Molecular and Cellular Mechanism of Cholera and Pertussis Toxin

CBMS 875
42530679 : Sreedevi Nandakumar

Introduction
Vibrio cholerae, a gram negative, straight or curved rod shaped bacterium with a single polar flagellum which belongs to the family of enterobacteriacea, is responsible for the disease called cholera. It affects the human intestinal system and causes severe diarrhoea and dehydration [1]. Vibrio cholerae releases cholera enterotoxin which is responsible for these symptoms.

Cholera Enterotoxin
Robert Koch in1884, suggested that Vibrio cholerae produces an enterotoxin a special poison that acts on the intestinal epithelium of human beings[2]. The basic properties of cholera toxin its structure and the mode of action were discovered by the scientists after the purification of the cholera toxin were made possible by Finkelstein and LoSpalluto. The cholera enterotoxin belongs to AB5 toxin family, which has a catalytic A subunit and five identical B subunits [3]. The five identical B subunits join together to form a pentamer via interactions between the sheets of each monomer; a pore of 1.1 -1.5 nm is present at the centre of the pentamer. a. Disruption of vital host functions by its specific enzymatic function ,is the major role of A subunit and the pentameric B subunit is responsible for the binding of the toxin to specific glycan receptors on the intestinal epithelial surface[3]. Catalytic A subunit is a single polypeptide chain that consists of two domains (A1 and A2) that are linked together by a disulphide bond. The toxicity to the host cell is due to A1 domain and the C-terminal end of A2 peptide binds to the B pentamer at the central pore and the N-terminal end is an helix interacts with the A1 domain by penetrating the central pore of B subunit. Thus A2 domain non covalently anchors A and B subunits together to form the holotoxin (Fig 1). Fig 1: Crystallographic Image of AB5

toxin[3] Intrachain disulphide bond is also present within A and B subunits and the formation of this bond is catalysed by the enzyme disulphide isomerase. Cholera toxin B (CTB) subunit when linked with glutamic acid decarboxylase (GAD) helps in the suppression of function of the dentritic cells and its maturation. This property of CTB-GAD fusion protein is utilised in the auto-antigen therapy against type 1 diabetes mellitus, which is an autoimmune disease. Autoimmune destruction of beta cells of the islets of Langerhans of the pancreas which produces insulin, results in an autoimmune disease called Type 1 Diabetes mellitus (juvenile diabetes, IDDM,). An increase in the amount of glucose in the blood (hyperglycemia) and urine takes place due to the lack of insulin. Initiation of type 1 diabetes mellitus is induced by the action of antigen presenting cells called dentritic cells. Development of antigen specific T lymphocytes will be stimulated by the recognition of the foreign antigens or self-antigens by the dentritic cells [4]. Dentritic cells when recognises a self-antigen induces an immune tolerance response, but in case of type 1 diabetes mellitus, the dentritic cells fail to recognise -cell autoantigens as self-antigens. Immune tolerance is the ability of the cells in the immune response to ignore self while reacting to non self.

The polarization and development of nave T helper cells or natural killer cell (Th0) into Th1 and Th2 is done by dentritic cells. Th1 helper T cells are responsible for the immune reactions against antigens and Th2 helper cells are responsible for the immune suppression in the body. In type 1 diabetes dentritic cells stimulate the Th1 autoreactive lymphocytes (CD4+ and CD8+) and they penetrate the pancreas. The CD4 + and CD8+ cells also helps to stimulate the secretion of interferon (INF ) and interleukin 2 (IL2) which belongs to the class of cytokines. Cytotoxic T lymphocytes (CTL) production is also stimulated by CD4+ and CD8+ cells. CTL releases cytotoxins (perforin and granulysin) which helps to make pores in the plasma membrane of the -cells of islet of Langerhans thus allowing ions and water to flow into the -cells and lyse the cells. CTL also release serine protease (granzyme) which enters through the pores and induces apoptosis of the -cells. Activation of bactericidal activities of macrophages is stimulated by INF and it also induces B cells to produce opsonizing antibodies thus creating cell mediated immunity. The cytokines also stimulate the secretion of inflammatory cytokines interleukin1 (IL1 ), tumour necrosis factor (TNF ) and TNF which are involved in the acute phase reaction of the systemic inflammation. This will result in the inflammation of Islet of Langerhans of the pancreas leading to a condition called insulitis .Destruction of -cells which produces insulin, results in the continuous decrease in the insulin production and also an increase in the blood glucose level (hyperglycemia).

Antigen Specific Immunotherapy

Islet autoantigens (insulin) when delivered orally in small amounts stimulates the dentritic cells to secrete anti-inflammatory cytokine interleukin 10 (IL10) which produces an antigen specific therapeutic effect; where as in Type 1 diabetes dentritic cells produce large amount of interleukin 12 which further stimulates Th0 cells to develop into autoreactive Th1 cells. IL10 excites nave Th0 cells to undergo morphogenesis into anti-inflammatory CD4+ cells which in turn produces IL10 and inhibits the development of autoreactive Th1 cells and it decreases the chance of insulitis development. In this case Th0 cells matures to T regulatory cells , which can prevent activation of dentritic cells, Th1 , Th2, and cytotoxic T lymphocytes thus preventing the insulitis and helps to continue insulin secretion. Thus antigen specific immune therapy helps to maintain the immunological homeostasis ( Fig 2)[5].

Fig 2: Antigen Specific Immunotherapy[5]

But the oral delivery of auto antigens given rise to only partial suppression of symptoms of juvenile diabetes in experimental animals and it also required repeated administration of autoantigens like insulin and glutamic acid decarboxylase(GAD)over for a long period of time. Adjuvants can acts as an immunological agent that helps to enhance the immune response to the supplied antigen in a drug or vaccine. Cholera enterotoxin B subunit (CTB) acts as an adjuvant which helps to stimulate the induction of immunological tolerance in type 1 diabetes when the B subunit is conjugated physically with autoantigens like insulin and GAD.

B subunit of cholera enterotoxin binds specifically to GM1 (Galb133GalNAcb1 (NeuAca233)3Glc-ceramide) ganglioside receptor which is present on the plasma membrane of epidermal cells. The holotoxin produced by the Vibrio cholerae enters the epidermal cells with the help of this binding of B subunit to the epidermal cells. This property of the B subunit of cholera enterotoxin is exploited in the immunological therapy so that the autoantigens linked to the B subunit easily binds to the epidermal cells and increases the chance of developing peripheral immunological tolerance. Dentritic cell interactions with the CTB- autoantigen fusion proteins were studied to investigate the mechanism of CTB autoantigen interactions that resulted in the partial suppression of type 1 diabetes. Although CTB-autoantigen fusion proteins are widely accepted as an immune suppressor of type1 diabetes, in some cases CTB-autoantigens stimulate autoimmunity rather than suppressing it. Thus this article not only deals with identifying the mechanism of CTBGAD autoantigen fusion protein and its interactions with dentritic cells but also help to develop a combinatorial CTB-GAD/ insulin protein therapy which helps to improve the diabetes suppression.

Pertussis toxin
Whooping cough or pertussis occurs due an infection in the upper respiratory tract of human beings caused by a bacterium called Bordetella pertussis. It is a gram negative, coccobacillus and aerobic bacterium. Its a serious disease and if untreated it may result in the death of the patient. The disease is highly contagious i.e. it easily spreads from one person to another through contaminated air [6]. Tiny droplets of bacteria easily spread in the air when a person with pertussis coughs or sneezes. The infection usually persists for six weeks. Bordetella pertussis can infect people of any age group but the disease was common among the children and infants before the development and extensive use of vaccine [7]. But the recent surveys suggest that, Bordetella pertussis can cause infection transiently among the vaccinated individuals and the individuals act as a carrier for the disease without even knowing that they are affected. The pertussis that usually develops among the vaccinated population will be in a mild form making it very difficult to diagnose. When an unvaccinated or immune-compromised individual comes in contact with the person, who is a carrier of the pertussis, they are more likely to get the disease. This ability of the bacterium to circulate among the vaccinated individuals has not been well studied even though the inefficiency of the vaccines was known clinically for many years. Although current vaccines against whooping cough are modified to induce strong serum antibody response, these vaccines cannot completely eliminate the transient infection caused by the bacterium. Many individuals among the vaccinated population still tests positive for the infection. Studies have found that B cells are necessary for the complete clearance of the Bordetella pertussis from the upper respiratory tract of patients having the infection. This process, the antibody mediated clearance does not occur rapidly but it takes place only after 7 days of infection even though the serum antibodies are supplied to the experimental mouse models at the time of inoculation with Bordetella pertussis. Thus for 7 days the bacteria will be viable in the upper respiratory tract and can spread the infection and also can intensify the existing infection. Both B cells and CD4 + T cells are required for the specific antibody production and immunity against Bordetella pertussis[8]. Mouse models were used to study the immune response to B. pertussis. Adult mice that are genetically incapable of producing B cells when infected with aerosols of B pertussis showed a persistent infection. It has also been found out that CD4 + cells stimulates the secretion of interleukin 2 (IL2) and interferon (INF ) and Th1 like cells helped in the clearance of bacteria in the absence of antibodies. The capability to resist the antibody mediated clearance of bacteria during the first seven days of infection is found only in the Bordetella pertussis and not in Bordetella bronchiseptica which is believed to be the ancestor of the former. Even though these species are closely related they do not show similarity in their response to the antibody mediated clearance. Bordetella bronchiseptica can be completely cleared from the upper respiratory tract within three days after the transfer of serum antibodies [9]. The antibody mediated clearance of Bordetella bronchiseptica requires neutrophils to phagocytose the entire bacterial cell. The neutrophil recruitment is induced by Toll like receptor 4(TLR4) and phagocytosis is done by FC receptors (FCI) or CR3. Pertussis toxin (PTx) like cholera enterotoxin belongs to the family of AB5 toxins. The structure and function of the pertussis toxin is similar to that of cholera toxin which contains catalytic A subunit and a pentameric B subunit. The pertussis toxin is produced only by Bordetella pertussis and not by Bordetella bronchiseptica, interferes with the chemokine receptors by inhibiting the pathway for G protein signalling. Pertussis toxin was also known to prevent the chemical induced

recruitment of neutrophils, macrophages and lymphocytes. The exact role of pertussis toxin is not clearly understood even though we know that the pathogenicity of Bordetella pertussis is the result of pertussis toxin. This is understood by the addition of inactivated pertussis toxin in the preparations of vaccine which resulted in the production of antibodies against PTx .The antibody mediated clearance of Bordetella pertussis is similar to that of its ancestor Bordetella bronchiseptica which require both neutrophils and Fc receptors. The difference in their responses to the antibody mediated clearance is the late elimination of B pertussis along with late recruitment of neutrophils to the cells. The late recruitment of the neutrophils will delay the immune response and hence delay the phagocytosis of the bacterium. This delayed response in B pertussis may be caused due to the presence of pertussis toxin, thus enhancing the life period of B pertussis in the immune hosts.

Cholera toxin B subunit linked to glutamic acid decarboxylase suppresses Dendritic cell maturation and function
Objective:
The main objective of the above mentioned article was to study the efficiency of adjuvant CTB (cholera toxin B subunit) linked with GAD (glutamic acid decarboxylase) acting as fusion proteins in the induction of immunological tolerance and in the suppression of autoimmunity, which will significantly reduce or block the action of dentritic cells which destroys the cells of islets of Langerhans causing type 1 diabetes mellitus. They also wanted to know whether they can use combinatorial CTB-GAD fusion proteins therapy for the enhancement of type 1 diabetes suppression. For this they have to 1. To establish and identify the mechanisms of dentritic cell interactions and the cholera enterotoxin B subunit glutamic acid decarboxylase (CTB-GAD).

Materials and Methods Used: Transformation of E.coli to produce CTB-GAD fusion proteins and purification of the Fusion proteins
1) Ecoli pRSET expression vector is used to encode the cDNA fragment of 5kDa of GAD linked to the Cterminal end of 309bp of cholera toxin B subunit (CTB). The expression vector used also consists of a T7 promoter from a bacteriophage; this is to generate high expression levels of the genes and also helps in the expression of a 6-histidine amino acid tag. E coli BL21 (DE 3)p Lys S producer strain is used in which the expression vectors are introduced. The expression vectors are introduced by electroporation. Purification of the gene products were done by the metal chelation chromatography. 250 ml Luria Broth medium with an addition of ampicillin (100mg/ml)is used to grow the transformed E coli B21 strain by continuous shaking at 370 C. A further addition of 90mg isopropyl- - D- 1-thiogalacto- pyranoside( IPTG) stimulated the protein production to the bacterial culture. After 6 hrs of growth, the bacterial culture was centrifuged at 5000rpm for 10 minutes and 40 C. The pellet obtained from the centrifugation is then resuspended in 10 mM of HEPES buffer that contains 100mM of imidazole. Cells are disrupted using sonicator and isolated for purification. Electrophoretic mobility analysis in 12% polyacrylamide gel is used to determine the purity of the fusion proteins. Immunoblot analysis using an anti polyhistidine primary antibody were used to confirm if the proteins are produced or not.

2) 3) 4) 5) 6) 7) 8) 9)

Monocytes derived dentritic cells from human cord blood is isolated and cultured
1) 2) 3) 4) 5) Freshly collected umbilical cord blood is the source of monocyte derived dentritic cells and is separated from red blood cells and platelets by gradient centrifugation. Incubation of this fraction at 40 C for 10 minutes with anti-CD4 PE (phycoerythrin ) followed by incubation at 40 C for 15 minutes with anti-PE micro beads. Cells are then separated using MACS column. RPMI 1640 culture medium is used to culture the isolated monocytes. The percentage of monocyte derived immature dentritic cells (M-iDC) was determined by flow cytometry.

Assay and phenotyping of DC maturation

1) 2) 3) Addition of CTB (10 g/ml), GAD35 (10 g/ml),CTB-GAD fusion proteins, phorbol myristate acetate and ionomycin stimulates the immature dentritic cells. It is followed by the incubation at 370C for 48 hrs. Surface markers expressed after the incubation (CD14, CD11c and HLA-DR) was determined by flow cytometry. Population of CD14-, HLA-DR+, CD11c+ was determined for the expression of activation markers like CD86, CD80, CD83 and Cd40.

Analysis by Flow cytometry

1) 2) 3) Antibodies conjugated to Phycoerythrin, Fluorescein isothiocyanate, allophycocyanin and peridinine chlorophyll protein complex were used to stain DC surface markers. The viability of the cells is measured by 7aminoactinomycin (7-AAD). Flow cytometric analysis of the cells was done after the incubation with 7-AAD.

Cytometric bead assay used to analyse the secreted cytokines

1) 2) 3) 4) 5) Cytokine based array kit ( CPA) is used to analyse the concentration of IL-6,IL10 and ILR/23 p 40 50l of 5 cytokine standards were incubated with capture antibodies in a dark room for 1 hr. After incubation 50l of phycoerythrin conjugated Abs were added to the samples and incubated again for 2 hrs. BD FACS calibur flow cytometer used to analyse the beads after washing away the impurities. FCAP array software is used to analyse the data.

Results The expression of CTB-GAD35 fusion proteins in E Coli

1) 2) Both monomeric and multimeric forms of CTB-GAD35 fusion proteins were identified. Immuno blot analysis confirmed this identification using anti polyhistidine primary antibody.

Monocyte derived dentritic cell activation is suppressed by CTB-GAD35

1) 2) 3) Surface expression of co-stimulating molecules CD86, CD83, CD80 and CD40 of immature dentritic cells were measured under the influence of CTB, GAD35, CTB-GAD35 and LPS Dentritic cell differentiation from monocytes is assessed using the expression of CD14-HLA-DR+CD11C+ surface markers, showed DC maturation. Stimulation of the culture with CTB, GAD35 and CTB-GAD35 fusion proteins followed by flow cytometry revealed that the expression of co stimulatory molecules is low in CTB-GAD35 when compared to CTB, GAD 35 and LPS alone.

PMA and ionomycin induced DC surface co-stimulatory molecule expression is down regulated by CTB-GAD35
1) PMA and ionomycin helps in the up regulation of CD86, CD83,and CD80,but CTB-GAD35 reduces the action of PMA and ionomycin.

Cytokine secretion of dentritic cells are also supressed by CTB-GAD35

1) When incubated with CTB-GAD35; the dentritic cells showed decrease in the production of inflammatory cytokines (IL-12, IL-6) and also increased secretion of anti-inflammatory cytokine IL-10.

Discussion
NOD mice, 15 weeks after the birth can prevent the onset of diabetes using CTS-insulin fusion protein.IN NOD mice chance of development of insulitis and autoimmune diabetes is decreased wit CTB auto antigen fusion proteins. Reduced levels of hyperglycemia and inflammation of -cells of islet of Langerhans in comparison with control animals were revealed with the help of experiments. Complete suppression of the early onset of diabetes is possible in NOD mice by using DNA vaccine therapy which consists of genes that codes for both CTB-GAD35 and IL-10.The anti-inflammatory interleukin10 formation helped to suppress the synthesis of pro-inflammatory cytokines.IL10 suppresses the synthesis of IL12 and inhibits dentritic cell maturation.IL-12 is responsible for the T cell development into TH1 cells that resulted in the secretion of inflammatory cytokines.CTB-GAD35 fusion proteins stimulates dentritic cells to synthesise high levels of IL-10.Cholera toxin B subunit when conjugated with autoantigens will increase the efficient of autoantigens and helps in the increased suppression of diabetes autoimmunity.

Pertussis toxin inhibits neutrophil recruitment to delay antibody-mediated

clearance of Bordetella pertussis

Objective:
The main aim of this study is to establish that pertussis toxin in Bordetella pertussis is responsible for the delayed immune response in clearing the bacteria from the upper respiratory tract.

Materials and Methods

A streptomycin resistant derivative BP536, B.pertussis strain and Pertussis enterotoxin (PTx) mutant of BP536BPH101 is used in this experiment. 1) 2) 3) Bordet Gengon (BG ) agar with 75%defibrinated sheeps blood and 20g/ml of streptomycin is used to grow the bacteria Stainer schotte broth (SS) with 20g/ml of streptomycin is used to culture bacteria and the bacterial growth was monitored till mid log phase. Mutant pertussis gene has two changes in its amino acids Arg 9-lys and Glu129-gly.Pertussis toxin gene used only shows immunogenic properties and is not toxigenic.

Inoculation
1) C57BL/6 mice are used in the experiment. Isoflurane is injected to sedate the mouse lightly and tips of its external nares were inoculated with 5*105 CFU of bacteria in 50l of phosphate buffer saline. 2) Determination of levels of colonisation is done by homogenisation of lungs in 1*PBS and aliquots are placed to count colonies. 3) Then the colonies are plated on to BG agar supplemented with streptomycin. 4) Counting of colonies was done after three days of incubation. 5) Mouse were inoculated either with B pertussis or with B pertussis PTx (with a mutant pertussis toxin gene)

Leukocytes Enumeration in the Lungs

1) 2) 3) Type 1 collagenase and DNAase I digestion is used to isolate leukocytes from the lungs. The lungs were sheared using scissors after perfusing it with phosphate buffer saline. After the enzymatic treatment for 3 hours the lungs homogenate is centrifuged for 30 minutes.

4) 5)

Total number of leukocytes was determined using haemocytometer. Staining of the isolated cells using Giesma stain helped to identify the cells individually.

Cytokine and Chemokine Estimation

1)

MH-S cells (alveolar cell line) were cultured in DMEM medium along with 10% FBC.Then the cells are incubated with B pertussis or with B pertussis PTx at 370C for 10-12 hrs. 2) Supernatants from the culture is stored at -800C 3) Lactate dehydrogenase assay is used to estimate the percentage of cell death. 4) ELISA is used to determine the concentration of cytokines and chemokines.

Migration Assay for Neutrophils

1) 2) 3) 4) 5) 3M transwells with DMEM medium along with 10% FCS is prepared to culture primary murine endothelial cells. Differential density separation is used to isolate peripheral blood PMNs from C57BL/6 mice. 200l of DMEM is used to layer around 105 PMNs on the endothelial cells. 100ng/ml of E.coli LPS, B pertussis or with B pertussis PTx incubated for 12 hours with 200l of macrophages The migration of neutrophils to the lower chamber was measured.

Depletion of Neutrophils
The depletion in the number of neutrophils were studied with the help of RB6-8C57 a kind of graft.

Results The serum antibodies used will not rapidly eliminate B pertussis
1) 2) 3) 4) 5*105 CFU of B pertussis was used to inoculate wild type mice , 200l of serum antibodies were also injected into the mice Studies have found that the bacteria obtained from the lungs on days1, 3 and 7 did not decrease in number. Significant reduction in bacterial count was observed after 7 days of inoculation with the serum antibodies. On 14th day after the inoculation, no bacteria could be detected in the lungs. The effect of antibody can be seen only after 1 week of inoculation with serum antibodies.

Serum Antibody Mediated Clearance require FC Rs, but not C3

1) Genetically modified mice with no C3 central complement compartment was inoculated with B pertussis. After 14 days of inoculation, the bacterial count was checked and it has been found that the bacterial count decreased even in the absence of C3 complement component. Hence C3 complement component is not involved in the serum mediated antibody clearance. FCR- mice is used to determine whether FC R has any role in the serum antibody mediated clearance. In contrast to the experiments using C3 component, the bacterial count remained the same even after 14 days of injection of serum antibody. This shows that in the absence of FC R the serum mediated clearance doesnt take place properly. FC R clear the antibodies by phagocytosis.

2)

Antibody Mediated Clearance of B.pertussis requires neutrophils.

1) 2) 1 mg of auto Ly-6 monoclonal antibodies was injected to C57BL/6 mice. This resulted in the depletion of the number of neutrophils in at least 2 weeks without affecting any other cells. After that C57BL/6 mice was inoculated with B pertussis.

3)

After 14 days the bacterial count was taken and it has been found that the antibody serum used to inoculate have no effect on B pertussis. Hence it is concluded that the neutrophils are required for the antibody mediated clearance of bacteria.

Antibody Mediated Clearance of B.pertussis is inhibited by Pertussis toxin (PTx)

1) 2) 3) It has been thought that PTx inhibits early recruitment of neutrophils making it difficult to clear B pertussis. In order to prove that, mice is inoculated with B pertussis or with B pertussis PTx which have a mutant PTx gene. Studies have found that mice injected with B pertussis PTx when treated with serum antibody were able to clear the bacteria in three days and complete clearance was observed after seven days. While in the other case, mice injected with B pertussis didnt show much difference in the bacterial count after 3 days.

Pertussis toxin (PTx) reduces the neutrophil recruitment into the lungs
1) 2) Mice were inoculated with B pertussis or with B pertussis PTx and recruitment of neutrophils to the lungs is calculated. The mice injected with B pertussis PTx and the leucocyte count after one day of inoculation of serum antibody was 5-6*106 leukocytes and on days 3 and 7 was 7-8*106. But mice injected with B pertussis showed an increase in the leucocyte recruitment of 10-12*106 leucocytes all most all recruited cells were neutrophils.

They have also concluded that the chemokine and cytokines produced by the alveolar macrophages are not modulated by pertussis toxin. The action of pertussis toxin inhibits the neutrophil recruitment.

Discussion
Early recruitment of neutrophils will be inhibited by the action of pertussis toxin which helps in the colonisation of B pertussis. Pertussis toxin also have a strong hand in preventing the antibody mediated clearance of the bacteria which Further benefits the bacteria to spread its infection from one host to another. PTx inhibits the recruitment of neutrophils by disrupting the chemokine receptor signalling. In order to overcome the effect of pertussis toxin, T cell dependent response is needed and it allows the antibody mediated bacterial clearance. Neutrophil recruitment is blocked by pertussis toxin 2 fold; this allows the bacterium to form colonies and gives ability to resist the effect of serum antibodies.

Conclusion
Pertussis toxin and cholera enterotoxin belongs to the family of AB 5 toxins and have similar structure. The subunit of cholera toxin can be used as an adjuvant in the antigen specific immunotherapy. The whooping cough caused by the bacterium Bordetella pertussis is endemic even after the vaccination. Pertussis toxin prevents the antibody mediated bacterial clearance thus preventing the complete elimination of the infecting agent from the upper respiratory tract. Both these findings will help in the further development of new techniques for the treatment of these diseases.

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