HA2-FimA DNA Vaccine Treats Experimental Periodontitis.

PURPOSE: To study the therapeutic effect of hemagglutinin-2 and fimbrial (HA2-FimA) vaccine on experimental periodontitis in rats. MATERIALS AND METHODS: The first batch of rats was divided into two groups and immunised with pure water or pVAX1-HA2-FimA at the age of 6, 7, and 9 weeks. After sacrificing the animals, total RNA was extracted from the spleens for RNA high-throughput sequencing (RNA-Seq) analysis. The second batch of rats was divided into four groups (A, B, C, D), and an experimental periodontitis rat model was established by suturing silk thread around the maxillary second molars of rats in groups B, C, and D for 4 weeks. The rats were immunised with pure water, pVAX1-HA2-FimA vaccine, empty pVAX1 vector, and pure water at 10, 11, and 13 weeks of age, respectively. Secretory immunoglobulin A (SIgA) antibodies and cathelicidin antimicrobial peptide (CAMP) levels in saliva were measured by enzyme-linked immunosorbent assay (ELISA). All rats were euthanised at 17 weeks of age, and alveolar bone loss was examined using micro-computed tomography (Micro-CT). RESULTS: Through sequencing analysis, six key genes, including Camp, were identified. Compared with the other three groups, the rats in the periodontitis+pVAX1-HA2-FimA vaccine group showed higher levels of SIgA and CAMP (p < 0.05). Micro-CT results showed significantly less alveolar bone loss in the periodontitis+pVAX1-HA2-FimA vaccine group compared to the periodontitis+pVAX1 group and periodontitis+pure water group (p < 0.05). CONCLUSION: HA2-FimA DNA vaccine can increase the levels of SIgA and CAMP in the saliva of experimental periodontitis model rats and reduce alveolar bone loss.

Bordetella pertussis isolates in Finland after acellular vaccination: serotype change and biofilm formation.

OBJECTIVES: In Finland, whole cell pertussis vaccine (wP) was introduced in 1952 and was replaced by acellular pertussis vaccine (aP) without fimbrial (FIM) antigen in 2005. We aimed to analyse the changes in serotypes of circulating Bordetella pertussis before and after acellular vaccination and to explore the relationship between biofilm formation and serotype diversity after the introduction of aP vaccine. METHODS: Serotyping of 1399 B. pertussis isolates collected at the Finnish National Reference Laboratory for Pertussis and Diphtheria in Turku, Finland, from 1974 to 2023 was performed by slide agglutination or indirect ELISA. Of 278 isolates collected after 2005, 53 were selected, genotyped for fim3 and fim2 alleles, and tested for biofilm formation. The selection criteria included maintaining a relatively equal distribution of isolates per time interval, ensuring approximately a 50:50 ratio of FIM2 (N = 26) and FIM3 (N = 27) serotypes. The reference strain Tohama I was used as a control. RESULTS: During the wP era, the majority of circulating B. pertussis exhibited the FIM2 serotype. However, FIM3 strains have appeared since 1999 and become prevalent. After the implementation of aP vaccines, the distribution of serotypes has exhibited substantial variability. FIM3 isolates displayed an enhanced biofilm formation compared to FIM2 isolates (Geometric mean value (95% CI): 0.90 (0.79-1.03) vs. 0.75 (0.65-0.85); p < 0.05). Of the 27 FIM3 isolates, 8 harboured fim3-1 and 19 fim3-2 alleles. FIM3 isolates with fim3-2 allele were significantly associated with increased biofilm formation when compared to those with fim3-1 (1.07 (0.96-1.19) vs. 0.61 (0.52-0.72); p < 0.0001). CONCLUSION: Following the implementation of aP vaccines, the distribution of serotypes in Finland has exhibited substantial variability. FIM3 isolates with the fim3-2 allele displayed an enhanced biofilm formation capability compared to FIM2 isolates.

Neutralizing Antibodies Against Allosteric Proteins: Insights From a Bacterial Adhesin.

Allosteric proteins transition between 'inactive' and 'active' states. In general, such proteins assume distinct conformational states at the level of secondary, tertiary and/or quaternary structure. Different conformers of an allosteric protein can be antigenically dissimilar and induce antibodies with a highly distinctive specificities and neutralizing functional effects. Here we summarize studies on various functional types of monoclonal antibodies obtained against different allosteric conformers of the mannose-specific bacterial adhesin FimH - the most common cell attachment protein of Escherichia coli and other enterobacterial pathogens. Included are types of antibodies that activate the FimH function via interaction with ligand-induced binding sites or by wedging between domains as well as antibodies that inhibit FimH through orthosteric, parasteric, or novel dynasteric mechanisms. Understanding the molecular mechanism of antibody action against allosteric proteins provides insights on how to design antibodies with a desired functional effect, including those with neutralizing activity against bacterial and viral cell attachment proteins.

Passive immunization with anti- chimeric protein PilQ/PilA -DSL region IgY does not protect against mortality associated with Pseudomonas aeruginosa sepsis in a rabbit model.

BACKGROUND: Pseudomonas aeruginosa sepsis is associated with unacceptably high mortality and, for many of those who survive, long-term morbidity. The aims of this study were to production of IgY against chimeric protein pilQ-pilA-DSL region and killed- whole cell Pseudomonas aeruginosa O1 (PAO1) strain and their efficacy for immunoprophylaxis of sepsis caused by P. aeruginosa in a rabbit model. METHODS: Specific IgY was obtained by immunization of hens. The purity of IgY was determined by SDS-PAGE analysis. The effect of IgY on growth and hydrophobicity of P. aeruginosa were performed through time-kill assay and microbial adhesion to hydrocarbons test (MATH), respectively. The efficacy of specific IgYs was examined against P. aeruginosa sepsis in a rabbit model. The rabbits were monitored for 72 h to record physiological characters and survival. Hematologic factors, C-reactive protein, pro-inflammatory cytokines, and bacterial count from blood and solid organs were measured, periodically. RESULTS: We found that the growth inhibitory effect of the anti- killed whole cell IgY was higher than anti-pilQ-pilA IgY (P < 0.001). The hydrophobicity effect of PAO1 increased when bacteria were opsonized by anti- killed whole cell IgY while the hydrophobicity activity was decreased following incubation of PAO1 with anti-pilQ-pilA IgY in a broth medium (P < 0.001). Following intravenous (IV) administration of produced IgYs, no significant difference was observed in the survival, decrease in inflammatory mediators and clinical symptoms between the groups 48h post infection (P > 0.05). Moreover, no considerable decrease was observed in the bacterial load of blood, lungs and kidneys in rabbits treated with specific IgYs and control groups (P > 0.05). No bacteria were found in the spleen and liver samples from infected rabbits. CONCLUSION: Although produced IgYs had a good immunoreactivity, IV immunization of IgYs was not protective against P. aeruginosa sepsis in the rabbit model. Further studies are needed to assess the immune response and decreasing mortality rate using the rabbit sepsis model.

Protective efficacy of a novel multivalent vaccine in the prevention of diarrhea induced by enterotoxigenic Escherichia coli in a murine model.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) infection is a primary cause of livestock diarrhea. Therefore, effective vaccines are needed to reduce the incidence of ETEC infection. OBJECTIVES: Our study aimed to develop a multivalent ETEC vaccine targeting major virulence factors of ETEC, including enterotoxins and fimbriae. METHODS: SLS (STa-LTB-STb) recombinant enterotoxin and fimbriae proteins (F4, F5, F6, F18, and F41) were prepared to develop a multivalent vaccine. A total of 65 mice were immunized subcutaneously by vaccines and phosphate-buffered saline (PBS). The levels of specific immunoglobulin G (IgG) and pro-inflammatory cytokines were determined at 0, 7, 14 and 21 days post-vaccination (dpv). A challenge test with a lethal dose of ETEC was performed, and the survival rate of the mice in each group was recorded. Feces and intestine washes were collected to measure the concentrations of secretory immunoglobulin A (sIgA). RESULTS: Anti-SLS and anti-fimbriae-specific IgG in serums of antigen-vaccinated mice were significantly higher than those of the control group. Immunization with the SLS enterotoxin and multivalent vaccine increased interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) concentrations. Compared to diarrheal symptoms and 100% death of mice in the control group, mice inoculated with the multivalent vaccine showed an 80% survival rate without any symptom of diarrhea, while SLS and fimbriae vaccinated groups showed 60 and 70% survival rates, respectively. CONCLUSIONS: Both SLS and fimbriae proteins can serve as vaccine antigens, and the combination of these two antigens can elicit stronger immune responses. The results suggest that the multivalent vaccine can be successfully used for preventing ETEC in important livestock.

Oral probiotic promotes indoleamine 2,3-dioxygenase- and TGF-ß-Producing plasmacytoid dendritic cells to initiate protection against type 1 diabetes.

Colonization factor antigen I (CFA/I) fimbria, an adhesin from enterotoxigenic Escherichia coli, confers protection in murine autoimmune models for type 1 diabetes (T1D), multiple sclerosis, and rheumatoid arthritis. Although CFA/I fimbriae's initial mode of action is in a bystander or in an antigen (Ag)-independent fashion, protection is ultimately dependent upon the induction and/or activation of auto-Ag-specific regulatory T cells (Tregs). However, little is known about how protection transitions from bystander suppression to Ag-specific Tregs. Since dendritic cells (DCs) play an integral role in fate decisions for T cells becoming inflammatory or tolerogenic, the described study tests the hypothesis that Lactococcus lactis expressing CFA/I (LL-CFA/I) stimulates DCs to establish a regulatory microenvironment. To this end, bone marrow-derived dendritic cells (BMDCs) were infected in vitro with LL-CFA/I. Results revealed increased production of IL-10, TGF-ß, and indoleamine 2,3-deoxygenase (IDO). Although co-culture of LL-CFA/I infected BMDCs with naïve T cells did not promote Foxp3 expression, TNF-α and IFN-γ production was suppressed. NOD mice orally dosed with LL-CFA/I showed an increase in regulatory plasmacytoid DCs (pDCs) expressing IDO and TGF-ß in pancreatic lymph nodes (PaLNs) and spleen three days post-treatment. However, Tregs did not appear in the mucosal inductive sites until much later. These findings show that LL-CFA/I influences specific DC populations to establish tolerance.

Recombinant FimH, a fimbrial tip adhesin of Vibrio parahaemolyticus, elicits mixed T helper cell response and confers protection against Vibrio parahaemolyticus challenge in murine model.

Vibrio parahaemolyticus causes vibriosis in wide range of marine organisms, and is responsible for food borne illnesses in humans through consumption of contaminated uncooked/partially cooked seafood. Continued and widespread antibiotics usage to increase the productivity has led to antibiotics resistance development. This has necessitated the need to develop alternative methods to control its infection. Use of safe and effective vaccines against the virulence factors not only protects from infection, it also minimizes antibiotic usage. The colonization of V. parahaemolyticus in the host and disease development requires several adhesins present on the cell surface, and thereby make them attractive vaccine candidates. V. parahaemolyticus produces extracellular type 1 fimbriae that have been shown to play a role in adhesion, biofilm formation and virulence. FimH is one of the minor components of the type 1 fimbriae occurring on its very tip. Being present on the cell surface, it is highly immunogenic, and can be targeted as a potential vaccine candidate. The present study describes the immunogenic and vaccine potential of recombinant V. parahaemolyticus FimH (rVpFimH) expressed in E. coli. Immunization of BALB/c mice with the rVpFimH elicited a strong mixed immune response, T-cell memory (evidenced by antibody isotyping, cytokine profiling and T-cell proliferation assay), and agglutination positive antibodies. FACS analysis and immunogold labeling showed that the polyclonal anti-rVpFimH antibodies were able to recognize the FimH on V. parahaemolyticus cells. In vivo challenge of the rVpFimH-immunized mice with 2×LD50 dose of live bacteria showed one hundred percent survival. Thus, our findings clearly demonstrate the potential of FimH as an effective vaccine candidate against V. parahaemolyticus.

Vibrio cholerae toxin coregulated pilus provokes inflammatory responses in Coculture model of Caco-2 and peripheral blood mononuclear cells (PBMC) leading to increased colonization.

The aim of this study was to assess the modulatory effect of TcpA in the expression of CEACAM1 adhesin molecule and IL-1, IL-8, and TNF-α pro-inflammatory cytokines in the Coculture model of Caco-2/PBMC (peripheral blood mononuclear cell) that can mimic the intestinal milieu. The TcpA gene from Vibrio cholerae ATCC14035 was cloned in pET-28a and transformed into Escherichia coli Bl-21. The recombinant TcpA-His6 protein was expressed and purified using Ni-column chromatography. The sequencing of transformed plasmid and Western blot analysis of purified protein confirmed the identity of rTcp. The cytotoxicity of different concentrations of recombinant protein for human colon carcinoma cell line (human colorectal adenocarcinoma cell [Caco-2 cell]) was assessed by MTT assay and showed viability of 92%, 82%, and 70%, for 10 µg/mL of TcpA after 24, 48, and 72 h, respectively. Co-cultures of Caco-2 and PBMCs were used to mimic the intestinal milieu and treated with different concentrations of rTcpA (1, 5, 10, and 50 µg/mL). Our data showed about 2.04-, 3.37-, 3.68-, and 42.7-fold increase in CEACAM1 gene expression, respectively, compared with the nontreated Caco-2/PBMC Coculture. Moreover, the expression of IL-1, IL-8, and TNF-α genes was significantly increased up to 15.75-, 7.04-, and 80.95-folds, respectively. In conclusion, V. cholerae TcpA induces statistically significant dose-dependent stimulatory effect on TNF-α, IL-,1, and IL-8 pro-inflammatory cytokines expression. Of these, TNF-α was much more affected which, consequently, elevated the CEACAM1 expression level in IECs. This suggests that TcpA protein is a critical effector as an inducer of increased adhesion potential of V. cholera as well as inflammatory responses of host intestinal tissue.

Intestinal and systemic inflammation induced by symptomatic and asymptomatic enterotoxigenic E. coli infection and impact on intestinal colonization and ETEC specific immune responses in an experimental human challenge model.

Recent studies have gained a better appreciation of the potential impacts of enteric infections beyond symptomatic diarrhea. It is recognized that infections by several enteropathogens could be associated with growth deficits in children and intestinal and systemic inflammation may play an important underlying role. With enterotoxigenic E. coli (ETEC) being one of the leading causes of diarrhea among children in the developing world and important contributor to stunting, a better understanding of the impact of ETEC infection beyond diarrhea is timely and greatly needed. To address this, we evaluated if ETEC infection induces intestinal and systemic inflammation and its impact on colonization and immune responses to ETEC vaccine-specific antigens in a dose descending experimental human challenge model using ETEC strain H10407. This study demonstrates that the concentrations of myeloperoxidase (MPO) in stool and intestinal fatty acid-binding protein (an indicator of compromised intestinal epithelial integrity) in serum, significantly increased following ETEC infection in both diarrhea and asymptomatic cases and the magnitudes and kinetics of MPO are dose and clinical outcome dependent. Cytokines IL-17A and IFN-γ were significantly increased in serum post-ETEC challenge. In addition, higher pre-challenge concentrations of cytokines IL-10 and GM-CSF were associated with protection from ETEC diarrhea. Interestingly, higher MPO concentrations were associated with higher intestinal colonization of ETEC and lower seroconversions of colonization factor I antigen, but the reverse was noted for seroconversions to heat-labile toxin B-subunit. Together this study has important implications for understanding the acute and long-term negative health outcomes associated with ETEC infection.

Respiratory FimA-Specific Secretory IgA Antibodies Upregulated by DC-Targeting Nasal Double DNA Adjuvant Are Essential for Elimination of Porphyromonas gingivalis.

Our previous studies showed that a combination of a DNA plasmid encoding Flt3 ligand (pFL) and CpG oligodeoxynucleotides 1826 (CpG ODN) (FL/CpG) as a nasal adjuvant provoked antigen-specific immune responses. In this study, we investigated the efficacy of a nasal vaccine consisting of FimA as the structural subunit of Porphyromonas gingivalis (P. gingivalis) fimbriae and FL/CpG for the induction of FimA-specific antibody (Ab) responses and their protective roles against nasal and lung infection by P. gingivalis, a keystone pathogen in the etiology of periodontal disease. C57BL/6 mice were nasally immunized with recombinant FimA (rFimA) plus FL/CpG three times at weekly intervals. As a control, mice were given nasal rFimA alone. Nasal washes (NWs) and bronchoalveolar lavage fluid (BALF) of mice given nasal rFimA plus FL/CpG resulted in increased levels of rFimA-specific secretory IgA (SIgA) and IgG Ab responses when compared with those in controls. Significantly increased numbers of CD8- or CD11b-expressing mature-type dendritic cells (DCs) were detected in the respiratory inductive and effector tissues of mice given rFimA plus FL/CpG. Additionally, significantly upregulated Th1/Th2-type cytokine responses by rFimA-stimulated CD4+ T cells were noted in the respiratory effector tissues. When mice were challenged with live P. gingivalis via the nasal route, mice immunized nasally with rFimA plus FL/CpG inhibited P. gingivalis colonization in the nasal cavities and lungs. In contrast, controls failed to show protection. Of interest, when IgA-deficient mice given nasal rFimA plus FL/CpG were challenged with nasal P. gingivalis, the inhibition of bacterial colonization in the respiratory tracts was not seen. Taken together, these results show that nasal FL/CpG effectively enhanced DCs and provided balanced Th1- and Th2-type cytokine response-mediated rFimA-specific IgA protective immunity in the respiratory tract against P. gingivalis. A nasal administration with rFimA and FL/CpG could be a candidate for potent mucosal vaccines for the elimination of inhaled P. gingivalis in periodontal patients.

Anti-CfaE nanobodies provide broad cross-protection against major pathogenic enterotoxigenic Escherichia coli strains, with implications for vaccine design.

Enterotoxigenic Escherichia coli (ETEC) is estimated to cause approximately 380,000 deaths annually during sporadic or epidemic outbreaks worldwide. Development of vaccines against ETEC is very challenging due to the vast heterogeneity of the ETEC strains. An effective vaccines would have to be multicomponent to provide coverage of over ten ETEC strains with genetic variabilities. There is currently no vaccine licensed to prevent ETEC. Nanobodies are successful new biologics in treating mucosal infectious disease as they recognize conserved epitopes on hypervariable pathogens. Cocktails consisting of multiple nanobodies could provide even broader epitope coverage at a lower cost compared to monoclonal antibodies. Identification of conserved epitopes by nanobodies can also assist reverse engineering of an effective vaccine against ETEC. By screening nanobodies from immunized llamas and a naïve yeast display library against adhesins of colonization factors, we identified single nanobodies that show cross-protective potency against eleven major pathogenic ETEC strains in vitro. Oral administration of nanobodies led to a significant reduction of bacterial colonization in animals. Moreover, nanobody-IgA fusion showed extended inhibitory activity in mouse colonization compared to commercial hyperimmune bovine colostrum product used for prevention of ETEC-induced diarrhea. Structural analysis revealed that nanobodies recognized a highly-conserved epitope within the putative receptor binding region of ETEC adhesins. Our findings support further rational design of a pan-ETEC vaccine to elicit robust immune responses targeting this conserved epitope.

Dust mite-derived Enterobacterial fimbriae H protein enforces the allergen specific immunotherapy in asthma mice

BACKGROUND: The mite alimentary canal contains plenty of microbiota. It is accepted that some of the microbial products function as adjuvants to speed up immune responses. OBJECTIVES: We identified five bacterial proteins from dust mite, and Enterobacterial fimbriae H (FimH) was one of them. This study aims to test a hypothesis that the FimH protein enforces immunotherapy in asthmatic mice. METHODS: Asthmatic mice were treated by allergen specific immunotherapy (ASIT) with rDer f1/f2 or rDer f1/f2 plus FimH. Changes in inflammatory cell infiltration, airway hyperreactivity, frequency of Tregs, splenic CD4+IFN-Gamma+ cells, and serum levels of TGF-Beta, IL-10, IL-13 and IL-17A of asthmatic mice were checked. RESULTS: ASIT with rDer f1/f2 plus FimH reduced inflammatory cell infiltration, airway hyperreactivity (AHR), and levels of IgE and IgG1 compared to ASIT with rDer f1/f2 alone, but the levels of IgG2a increased. Asthmatic mice that underwent ASIT with rDer f1/f2 plus FimH showed increased frequency of Tregs, splenic CD4+IFN-Gamma+ cells, serum levels of TGF-Beta and IL-10; and deceased splenic CD4+IL-4+ cells, and serum levels of IL-13 and IL-17A. In vitro study showed FimH triggered IL-10 expression in a concentration dependent manner and facilitated the differentiation of Tregs. CONCLUSION: Used as an adjuvant, FimH enforces the effect of ASIT in asthmatic mice via augmenting Tregs

No disponible

Protective effects of egg yolk immunoglobulins (IgYs) developed against recombinant immunogens CtxB, OmpW and TcpA on infant mice infected with Vibrio cholerae.

Vibrio cholerae causes cholera and other infections, especially in children under five years of age. Cholera toxin (CT), toxin-coregulated pilus (TCP) and outer membrane protein W (OmpW) are three major virulence factors of this bacterium. The emergence of antimicrobial-resistant (AMR) strains and the absence of a comprehensive and flawless vaccine, has prompted other treatments. There are several advantages of egg yolk antibodies (IgY) over other immunotherapy agents, such as economic feasibility, high yield simple production, and better immune responsiveness to mammalian antigens due to phylogenetic distance. Accordingly, in the present study, IgYs against recombinant proteins CtxB (responsible for the CT binding to eukaryotic cells), TcpA (enhances bacterial attachment to enterocytes) and OmpW were produced, in single, coupled or combined forms, to evaluate and compare their protectivity potency. Immunoreactivity of IgYs were examined through protein and whole cell ELISA, their specificity was confirmed by western blotting, and their neutralizing effects on CT was evaluated in Y1 cell culture. Produced IgYs were gavage administered to different groups of infant mice infected with V. cholerae. The results indicated that IgYs produced against CtxB had the highest titers, and were able to neutralize cytotoxicity effects in Y1 cell culture, while the highest protection in the mice challenge was obtained by IgY-TcpA. No considerable increase was observed in immunoreactivity or protectivity of antibodies produced against combined antigens. The produced IgYs showed a good antigen-specificity and protectivity which can be used in passive immunotherapy against cholera.

Generating mucosal and systemic immune response following vaccination of vibrio cholerae adhesion molecule against shigella flexneri infection.

Introduction: Previous studies have shown 37.8 kDa pili subunit protein of Vibrio cholerae and 49.8 kDa pili subunit protein of Shigella flexneri can act as an adhesion molecule to initiate infection. These molecules also have the ability to agglutinate blood. The present study assessed mucosal and systemic immunity following vaccination using 37.8 kDa V. cholerae and protection against S. flexneri. Subjects and Methods: Haemagglutination test was performed after purification of V. cholerae protein, followed by an anti-haemagglutination test. The intestinal weight and colony count were used to validate the protective effect on balb/c mice which were divided into the naive group, Shigella-positive control group, Vibrio-positive control group, V. cholerae infected-Vibrio-vaccinated group and S. flexneri-infected-Vibrio-vaccinated group. Th17, Treg, interleukin (IL) IL-17A, ß-defensin and secretory-immunoglobulin A (s-IgA) were also measured to determine the systemic and mucosal immunity after vaccination. Results: The haemagglutination and anti-haemagglutination tests showed that the 37.8 kDa protein could inhibit 49.8 kDa of the S. flexneri pili subunit. Decreased intestinal weight and colony count of vaccinated group compared to naive group also support cross reaction findings. Vaccination also generates higher level of Th17, Treg, IL-17A, ß-defensin and s-IgA significantly. Conclusions: 37.8 kDa subunit pili can act as a homologous vaccine candidate to prevent V. cholerae and S. flexneri infection.

Nontypeable Haemophilus influenzae Type IV Pilus Mediates Augmented Adherence to Rhinovirus-Infected Human Airway Epithelial Cells.

Human rhinovirus (hRV) is frequently detected in the upper respiratory tract, and symptomatic infection is associated with an increased nasopharyngeal bacterial load, with subsequent development of secondary bacterial diseases. Nontypeable Haemophilus influenzae (NTHI) is a commensal bacterial species of the human nasopharynx; however, in the context of prior or concurrent upper respiratory tract viral infection, this bacterium commonly causes multiple diseases throughout the upper and lower respiratory tracts. The present study was conducted to determine the mechanism(s) by which hRV infection promotes the development of NTHI-induced diseases. We showed that hRV infection of polarized primary human airway epithelial cells resulted in increased adherence of NTHI, due in part to augmented expression of CEACAM1 and ICAM1, host cell receptors to which NTHI binds via engagement of multiple adhesins. Antibody blockade of these host cell receptors significantly reduced NTHI adherence. With a specific focus on the NTHI type IV pilus (T4P), which we have previously shown binds to ICAM1, an essential adhesin and virulence determinant, we next showed that T4P-directed antibody blockade significantly reduced NTHI adherence to hRV-infected airway cells and, further, that expression of this adhesin was required for the enhanced adherence observed. Collectively, these data provide a mechanism by which "the common cold" promotes diseases due to NTHI, and they add further support for the use of PilA (the majority subunit of T4P) as a vaccine antigen, since antibodies directed against PilA are expected to limit the notably increased bacterial load associated with hRV coinfection and thereby to prevent secondary NTHI-induced diseases of the respiratory tract.

Recombinant PAL/PilE/FlaA DNA vaccine provides protective immunity against Legionella pneumophila in BALB/c mice.

BACKGROUND: Legionella pneumophila (L.pneumophila), a Gram-negative small microorganism, causes hospital-acquired pneumonia especially in immunocompromised patients. Vaccination may be an effective method for preventing L.pneumophila infection. Therefore, it is necessary to develop a better vaccine against this disease. In this study, we developed a recombinant peptidoglycan-associated lipoprotein (PAL)/type IV pilin (PilE)/lagellin (FlaA) DNA vaccine and evaluated its immunogenicity and efficacy to protect against L.pneumophila infection. RESULTS: According to the results, the expression of PAL, PilE, FlaA proteins and PAL/PilE/FlaA fusion protein in 293 cells was confirmed. Immunization with PAL/PilE/FlaA DNA vaccine resulted in highest IgG titer and strongest cytotoxic T-lymphocyte (CTL) response. Furthermore, the histopathological changes in lung tissues of mice challenged with a lethal dose of L.pneumophila were alleviated by PAL/PilE/FlaA DNA vaccine immunization. The production of T-helper-1 (Th1) cytokines (IFNγ, TGF-α, and IL-12), and Th2 cytokines (IL-4 and IL-10) were promoted in PAL/PilE/FlaA DNA vaccine group. Finally, immunization with PAL/PilE/FlaA vaccine raised the survival rate of mice to 100% after challenging with a lethal dose of L.pneumophila for 10 consecutive days. CONCLUSIONS: Our study suggests that the newly developed PAL/PilE/FlaA DNA vaccine stimulates strong humoral and cellular immune responses and may be a potential intervention on L.pneumophila infection.

Escherichia coli adhesion portion FimH functions as an adjuvant for cancer immunotherapy.

Induction of antigen-specific immune activation by the maturation of dendritic cells (DCs) is a strategy used for cancer immunotherapy. In this study, we find that FimH, which is an Escherichia coli adhesion portion, induces toll-like receptor 4-dependent and myeloid differentiation protein 2-independent DC maturation in mice in vivo. A combined treatment regimen with FimH and antigen promotes antigen-specific immune activation, including proliferation of T cells, production of IFN-γ and TNF-α, and infiltration of effector T cells into tumors, which consequently inhibits tumor growth in mice in vivo against melanoma and carcinoma. In addition, combined therapeutic treatment of anti-PD-L1 antibodies and FimH treatment efficiently inhibits CT26 tumor growth in BALB/c mice. Finally, FimH promotes human peripheral blood DC activation and syngeneic T-cell proliferation and activation. Taken together, these findings demonstrate that FimH can be a useful adjuvant for cancer immunotherapy.

Construction and development of FimH lectin domain for rising immune response after injection by uropathogenic E. coli.

Uropathogenic E. coli is one of the major agents of urinary tract infection. Today, no effective treatment or vaccine against this infection is exist. Accordingly, in the present study, a genetic constrruct for inducing of cellular immune system was designed. At first, fimH gene from E. coli 35218 was amplified using PCR. PCR product inserted into pET23a expression vector and the recombinant vector was analysed by sequencing. The vector was transformed to E. coli strain Origami and the protein was expressed under the 1 mM IPTG. FimH was purified with Ni-NTA column and the purified protein was used for immunization of BALB/c. Two weeks after the last injection, lymphocyte proliferation assay was carried out. In addition, IL-4 and IFN-γ cytokines, total antibody serum, IgG1 and IgG2a isotypes were quantified. Finally, protection ability of the vaccine in bladder and kidney infection of mice was evaluated.The results indicated that cellular immune response has a main protective role against UTI and FimH, as a vaccine candidate, significantly increase lymphocyte proliferation, IFN-γ response and total antibody amount. Immunization of mice with FimH conferred effective protection of kidney and bladder against urinary tract infection by uropathogenic E. coli (P< 0.002). It can be concluded that, the current FimH will be valuable for more trying to prepare a new vaccine against UTI.

Biochemical and immunological characterization of an ETEC CFA/I adhesin cholera toxin B subunit chimera.

Surface-expressed colonization factors and their subunits are promising candidates for inclusion into a multivalent vaccine targeting enterotoxigenic Escherichia coli (ETEC), a leading cause of acute bacterial diarrhea in developing regions. However, soluble antigens are often poorly immunogenic in the absence of an adjuvant. We show here that the serum immune response to CfaE, the adhesin of the ETEC colonization factor CFA/I, can be enhanced in BALB/c mice by immunization with a chimeric antigen containing CfaE and pentameric cholera toxin B subunit (CTB) of cholera toxin from Vibrio cholerae. We constructed this antigen by replacing the coding sequence for the A1 domain of the cholera toxin A subunit (CTA) with the sequence of donor strand complemented CfaE (dscCfaE) within the cholera toxin operon, resulting in a dscCfaE-CTA2 fusion. After expression, via non-covalent interactions between CTA2 and CTB, the fusion and CTB polypeptides assemble into a complex containing a single dscCfaE-CTA2 protein bound to pentameric CTB (dscCfaE-CTA2/CTB). This holotoxin-like chimera retained the GM1 ganglioside binding activity of CTB, as well as the ability of CfaE to mediate the agglutination of bovine red blood cells when adsorbed to polystyrene beads. When administered intranasally to mice, the presence of CTB in the chimera significantly increased the serum immune response to CfaE compared to dscCfaE alone, stimulating a response similar to that obtained with a matched admixture of dscCfaE and CTB. However, by the orogastric route, immunization with the chimera elicited a superior functional immune response compared to an equivalent admixture of dscCfaE and CTB, supporting further investigation of the chimera as an ETEC vaccine candidate.

Immunogenicity of 987P fimbriae of enterotoxigenic Escherichia coli surface-displayed on Lactobacillus casei.

As most pathogens invade the bodies through the mucosa, it is crucial to develop vaccines that induce mucosal immunity. To this end, we generated a safe and effective vaccine candidate that displayed fimbrial protein 987P of enterotoxigenic Escherichia coli (ETEC) on the surface of Lactobacillus casei (L.casei) CICC 6105 by using poly-γ-glutamate synthetase A (PgsA) as an anchoring matrix. After gavage inoculation of the recombinant strain pLA-987P/L.casei into specific-pathogen-free (SPF) BALB/c mice, high levels of mucosal immunoglobulin A (IgA) were induced in fecal samples, intestine and lung lavage fluids and systemic immunoglobulin G of IgG subclasses (IgG1, IgG2b, and IgG2a) was produced in serum. T-cell proliferation assays showed the stimulation index (SI) of the groups immunized with pLA-987P/L.casei to be significantly higher than that of the control group. The recombinant L.casei promoted T cells to produce both Th1 and Th2 cytokines, while the number of splenic IL-4 Spot forming cells (SFC) exceeded the number of IFN-γ SFC by 2.26-fold (P < .01). >83.3% of the vaccinated mice were protected from challenge with a lethal dose of virulent strain C83916. These results indicate that the recombinant L.casei expressing ETEC 987P fimbrial protein could elicit a protective immune response against ETEC 987P infection effectively.