Animals were maintained in pathogen-free housing and experiments

Animals were maintained in pathogen-free housing and experiments were carried out in accordance to federal, state, and institutional guidelines. Colitis was induced by administration of 3% (w/v) Dextran Sulfate Sodium (36–50 kDa, MP Biomedicals; Santa Ana, CA, USA) in drinking water for 7 days, followed by 3 days with normal water. CD4+ cells were purified from pooled lymph nodes and spleens of WT or gene-deficient DO11.10+/− Rag2−/− mice using magnetic beads (>96% purity). A total of 3–5 × 105 were intravenously injected per recipient in 400 μL PBS. Where noted, recipient

mice were treated with 500 μg of anti-IL-4 mAb on days 0, 3, and 6 (Clone: 11B11). For immunizations, WT donor T cells were transferred into Balb/c mice and, 24 h later, these were intravenously injected with 1–2 × 105 congenic, INK 128 mw bone marrow-derived dendritic cells (BM-DCs) that were preactivated with LPS and loaded with Ova peptide (1 μg/mL each).

For sOva Rag2−/− hosts, single cell suspensions were made from peripheral lymph nodes and restimulated overnight with Ova-pulsed BM-DCs (5:1 lymphocyte to DC ratio). For immunized hosts, CD4+ cells were purified from pooled LNs and spleens, then restimulated overnight (10:1 T to DC ratio). For DSS colitis experiments, single cell suspensions were made from mesenteric lymph nodes (mLNs) and restimulated overnight with platebound antiCD3 mAb (10 ug/mL, clone 145–2C11). All cultures were treated with Brefeldin A (10 μg/m) for the final 2 h, fixed, permiabilized, and stained with anti-CD4 and anti-DO11.10 in combination with anti-cytokine and HIF inhibitor or anti-TF antibodies. Gating strategies for

all intracellular flow cytometry experiments are shown in Supporting Information Fig. 1. Cell sorting was used to purify CD4+ DO11+ CD44high cells from adoptively transferred hosts (5–10 × 104 cells/group). Naïve, CD4+ DO11+ CD25− CD44low controls were purified from DO11.10 Rag2+/+ mice. Real-time PCR protocol and primer sequences have been reported ZD1839 in vivo [15]. Data are presented as fold induction (n > 1) or reduction (n < 1) compared to naïve controls (n = 1). Student's t-test was used to quantify statistical deviation. In all figures, error bars denote standard deviation and asterisks represent significant differences (p < 0.05). The authors thank Dr. Abul Abbas (UCSF) for mice, reagents, and advice. We also thank Dr. J. O'Shea (NIH) and members of the O'Shea laboratory for discussions. Research supported by NIH grant RO1AI64677 and a minority supplement to A.V.V. (PA-05-015). The authors declare no financial conflict of interests. Disclaimer: Supplementary materials have been peer-reviewed but not copyedited. "
“The pattern-recognition molecules mannan-binding lectin (MBL) and the three ficolins circulate in blood in complexes with MBL-associated serine proteases (MASPs).

[32] For histological analysis, colons were fixed, sectioned and

[32] For histological analysis, colons were fixed, sectioned and stained with haematoxylin & eosin. Histological changes were graded from 0 to 4 in a blind fashion according to previously described

criteria as follows: 0, no signs of inflammation; 1, very low level of leucocyte infiltration; 2, low level of leucocyte infiltration; 3, high level of leucocyte infiltration, high vascular density, and thickening of the colon wall; 4, transmural leucocyte infiltration, loss of goblet cells, high vascular density and PS-341 manufacturer thickening of the colon wall.[32] Myeloperoxidase (MPO) activity of the colon was measured according to the method described previously.[33] Briefly, tissues were homogenized and centrifuged (30 000 g, 30 min at

4°). Pellets were resuspended in hexadecyltrimethylammonium bromide in 50 mm potassium phosphate buffer and then freeze–thawed three times. The supernatants were diluted in potassium phosphate buffer (pH 6·0) containing 0·167 mg O-dianisidine dihydrochloride (Sigma-Aldrich) and 0·0006% (vol/vol) H2O2. Changes in absorbance at 460 nm were recorded with kinetic readings over 3 min. Sample protein concentrations were determined (bicinchoninic acid assay), and the results are presented as MPO units per milligram selleck chemicals llc of protein. Mesenteric lymph node (MLN) cells were isolated and incubated in complete RPMI-1640 with 10% fetal calf serum at a concentration of 1 × 106 cells/ml for 48 hr in the presence or absence of PMA (10 ng/ml) and concanavalin

A (Con A; 2 μg/ml) selleck screening library (Sigma-Aldrich). Cytokine production in culture supernatants was determined by ELISA. The levels of IL-6, IL-17A and transforming growth factor-β (TGF-β) in MLN cell culture supernatants were determined by sandwich ELISA using the kits supplied by eBioscience (San Diego, CA). ELISA was performed according to the manufacturer’s instructions. Mesenteric lymph node cells were isolated and suspended in complete RPMI-1640 with 10% fetal calf serum at a density of 1 × 106/ml. The cell suspensions were re-stimulated with PMA (20 ng/ml), ionomycin (1 μg/ml) and 2 μm of monensin (Sigma-Aldrich) for 4 hr. Cells were harvested, blocked with rat anti-mouse CD16/32 antibodies, and stained with phycoerythrin-cy5-conjugated anti-mouse CD4 antibody (BD Pharmingen, San Jose, CA). Cells were then fixed and permeabilized with Cytofix/Cytoperm (BD Pharmingen) and stained with phycoerythrin-conjugated anti-mouse IL-17A antibody. Intracellular FoxP3 was determined according to the manufacturer’s instructions. Data were acquired on a FACScalibur (BD Biosciences, San Jose, CA) and analysed with the CellQuest v3.3 software as instructed.

Long-term success can be secured only by adaptability It is incr

Long-term success can be secured only by adaptability. It is increasingly clear that to cope with our expanding knowledge of T cell biology, immunologists must be as flexible as the cells they love to study. S. M. A. and R. A. O. are supported by grants from the UK Medical Research Council, the Wellcome Trust and the UK Multiple

Sclerosis Society. S. M. A. holds a Research Councils UK fellowship in translational medicine. L. S. T. is supported check details by MRC- and BBSRC-funded PhD studentships and by financial support from the Department of Health via the National Institute for Health Research (NIHR) comprehensive Biomedical Research Centre award to Guy’s & St Thomas’ NHS Foundation Trust in partnership with King’s College

London and King’s College Hospital NHS Foundation Trust. The authors declare no conflict of interest. “
“Interleukin (IL)-17A is increased both in serum and in kidney biopsies from patients with lupus nephritis, but direct evidence of pathogenicity is less well established. Administration of pristane to genetically intact mice results in the production of autoantibodies and proliferative glomerulonephritis, resembling human lupus nephritis. These studies sought to define the role of IL-17A in experimental lupus induced by pristane administration. Pristane was administered to wild-type (WT) and IL-17A−/− mice. Local and systemic immune responses were assessed after 6 days and 8 weeks, and autoimmunity, glomerular inflammation and renal 17-DMAG (Alvespimycin) HCl injury were measured at 7 months. IL-17A production increased significantly 6 days after pristane Dasatinib supplier injection, with innate immune cells, neutrophils (Ly6G+) and macrophages (F4/80+) being the predominant source of IL-17A. After 8 weeks, while systemic IL-17A was still readily detected

in WT mice, the levels of proinflammatory cytokines, interferon (IFN)-γ and tumour necrosis factor (TNF) were diminished in the absence of endogenous IL-17A. Seven months after pristane treatment humoral autoimmunity was diminished in the absence of IL-17A, with decreased levels of immunoglobulin (Ig)G and anti-dsDNA antibodies. Renal inflammation and injury was less in the absence of IL-17A. Compared to WT mice, glomerular IgG, complement deposition, glomerular CD4+ T cells and intrarenal expression of T helper type 1 (Th1)-associated proinflammatory mediators were decreased in IL-17A−/− mice. WT mice developed progressive proteinuria, but functional and histological renal injury was attenuated in the absence of IL-17A. Therefore, IL-17A is required for the full development of autoimmunity and lupus nephritis in experimental SLE, and early in the development of autoimmunity, innate immune cells produce IL-17A. “
“A bacteriophage lambda DNA vaccine expressing the small surface antigen (HBsAg) of hepatitis B was compared with Engerix B, a commercially available vaccine based on the homologous recombinant protein (r-HBsAg).

aeruginosa and S aureus grown in a flow-chamber system We demon

aeruginosa and S. aureus grown in a flow-chamber system. We demonstrated how adaptive mutations in regulator genes of P. aeruginosa affect interactions between P. aeruginosa and S. aureus in co-culture biofilms. Pseudomonas aeruginosa

wild-type PAO1 (Holloway & Morgan, 1986), P. aeruginosa mucA mutant (Hentzer et al., 2001), Selumetinib manufacturer P. aeruginosa rpoN mutant (Webb et al., 2003), P. aeruginosa pilA mutant (Klausen et al., 2003b), P. aeruginosa pilH mutant (Barken et al., 2008), P. aeruginosa pqsA mutant (D’Argenio et al., 2002), S. aureus MN8 (Yarwood et al., 2004), S. aureus ISP479 (Toledo-Arana et al., 2005) and S. aureus 15981 (Toledo-Arana et al., 2005) were kindly provided by the cited authors and used in the present study. The pDA2 plasmid (An et al., 2006) was used Selleckchem KPT330 to complement the pilA mutant. Fluorescence-tagged strains were constructed by the insertion of a mini-Tn7-eGFP-Gmr cassette as described (Koch et al., 2001; Klausen et al., 2003b). Escherichia coli strains MT102 and DH5α were used for standard DNA manipulations. Luria–Bertani medium (Bertani, 1951) was used to cultivate E. coli strains. A modified FAB medium (Qin et al., 2007) supplemented with 0.3 mM glucose and 3% of Tryptic Soy Broth (TSB, BD Diagnostics) was used for biofilm cultivation. Selective media were supplemented with ampicillin (100 mg L−1), gentamicin (60 mg L−1) or carbenicillin

(200 mg L−1). Biofilms were grown in flow chambers

with individual channel dimensions of 1 × 4 × 40 mm at 37 °C. The flow system was assembled and prepared as described previously (Sternberg & Tolker-Nielsen, 2006). Overnight cultures of P. aeruginosa and S. aureus were diluted to an OD600 nm of 0.001. The flow chambers were inoculated by injecting 350 μL of monospecies diluted cultures or P. aeruginosa–S. aureus 1 : 1 mixed-species diluted cultures into each flow channel with a small syringe. After inoculation, flow channels were left without flow for 1 h, after which medium flow (0.2 mm s−1) was started using a Watson Marlow 205S peristaltic pump. For DNase I treatment, biofilm medium was supplemented with 20 μg mL−1 bovine DNase I (Sigma) from the beginning of cultivation. All microscopic observations and image acquisitions were performed using a Zeiss LSM 510 confocal laser scanning microscope (Carl Zeiss, Jena, DNA ligase Germany) equipped with detectors and filter sets for monitoring of green and red fluorescence from general nucleic acid staining SYTO 9 (Invitrogen) and gram-positive specific staining hexidium iodide (Invitrogen) (Mason et al., 1998), respectively. BacLite Live/Dead viability stain (Molecular Probes, Eugene, OR) was used to visualize dead and live cells in co-culture biofilms. Images were obtained using a × 40/1.3 objective. Simulated three-dimensional images and sections were generated using the imaris software package (Bitplane AG, Zürich, Switzerland).

Junctional ectopic tachycardia may be a spectrum of injury to the

Junctional ectopic tachycardia may be a spectrum of injury to the AV node in which

partial injury may be associated with increased automaticity and more complete injury with AVB. Although to date there has been no report documenting an association between congenital Silmitasertib solubility dmso junctional ectopic tachycardia in the absence of AVB and maternal autoantibodies, given the lack of symptoms among otherwise healthy women with infants who have complete AVB and/or maternal autoimmune-mediated cardiomyopathy, prospective serological evaluation of the mothers of affected infants should be considered. A spectrum of structural heart disease has been reported among foetuses and infants with maternal autoimmune-mediated cardiovascular disease. In children with maternal autoimmune-mediated learn more congenital AVB, structural congenital heart disease has been reported in 16–42% [22, 38]. These lesions have included persistent ductus arteriosus most of which have required intervention, and atrial and ventricular septal defects. Of greater interest, semilunar and atrioventricular valve abnormalities have also been described in association with AVB, including

stenosis, regurgitation and dysplasia without functional changes (Fig. 4) [22, 38, 54]. Inflammation and fibrosis as well as haemodynamic changes could potentially contribute to the evolution of at least some of these lesions, as suggested in one case of acute chordal rupture with moderate mitral insufficiency in 7-week old infant with echocardiographic evidence of EFE involving left ventricular papillary muscles and chordae [54]. The incidence of structural and even functional heart disease among infants of mothers

with autoantibodies in the absence of AVB is still not certain. In one study that assessed structural abnormalities in a series of 165 pregnancies with autoimmune disease and anti-Ro antibodies, four offspring had structural heart disease suggesting a potential incidence of 2.8%, which represents an increase over that of the general Calpain population [51]. Maternal autoimmune-mediated pathology could be aetiological in the evolution of other forms of congenital heart disease, as suggested in a recent case of prenatally diagnosed hypoplastic left heart syndrome [55]. Further prospective longitudinal investigations of pregnancies (and offspring) in women with anti-Ro and anti-La antibodies with and without autoimmune disease are necessary at this time to determine the true incidence of congenital structural, functional and rhythm-related cardiovascular disease associated with maternal autoantibodies.

45 Mouse labyrinthine

45 Mouse labyrinthine Dabrafenib clinical trial trophoblasts express paternal MHC class I.46 The interplacentomal trophoblasts of the cow express both classical and non-classical MHC class I genes late in pregnancy.47 As in other species, MHC class II molecules are not expressed by any equine trophoblast populations.36,48

While the pregnant mare is capable of mounting a robust and reproducible humoral immune response against paternal MHC class I antigens, this is not the case with the cell-mediated immune response. Equine pregnancy appears to induce a state of ‘split tolerance’ to trophoblast – a situation where one compartment of the immune system responds to an antigen, while another is tolerant.49–51 In the pregnant mare, this presents as a dramatic allospecific anti-paternal humoral immune response with a simultaneous dampening of certain T-cell-mediated responses. Peripheral blood lymphocytes isolated from pregnant mares demonstrate

a reduced capacity to develop into effective cytotoxic T lymphocytes (CTL) capable of lysing target cells from the breeding stallion.52 This reduction Olaparib nmr in T-cell-mediated alloreactivity reverts after parturition or pregnancy termination, and it is not observed in males or non-pregnant females. This phenomenon seems logical, as the formation of anti-paternal cytotoxic cells during pregnancy could be disastrous for the semi-allogeneic fetus. However, a generalized reduction Guanylate cyclase 2C in cell-mediated immunity would make the mother susceptible to certain types of infections. It has not yet been

determined whether the alteration in the CTL activity of pregnant mares is limited to responses against paternal alloantigens. Studies using transgenic mice have demonstrated that peripheral maternal lymphocytes specific for paternal antigens may be inactivated or deleted during pregnancy.53–55 Studies of infectious diseases in conventional pregnant mice suggest broader antigen-independent mechanisms.56,57 Likewise, pregnant women appear to experience an increased susceptibility to infections such as Listeria and Toxoplasma.58,59 While mares are vulnerable to a number of pregnancy-associated abortogenic infections,60–62 it is not clear whether this is attributable to a general systemic immune tolerance or pregnancy-associated tissue tropism. The peripheral lymphocyte populations of pregnant mares have demonstrated a few significant detectable alterations in phenotype. A modest increase in the number of circulating lymphocytes that express the TH2 cytokine IL-4 has been demonstrated during pregnancy.49 This finding is consistent with the high levels of paternal alloantibodies observed during pregnancy, as the presence of IL-4 favors a humoral immune response. The maternal leukocytes that accumulate around the equine endometrial cups represent one of the most dramatic examples of a local cellular immune response to the conceptus.

Mice immunized with either recombinant proteins or plasmid DNA we

Mice immunized with either recombinant proteins or plasmid DNA were infected with blood trypomastigotes. The recombinant protein-immunized mice showed a variable reduction in peak parasitemia, and most died by day 60. Only the pBKTcSPR-immunized mice exhibited a significant reduction in peak parasitemia and survived the lethal challenge. DNA-based immunization with DNA coding for the repeats

domain of TcSP is a good candidate for the development of a vaccine against experimental T. cruzi infection. Chagas disease, caused by Trypanosoma cruzi, continues to be a major health problem in South and Central America, although the estimated number of infected people has fallen from approximately 20 million in 1981 to approximately 10 million in 2009 due to the implementation of vector control measures and

this website safer blood transfusions [1, 2]. Urbanization and migratory population movements from endemic countries have led to diagnosis of the disease even in nonendemic areas [3]. Although the transmission of this disease has diminished recently [4, find more 5], it is still a major problem, and currently, there are neither effective drugs nor vaccines for the treatment or prevention of the disease. The infection results in an acute parasitemic phase, followed by a chronic indeterminate phase during which parasitemia is generally undetectable and most patients remain asymptomatic. Approximately 30% of individuals in the chronic indeterminate phase progress to a chronic symptomatic phase involving severe cardiomyopathy or Lumacaftor gastrointestinal pathology. Several studies have examined

the protective roles of antibodies [6], Th1-type cytokines [7, 8] and cytotoxic T cells (CTL) [9, 10] in experimental models. A better understanding of the host immune response to parasite antigens will allow the development of effective vaccines to control T. cruzi infection. Towards this goal, a number of parasite antigens have been tested for their effectiveness in controlling parasite infection, including cruzipain [11], trans-sialidase (TS) [12], amastigote surface protein-2 [13], trypomastigote surface antigen-1 [14] and paraflagellar rod protein [15], among others. These antigens are located on the parasite surface and induce strong cellular and humoral responses during infection in mice. The T. cruzi genome contains 1430 gene members of the TS superfamily [16]. Members of the TS superfamily show at least 30–40% homology with the unique TS enzyme sequence. The importance of TS enzymatic activity for T. cruzi virulence [17, 18] and the large number of TS homologues suggest that this gene family may be involved in mechanisms of immune escape in the murine model of Chagas disease [19].

A total of 157 peptides were

found to bind to one of the

A total of 157 peptides were

found to bind to one of the 12 HLA molecules with a measured KD ≤ 500 nm, which is the normally accepted threshold36–38 for being a potential antigenic epitope. The numbers of binding peptides for the individual supertypes are: HLA-A1 (11 peptides), HLA-A2 (15 peptides), HLA-A3 (four peptides), HLA-A24 (14 peptides), HLA-A26 (15 peptides), HLA-B7 (18 peptides), HLA-B8 (seven peptides), HLA-B27 (eight peptides), HLA-B39 (17 peptides), HLA-B44 (20 peptides), HLA-B58 (14 peptides) and HLA-B62 (14 peptides). Consistent with previous classifications, the binding affinity (KD) of the 157 binding peptides can be divided into groups of high-affinity binders (n = 83; KD ≤ 50 nm) and intermediate-affinity binders (n = 74; 50 nm < KD ≤ 500 nm). The 157 HLA-I binding peptides were tested for their ability to stimulate T cells from a cohort of healthy PPD+ Danish subjects aged 35–65 years. The peptides were evaluated for their ability to stimulate IFN-γ production

in an ELISPOT assay by PBMC from those HLA-matched donors who reacted most strongly with PPD. Since many donors’ PBMC failed to respond after 2 days of peptide exposure, the Gefitinib supplier sensitivity of the procedure was increased by exposing PBMC for 10 days to peptides before performing the ELISPOT assays. Positive reactivity towards peptides was confirmed at least twice in the same donor as well as in other HLA supertype matched donors. According to this criterion eight peptides (5%)

belonging to five different supertypes (A1, A26, B7, B44 and B62) were found to be antigenic. An overview of peptide-reactive donors, their HLA class I type, and their reactivity according to ELISPOT data is shown in Table 1. The number of reactive donors and the actual ELISPOT data are shown in Table 2. Each RANTES of the eight antigenic peptides was also tested in 10 donors with low PPD reactivity. Only four of these donors showed reactivity against one or more of the eight antigenic peptides, an observation, which strongly underscores the M. tuberculosis specificity of the responses observed in the present study. We have previously demonstrated that variola virus-derived 9mer peptides with high HLA-I binding affinity (KD ≤ 5 nm) are able to induce CD4+ T-cell responses from PBMC of vaccinated donors.39 Likewise, we showed that influenza A virus-derived 9mer peptides with binding affinities for HLA-I allele are capable of stimulating strong CD4+ T-cell responses.28 To ascertain whether, or not, CD4+ T cells are involved in the anti-M. tuberculosis responses documented above, a pan-specific anti-HLA-II blocking antibody IVA12 as well as anti-DP, -DQ and -DR blocking antibodies were added into ELISPOT microcultures (see Materials and methods section). Similarly, cultures were exposed to the pan-specific anti-HLA class I antibody W6/32. As shown in Fig.

All procedures with animals were approved by the Committee for An

All procedures with animals were approved by the Committee for Animal Protection and Use of the Institute of Microbiology. PR4 is a commensal strain of B. choerinum isolated from fecal flora of 8-week-old pigs of (LW × L) × Pn breed using modified trypticase–phytone–yearst (MTPY) agar [30]. The isolate was identified using the random amplified polymorphic DNA–polymerase chain reaction (RAPD-PCR) procedure according to Sakata

et al. [31] and compared with porcine bifidobacteria strains from the German Resource Centre Cytoskeletal Signaling inhibitor for Biological Material. EcN is E. coli Nissle 1917 (EcN, serovar O6:K5:H1). This serum-sensitive non-virulent E. coli strain is used as a human and veterinary probiotic [16]. LT2 is a serum-resistant LT2 strain of S. enterica serovar Typhimurium causing lethal sepsis in germ-free piglets [26]. Fresh cultures of bacteria were prepared for each experiment by cultivation at 37°C overnight. PR4 was cultivated in an anaerobic chamber in 10 ml TPY

broth (Scharlau, Barcelona, Spain). The cells were harvested by centrifugation at 4000 g for 10 min. The pellet was washed twice with 0·05 M phosphate buffer, pH 6·5 containing 500 mg/l cysteine find more (PBC). EcN and LT2 were cultivated on meat-peptone agar slopes (blood agar base; Oxoid, Basingstoke, UK). Bacteria were resuspended to the density of 5 × 108 colony-forming units (CFU)/ml and given to gnotobiotic pigs in milk diet. The number of CFU estimated by spectrophotometry at 600 nm was verified by a cultivation method. One-week-old germ-free pigs were orally associated/infected

with 1 × 108 CFU of bacteria in 5 ml of milk diet. Di-associated pigs were infected with S. Typhimurium 24 h after the association with PR4 or EcN, respectively. All experimental pigs were euthanized 24 h after the last bacteria treatment by exsanguination TCL under halothane anaesthesia. The germ-free control group was euthanized at the same age. Six experimental groups of 1-week-old gnotobiotic pigs (five pigs in each group) from five hysterectomies of miniature sows were investigated: (i) germ-free piglets, (ii) pigs mono-associated with LT2 (LT2 strain of S. enterica serovar Typhimurium), (iii) pigs mono-associated with PR4 (B. choerinum strain PR4), (iv) pigs mono-associated with EcN (E. coli strain Nissle 1917), (v) pigs di-associated with PR4+LT2 and (vi) LT2 pigs di-associated with EcN+ LT2. Experimental animals were euthanized and samples of peripheral blood, intestinal lavages and homogenized tissues (from the spleen, mesenteric lymph nodes and liver) were serially diluted in PBC. Appropriate dilutions were transferred to sterile 60 mm Petri dishes, which were immediately filled with the media for bifidobacteria (TPY agar; Scharlau) supplemented with 100 mg/l mupirocin and 1 ml/l of concentrated glacial acetic acid [30]. Bifidobacteria were incubated in an anaerobic jar (Anaerobic Plus System; Oxoid) in CO2/H2 (90/10%) atmosphere at 37°C for 3 days. E.

This translocation process is facilitated by the binding of PA to

This translocation process is facilitated by the binding of PA to common regions within the N-terminal domains of LF (LFn) and EF and occurs in the absence of the toxic C-terminal domains of either protein. Indeed, it has been demonstrated that the coadministration of PA and LFn enhances the uptake of both antigens to heighten the magnitude Selleckchem SB431542 of PA- and LFn-specific antibody responses and protect during a lethal anthrax spore infection (Price et al., 2001). The combination of PA and LFn as a molecular syringe has been used to deliver antigens from HIV

and Listeria monocytogenes fused to LFn to the cytoplasm of antigen-presenting cells (APCs; Ballard et al., 1996; Lu et al., 2000). This approach effectively enhanced CD8+ and CD4+ T cell responses to the foreign antigens, highlighting its potential as a multi-agent vaccine delivery system for intracellular pathogens. Multi-agent vaccines that confer protection against two or more diseases are highly desirable for biodefense applications because they reduce the number of vaccines an individual must receive resulting in increased compliance to a vaccination schedule. Like anthrax, immunization against Y. pestis requires an antibody response to two key antigens: Fraction 1 (F1, a component of

the bacteria’s capsule) and LcrV (V, involved in plague’s type III secretion apparatus). In a previous study, we reported that the coadministration of a plasmid encoding PA enhanced the magnitude of the antibody response to V when it was expressed from a second plasmid and concluded that this effect selleck was probably due to the presence of CpG motifs within the PA plasmid because V is not known to bind directly to PA (Williamson et al., 2002). In the present study, we build upon this VAV2 work by determining whether the protective immune response to anthrax and plague could be further enhanced by DNA vaccines expressing the PA/LFn molecular syringe and a V-LFn fusion. As antibody titers to F1 have been correlated with plague survival (Williamson et al., 1999), we also constructed and evaluated a second fusion gene of LFn-F1. Comparison of dissimilar vaccines often requires multiple

animal models to bridge the results from multiple studies. Some of these animal models may not be optimal surrogates for the human disease or are not responsive to treatment (Riemenschneider et al., 2003). To avoid the issue of animal model variability and demonstrate the combined efficacy of both the anthrax and plague DNA vaccine components during pathogen challenge, a common infection model was needed. A/J mice have been identified as an acceptable model for evaluating anthrax vaccines, while BALB/c mice are traditionally the strain of choice for Y. pestis challenge (Griffin et al., 2005). However, unlike A/J mice, BALB/c mice are not susceptible to B. anthracis challenge in a clear dose-dependent manner (Beedham et al., 2001). To establish the utility of A/J mice during Y.