annual index angiogram rates for FN and non-FN populations and among those rates of 'urgent' angiograms based on acute myocardial infarction (AMI)-related hospitalisations during the previous 7 days. (3) Proportions of people who did not receive an angiogram in the 20 years preceding an ischaemic heart disease (IHD) diagnosis or a cardiovascular death; stratified by age (<65 or ≥65 years old). RESULTS: FN recipients were younger (56.3vs63.8 years; p<0.0001) and had higher Charlson Comorbidity scores (1.32vs0.78; p<0.001). During all years examined, index angiography rates were lower among FN people (2.67vs3.33 per 1000 population per year; p<0.001) with no notable temporal trends. Among the index angiogram recipients, a higher proportion was associated with an AMI-related hospitalisation in the FN group (28.8%vs25.0%; p<0.01) and in both groups rates significantly increased over time. FN people who died from cardiovascular disease or were older (65+years old) diagnosed with IHD were more likely to have received an angiogram in the preceding 20-30 years (17.8%vs12.5%; p<0.01 and 50.9%vs49.5%; p<0.03, respectively). FN people diagnosed with IHD who were under the age of 65 were less likely to have received an angiogram (47.8%vs53.1%; p<0.01) CONCLUSIONS: Index angiogram use differences are suggested between FN and non-FN populations, which may contribute to reported IHD disparities. Investigating factors driving these rates will determine any association between ethnicity and angiography services.

Vaccination with a human parainfluenza virus type 3 chimeric FHN glycoprotein formulated with a combination adjuvant induces protective immunity.

Human parainfluenza virus type 3 (PIV3) is a major cause of lower respiratory disease i.e. bronchitis, bronchiolitis or pneumonia, in infants and young children. Presently there is no licensed vaccine against PIV3. To produce an effective subunit vaccine, a chimeric FHN glycoprotein consisting of the N-terminal ectodomain of the fusion (F) protein linked to the haemagglutinin-neuraminidase (HN) protein without transmembrane domain, and secreted forms of the individual F and HN glycoproteins, were expressed in mammalian cells and purified. Mice and cotton rats were immunized intramuscularly (IM) with FHN or both F and HN proteins (F + HN), formulated with poly(I:C) and an innate defense regulator peptide in polyphosphazene (TriAdj). Significantly higher levels of systemic virus-neutralizing antibodies were observed in mice and cotton rats immunized with FHN/TriAdj when compared to animals immunized with the combination of F and HN proteins (F + HN/TriAdj). As PIV3 is a pneumotropic virus, another goal is to produce an effective mucosal subunit vaccine. Intranasal (IN) administration with FHN/TriAdj resulted in mucosal IgA production in the lung and virus neutralizing antibodies in the sera. After PIV3 challenge no virus was detected in cotton rats immunized with FHN/TriAdj regardless of the route of delivery. Protective immunity against PIV3 was also induced by FHN/TriAdj in hamsters. In conclusion, the FHN protein formulated with TriAdj has potential for development of a safe and effective vaccine against PIV3.

Enhanced immune responses and protection by vaccination with respiratory syncytial virus fusion protein formulated with CpG oligodeoxynucleotide and innate defense regulator peptide in polyphosphazene microparticles.

Although respiratory syncytial virus (RSV) is the leading cause of serious respiratory tract disease in children, to date no RSV vaccine is available. To produce an effective subunit vaccine, a truncated secreted version of the F protein (ΔF) was expressed in mammalian cells, purified and shown to form trimers. The ΔF protein was then formulated with a CpG oligodeoxynucleotide (ODN) and an innate defense regulator (IDR) peptide in polyphosphazene microparticles (ΔF-MP). Mice immunized either intramuscularly (IM) or intranasally (IN) with ΔF-MP developed significantly higher levels of virus-neutralizing antibodies in the sera and lungs, as well as higher numbers of IFN-γ secreting cells than mice immunized with the ΔF protein alone. In contrast, the IM delivered ΔF induced high production of IL-5 while the IN delivered ΔF did not elicit a measurable immune response. After RSV challenge, essentially no virus and no evidence of immunopathology were detected in mice immunized with ΔF-MP regardless of the route of delivery. While the mice immunized IM with ΔF alone also showed reduced virus replication, they developed enhanced levels of pulmonary IgE, IL-4, IL-5, IL-13 and eotaxin, as well as eosinophilia after challenge. The level of protection induced by the ΔF-MP formulation was equivalent after IM and IN delivery. The efficacy and safety of the ΔF-MP formulation was confirmed in cotton rats, which also developed enhanced immune responses and were fully protected from RSV challenge after vaccination with ΔF-MP. In conclusion, formulation of recombinant ΔF with CpG ODN and IDR peptide in polyphosphazene microparticles should be considered for further evaluation as a safe and effective vaccine against RSV.

Effect of high-fat diet on glucose homeostasis and gene expression in glucokinase knockout mice.

AIM: We have generated a heterozygous glucokinase knockout mouse (gk(del/wt)), upon which we investigated the effect of high-fat diet (HFD) with respect to metabolic control and both hepatic and beta-cell gene expression. We also investigated the in vitro efficacy of a glucokinase activator (GKA) on glucose-stimulated insulin secretion (GSIS) in gk(del/wt)mouse islets. METHODS: Male gk(del/wt)and gk(wt/wt)mice were grouped (n = 8-10) at 10 weeks of age and fed HFD or chow diet (CD) for 10 weeks. Multiple parameters including blood glucose, plasma insulin and glucose tolerance were assessed. Further animal groups were used for in vitro GSIS and islet and liver gene expression analysis. RESULTS AND CONCLUSIONS: gk(del/wt)mice showed early-onset persistent hyperglycaemia, raised glycated haemoglobin levels, impaired GSIS and glucose tolerance but no change in plasma cholesterol, non-esterified fatty acids or triglyceride levels. After HFD feeding, insulin levels of gk(del/wt)mice were less than half that of gk(wt/wt)mice, although they were equivalent to gk(wt/wt)mice on CD. While gk(wt/wt)mice maintained moderate hyperglycaemia, gk(del/wt)mice became overtly diabetic, with worsened glucose tolerance. A GKA (GKA50) increased GSIS, at 10 mM glucose, in gk(del/wt)mice to an extent at least as great as that seen in gk(wt/wt)mice on both CD and HFD. gk(del/wt)mice showed only a small number of changes in gene expression compared with gk(wt/wt)mice. We propose the high fat-fed gk(del/wt)mouse as a model of type 2 diabetes and report retained efficacy of a GKA on in vitro GSIS.

Intercellular trafficking of the major tegument protein VP22 of bovine herpesvirus-1 and its application to improve a DNA vaccine.

Intercellular spread of bovine herpesvirus-1 (BHV-1) VP22 was demonstrated in living COS-7 cells transfected with a plasmid expressing VP22-YFP (yellow fluorescence protein) and CFP (cyan fluorescence protein) bicistronically. The intercellular trafficking property of VP22 was localized to the C-terminal portion of the molecule (amino acids 121-258; VP22-C). Plasmids encoding a truncated form of BHV-1 glycoprotein D (tgD) fused to VP22, VP22-C, or the N-terminal portion of VP22 (amino acids 1-120; VP22-N) were constructed. Mice immunized with plasmid encoding tgD-VP22 or tgD-VP22-C developed stronger immune responses when compared to animals immunized with plasmid encoding tgD or tgD fused to tgD-VP22-N.

Infection of newborn piglets with Bordetella pertussis: a new model for pertussis.

Bordetella pertussis is the causative agent of pertussis or whooping cough. This bacterium is a human pathogen that under experimental conditions also infects selected rodents and primates. Here, we show for the first time that newborn piglets can be infected with B. pertussis when it is delivered intrapulmonarily. Infected piglets displayed fever and respiratory symptoms, such as nasal discharge, nonparoxysmal coughing, and breathing difficulties. Eventually, all infected animals developed severe bronchopneumonia, which in some cases was combined with a fibrinous pleuritits. Immunohistochemical staining revealed the presence of large numbers of B. pertussis cells within airways, adhering to the epithelial lining or phagocytosed by macrophages and neutrophils. Viable bacteria were reisolated from bronchoalveolar lavages and lung lesions for more than 10 days postinfection. The systemic presence of pertussis toxin was shown by hypoglycemia, lymphocytosis, and induction of a clustered pattern of CHO cells by serum and bronchoalveolar lavage samples. Thus, a large-animal model for pertussis was developed, which should complement existing rodent models for identifying the immune responses relevant to the design of new vaccines. In particular, this model should help researchers analyze the roles of both maternal and mucosal immunity in disease protection against pertussis and should ultimately assist in the design of new vaccines for early life protection.

Differential display analysis of gene expression during the induction of mucosal immunity.

One approach to understanding the physiologically relevant events during the induction of an immune response is to identify genes that are expressed when the immune system first encounters antigen. Such an investigation requires a naive but fully functional immune system, and the fetal lamb provides these conditions during the last trimester of gestation. 'Intestinal segments,' containing a jejunal Peyer's patch, were surgically prepared in fetal lambs (>120 days gestation) and individual 'intestinal segments' were injected with either culture medium or infectious bovine rotavirus. Peyer's patch tissue was collected 18 h postinfection. Histology and virus culture confirmed that bovine rotavirus had infected the mucosal epithelium. RNA was extracted from jejunal Peyer's patch tissue and mRNA differential display was used to identify genes expressed following rotavirus infection. Ten cDNAs were identified by differential display and these cDNAs were isolated, cloned, and sequenced. One of the cDNAs sequenced, displayed homology to the gene encoding the sperm surface protein Sp17. Differential expression of this gene in antigen-exposed jejunal Peyer's patches was confirmed by Northern blot and RT-PCR. The complete sequence for sheep Sp17 mRNA was obtained from a lambda cDNA library, prepared from the jejunal Peyer's patch of a young lamb. Sp17 expression was detected by RT-PCR in a variety of mucosa-associated lymphoid tissues but not in primary or other secondary lymphoid tissues. Thus, the fetal lamb model may be appropriate for identifying genes relevant to mucosal immunity.

Superficial urothelial (umbrella) cells. A potential cause of abnormal DNA ploidy results in urine specimens.

OBJECTIVE: To determine the DNA ploidy distribution in urothelial superficial (umbrella) cells and to assess the value of the image analysis operator's experience. STUDY DESIGN: DNA ploidy was assessed in 12 cytologically negative bladder washes stained with Feulgen stain. All 12 cases were evaluated independently by three operators with different levels of cytopathology experience and different goals. Operator 1 (experienced) selected only nuclei of urothelial cells, avoiding nuclei of superficial cells; operator 2 (experienced) selected only nuclei of superficial cells; operator 3 (inexperienced) selected the largest and most-atypical-looking nuclei. Each operator measured a total of 100 nuclei per case. RESULTS: Operator 1 found all cases to be diploid (97% of nuclei on average). Operators 2 and 3 showed a wide range of results. Almost half the nuclei (47%) analyzed by operator 2 were in the diploid region, a third (35%) were in the tetraploid region, and the remaining (18%) ones had a DNA index (DI) in the range of 1.2-1.8 or > 2.5. Operator 3 obtained the most abnormal results. Only 9% of the nuclei were diploid, while 37% were in the tetraploid region, 18% were in the hyperploid region, and 35% had a DI in the range of 1.2-1.8. Differences among results obtained by each operator were statistically significant. CONCLUSION: The nuclei of superficial (umbrella) cells often have abnormal DNA content, which may cause abnormal DNA ploidy results in cytomorphologically normal bladder washes. Consequently, the nuclei of superficial cells should be avoided in the evaluation of urine samples. DNA analysis of urine specimens requires selection of nuclei only of deep urothelial cells by an experienced operator.

ZD9583, an orally effective thromboxane A2 synthase inhibitor and receptor antagonist with a sustained duration of action in rat and dog.

The thromboxane A2 (TXA2) synthase inhibitory activity and the TXA2 receptor (TP-receptor) blocking action of ZD9583 ((4Z)-6-[(2S,4S,5R)-2-(1-[2-cyano-4-methylphenoxy]-1-methylethyl) -4-(3-pyridyl)-1,3-dioxan-5-yl]hex-4-enoic acid) has been evaluated in-vitro by use of whole blood and platelets from man, and ex-vivo by use of platelets and whole blood from rats and dogs. ZD9583 caused concentration-dependent inhibition of human platelet microsomal TXA2 production with an IC50 of 0.017 +/- 0.003 microM; this inhibition was associated with an increase in prostaglandin E2 (PGE2) and prostaglandin F2 alpha (PGF2 alpha) formation. ZD9583 also inhibited collagen-stimulated TXA2 synthesis in whole blood from man, rat and dog giving IC50 values of 0.027 +/- 0.005, 0.02 +/- 0.006 and 0.013 +/- 0.01 microM, respectively. The drug did not modify platelet cyclooxygenase activity as inhibition of thromboxane B2 (TXB2) formation was associated with a concomitant increased synthesis of prostaglandin D2 (PGD2), PGE2 and PGF2 alpha. ZD9583 had little effect on cultured human umbilical vein endothelial cell prostacyclin synthase giving an IC50 of 24.2 +/- 4.9 microM. In-vitro ZD9583 caused concentration-dependent inhibition of U46619-induced aggregation responses of platelets from man, rat and dog, yielding apparent log A2 values of 8.7 +/- 0.12, 8.8 +/- 0.2 and 9.3 +/- 0.2, respectively. The drug was selective; at concentrations up to 100 microM it did not affect 5-hydroxytryptamine or the primary phases of adenosine diphosphate and adrenaline-induced aggregation. ZD9583 (100 microM) did not, furthermore, modify the platelet inhibitory effects of PGD2, prostaglandin E1 (PGE1) and prostacyclin. Oral administration of ZD9583 (3-10 mg kg-1) to both rats and dogs caused dose-dependant inhibition of collagen-stimulated TXA2 production ex-vivo which persisted for up to 12 h. The drug also caused profound TXA2 receptor blockade in both species for in excess of 12-h after an oral dose of 3 mg kg-1. ZD9583 (3 mg kg-1, p.o.), when administered to dogs over a five-day period at 12 h intervals, did not cause either tachyphylaxis or an accumulation of effect. We conclude that ZD9583 is a potent, selective, orally active thromboxane synthase inhibitor and TXA2 receptor antagonist.

The relationship between blood flow and diameter in the iliac artery of the anaesthetized dog: the role of endothelium-derived relaxing factor and shear stress.

The quantitative relationship between, increase in blood flow and arterial diameter was determined in an anaesthetized dog preparation (pentobarbitone, induction 30 mg kg-1 i.v., maintenance 3 mg kg-1 i.v. every 30 min). Changes in external iliac artery diameter were measured using piezoelectric ultrasound transducers capable of measuring diameters within the range of 2-20 mm with a resolution of +/- 0.005 mm. The diameter of the artery was measured at two sites, at one of which the endothelium was damaged using a balloon angioplasty catheter. Increases in blood flow were brought about by a combination of vasodilatation and cardiac stimulation (intra-arterial administration of acetylcholine, downstream to the sites of diameter measurement, and electrical stimulation of the left ansa subclavia), thereby preventing large changes in blood pressure. The effects of both transient and maintained increases in blood flow on mean arterial diameter in the section of artery with intact endothelium were measured. Transient increases in mean flow from 147 +/- 0.21 to 611 +/- 80.0 ml min-1 caused increases in diameter of 0.12 +/- 0.02 mm from a control of 5.42 +/- 0.19 mm. The mean delay between maximum flow and maximum diameter was 24.51 +/- 1.1 s and the half-time for the return to control diameter was 82.0 +/- 9.6 s, compared with 12.1 +/- 1.5 s for the return to control flow. Maintained (3-4 min) increases in mean blood flow (from 104.7 +/- 15.1 to 694.7 +/- 135.1 ml min-1) produced larger increases in diameter of 0.48 +/- 0.30 mm from a control diameter of 4.89 +/- 0.12 mm. These changes in diameter were abolished by N omega-nitro-L-arginine methyl ester (L-NAME. 10-100 mg kg-1 i.v.). In the section of artery with damaged endothelium, changes in diameter were relatively small and associated with small changes in blood pressure. This effect of a nearly 7-fold increase in flow on arterial diameter is dependent upon the integrity of the endothelium and the release of endothelium-derived relaxing factor and causes a 29% reduction in calculated boundary wall shear stress.

ZD1542, a potent thromboxane A2 synthase inhibitor and receptor antagonist in vitro.

1. The thromboxane A2 synthase (TXS) inhibitory activity and the thromboxane A2 (TP)-receptor blocking action of ZD1542 (4(Z)-6-[2S,4S,5R)-2-[1-methyl-1-(2-nitro-4-tolyloxy)ethyl]-4-(3- pyridyl)-1,3-dioxan-5-yl]hex-4-enoic acid) has been evaluated in vitro on platelets and whole blood from a range of species including man. Antagonist activity has also been investigated in vascular and pulmonary smooth muscle preparations in vitro. 2. ZD1542 caused concentration-dependent inhibition of human platelet microsomal thromboxane B2 (TXB2) production in vitro (IC50 = 0.016 microM); this inhibition was associated with an increase in prostaglandin E2 (PGE2) and PGF2 alpha formation. 3. ZD1542 also inhibited collagen-stimulated TXS in human, rat and dog whole blood giving IC50 values of 0.018, 0.009 and 0.049 microM respectively. The drug did not modify platelet cyclo-oxygenase activity as inhibition of TXB2 formation was associated with a concomitant increase in the levels of PGD2, PGE2 and PGF2 alpha. ZD1542 had little if any effect against cultured human umbilical vein endothelial cell (HUVEC) cyclo-oxygenase (IC50 > 100 microM) and prostacyclin (PGI2) synthase (IC50 = 18.0 +/- 8.6 microM). 4. ZD1542 caused concentration-dependent inhibition of U46619-induced aggregation responses of human, rat and dog platelets yielding apparent pA2 values of 8.3, 8.5 and 9.1 respectively. The drug was selective as, at concentrations up to 100 microM, it did not modify 5-hydroxytryptamine (5-HT) or the primary phases of adenosine diphosphate (ADP) and adrenaline-induced aggregation. Furthermore, ZD1542 (100 microM) modified only weakly the platelet effects of PGD2, PGE1 and PGI2. 5. ZD1542 also caused concentration-dependent inhibition of U46619-mediated contractions of rat thoracic aorta, guinea-pig trachea and lung parenchyma preparations giving apparent pA2 values of 8.6,8.3 and 8.5 respectively. At concentrations approaching three orders of magnitude greater than those required to block U46619-mediated contractions, the drug did not affect the actions of non-prostanoid agonists or exhibit agonist activity in any of the smooth muscle preparations employed; neither did it interact at EP- or FP-receptors.6. In conclusion, the present study demonstrates that ZD1542 is a drug that exhibits both potent,selective TXS inhibition and TXA2 receptor antagonism.

Stimulation of secretory antibodies against Bordetella pertussis antigens in the lungs of mice after oral or intranasal administration of liposome-incorporated cell-surface antigens.

Lipopolysaccharide (LPS) and outer membrane protein (OMP) preparations of Bordetella pertussis were incorporated into multilamellar liposomes composed of soya bean-derived phospholipids which were then used for oral and intranasal immunization of mice. Specific antibody responses of animals immunized by either route were measured in lung washes. A specific IgA response to LPS was detected after immunization with the OMP-containing liposomes but not with the LPS-containing liposomes, indicating adjuvant activity of the proteins. The OMP-containing liposomes were significantly more effective in inducing immune responses than the OMP preparation alone. Responses were highest when mice were given a booster 30 days after primary immunization. Maximum responses occurred 20 days after the booster but specific antibody was still detected 75 days after the secondary immunization. These results suggest that this liposome antigen delivery system has potential in stimulating secretory antibody responses which may be helpful in protecting against infection from B. pertussis.

Antibody responses in the lungs of mice following oral immunization with Salmonella typhimurium aroA and invasive Escherichia coli strains expressing the filamentous hemagglutinin of Bordetella pertussis.

The filamentous hemagglutinin (FHA) of Bordetella pertussis was expressed in the attenuated aroA mutant of Salmonella typhimurium, SL3261, and in a strain of Escherichia coli harboring Shigella flexneri plasmid pWR110, which encodes bacterial invasiveness for epithelial cells. Expression of FHA in these strains did not interfere with their ability to invade Henle cells. Immunoglobulins A and G specific for FHA were detected in lung washes of mice following oral immunization with the live recombinant organisms; antibody levels were significantly higher than those in mice immunized with killed bacteria administered orally or intraperitoneally. Live oral vaccines carrying protective antigens of B. pertussis may be an important alternative to new-generation component vaccines against whooping cough.

Engineering upstream transcriptional and translational signals of Bordetella pertussis serotype 2 fimbrial subunit protein for efficient expression in Escherichia coli: in vitro autoassembly of the expressed product into filamentous structures.

Escherichia coli containing a cloned gene encoding the Bordetella pertussis serotype 2 fimbrial subunit failed to produce detectable levels of the gene product in whole-cell extracts. To engineer plasmids capable of directing the expression in E. coli of high levels of this product, both as a pre-protein and as a methionylated mature form the upstream signals of the fimbrial subunit gene were replaced by the lambda P(L) and P(R) promoters and the E. coli atpE translational initiation region. These constructs did not result in the expression of fimbrial subunit at detectable levels in several E. coli strains including DH5. However, they did in E. coli CAG629, which is lon protease and heat shock protein deficient. Both pre-protein and methionylated mature protein had molecular weights of 25.0 kD, which indicated that correct processing of the leader sequence had occurred and thus that it was transposed across the inner membrane. Electron microscopic investigation of the cell surface of E. coli cells expressing either form of the fimbrial gene failed to detect the presence of filamentous structures. The methionylated mature form of the recombinant fimbrial subunit was purified to apparent homogeneity. After dialysis in appropriate conditions it was seen to autoassemble into protein polymers. Antibodies raised against polymerized recombinant subunit reacted weakly with whole B. pertussis serotype 2 fimbriae in immunodot blot assays. However, such antibodies reacted in Western blots equally well with the recombinant and wild-type form of the fimbrial subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

Bordetella pertussis adenylate cyclase: purification and characterization of the toxic form of the enzyme.

Bordetella pertussis produces a calmodulin-sensitive adenylate cyclase (AC) which is an essential virulence factor in mammalian pertussis. Here we report the purification and characterization of the toxic form of the enzyme, which penetrates eukaryotic cells and generates high levels of intracellular cAMP. This form was purified from an extract of B.pertussis strain carrying a recombinant plasmid which over-produced both enzymatic and toxic activities of the enzyme. Western blot analysis of the extract using anti-B.pertussis AC antibodies detected only one protein of 200 kd. However, gel filtration of the extract resolved two peaks of enzymatic activity. The first peak of aggregated material contained greater than 70% of the total enzymatic activity, and the second peak contained the majority of the toxic activity. Purification of the enzyme from both peaks yielded proteins of 200 kd, with similar biochemical and immunological properties. Yet only the enzyme purified from the second peak could penetrate human lymphocyte and catalyse the formation of intracellular cAMP. B.pertussis AC gene expressed in Escherichia coli produced a calmodulin-dependent enzyme of 200 kd, which lacked lymphocyte penetration capacity. It is proposed that a post-translational modification that occurs in B.pertussis but not in E.coli confers upon the 200 kd protein of B.pertussis AC the toxic properties.

Cloning of the adenylate cyclase genetic determinant of Bordetella pertussis and its expression in Escherichia coli and B. pertussis.

A recombinant plasmid, pRMB1, identified from a gene library of B. pertussis, restored adenylate cyclase (AC) and haemolysin (HLY) activities to B. pertussis BP348 (a Tn5-insertion mutant deficient in both these activities). B. pertussis BP348 was considerably less virulent than wild type strains of B. pertussis when 3-week-old mice were challenged intranasally; possession of pRMB1 restored virulence. Neither AC nor HLY activities were expressed in E. coli harbouring pRMB1. However, expression of calmodulin-responsive AC was obtained in E. coli when restriction fragments of pRMB1 were subcloned into other vectors; expression depended on the lac and tac promoters of these vectors. The enzyme was not readily solubilized from urea extracts of E. coli and required sonication for efficient release. One plasmid conferred a specific AC enzymic activity to E. coli which was greater than that for wild type B. pertussis strains. Unlike extracts of B. pertussis, extracts from E. coli expressing enzymic AC activity, did not elevate cAMP levels in S49 lymphoma cells. A second plasmid, pRMB2, was identified from the gene library, which contained a trans-acting regulatory determinant required for expression of AC, HLY and other virulence-associated factors in B. pertussis.

Complementation of mutations in Escherichia coli and Bordetella pertussis by B. pertussis DNA cloned in a broad-host-range cosmid vector.

A gene library of Bordetella pertussis DNA was constructed in Escherichia coli using the broad-host-range cosmid vector pLAFR1. The average insert size was 24.9 kb. From 500 members of the gene library, clones were identified which complemented trpE, glnA and Thr- mutations in E. coli but none which complemented trpD, trpC, trpB, trpA, proA or Leu- mutations. Four clones were identified which complemented trpE in E. coli. Anthranilate synthase activity was detected in a trpE strain only when it harboured a plasmid from one of these clones; activity was repressed when tryptophan was included in the growth medium. Two clones were identified which complemented glnA of E. coli. A recombinant plasmid from one of these clones also restored some of the nitrogen acquisition functions of glnG and glnL in E. coli. Expression of several B. pertussis virulence-associated products (haemolysin, heat-labile toxin, adenylate cyclase, filamentous haemagglutinin, and the cell-envelope polypeptide of Mr 30,000) was not detected in 500 independent clones. However, by transferring the recombinant plasmids to a mutant of B. pertussis deficient in haemolysin and adenylate cyclase, a plasmid was identified which restored both these activities.

The effect of growth conditions on adenylate cyclase activity and virulence-related properties of Bordetella pertussis.

Growth of Bordetella pertussis in Stainer & Scholte medium in which the NaCl had been replaced by one of several inorganic or organic salts resulted in a large decrease in adenylate cyclase activity, histamine-sensitizing activity and in the amounts of two cell-envelope polypeptides of Mr 28000 and 30000. Although some variation between strains was observed, there was never a case where one of these properties was lost independently of the others. Cultures in which these properties were lost had decreased amounts of extracellular cAMP when compared to NaCl-grown cultures. Adenylate cyclase activity was detected in three locations of B. pertussis cultures (extracellular, extracytoplasmic but cell-associated, and cytoplasmic). After growth in medium containing high concentrations of MgSO4, enzyme activity was decreased to a similar extent in all three locations.

Adenylate cyclase activity during phenotypic variation of Bordetella pertussis.

During MgSO4-induced modulation of Bordetella pertussis, adenylate cyclase activity, histamine-sensitizing activity (HSA) and the major cell-envelope polypeptides with Mr 28000 and 30000 (X polypeptides) were lost synchronously at a rate which could be accounted for by a simple growth-dilution effect. MgSO4 and other compounds which induced the above phenotypic change caused little inhibition of adenylate cyclase activity. Nicotinic acid was the sole exception and at 4.1 mM-caused 60% inhibition of activity. Lysates of modulated cells, mixed with lysates of unmodulated cells, had no effect on either adenylate cyclase activity or HSA. Protein synthesis was a prerequisite for MgSO4-induced modulation and also for the reversal of this process. Exogenous cAMP and dibutyryl cAMP (5 mM) had no counteracting effect on MgSO4- or nicotinic acid-induced modulation. The concentration of MgSO4 required to induce loss of the X polypeptides (10 to 11 mM) was not altered by promoting adenylate cyclase activity by including an activator in the growth medium. In one culture containing 10 mM-MgSO4 and activator, partial loss of the X polypeptides occurred and yet the extracellular cAMP concentration was twice that of cultures without activator and where full expression of the X polypeptides occurred. [3H]cAMP-binding activity was detected in cell extracts of several strains of B. pertussis, but antiserum against purified Escherichia coli catabolite repressor protein gave no reaction with B. pertussis cell extracts. Respiration rates with amino acids were similar for modulated and unmodulated variants and an avirulent strain of B. pertussis. These results are discussed in relation to a possible causal role for adenylate cyclase in modulation of B. pertussis.