RIPK3 Promotes Mefv Expression and Pyrin Inflammasome Activation via Modulation of mTOR Signaling.

Mutations in MEFV, the gene encoding pyrin in humans, are associated with the autoinflammatory disorder familial Mediterranean fever. Pyrin is an innate sensor that assembles into an inflammasome complex in response to Rho-modifying toxins, including Clostridium difficile toxins A and B. Cell death pathways have been shown to intersect with and modulate inflammasome activation, thereby affecting host defense. Using bone marrow-derived macrophages and a murine model of peritonitis, we show in this study that receptor-interacting protein kinase (RIPK) 3 impacts pyrin inflammasome activation independent of its role in necroptosis. RIPK3 was instead required for transcriptional upregulation of Mefv through negative control of the mechanistic target of rapamycin (mTOR) pathway and independent of alterations in MAPK and NF-κB signaling. RIPK3 did not affect pyrin dephosphorylation associated with inflammasome activation. We further demonstrate that inhibition of mTOR was sufficient to promote Mefv expression and pyrin inflammasome activation, highlighting the cross-talk between the mTOR pathway and regulation of the pyrin inflammasome. Our study reveals a novel interaction between molecules involved in cell death and the mTOR pathway to regulate the pyrin inflammasome, which can be harnessed for therapeutic interventions.

Murine Intrarectal Instillation of Purified Recombinant Clostridioides difficile Toxins Enables Mechanistic Studies of Pathogenesis.

Clostridioides difficile is linked to nearly 225,000 antibiotic-associated diarrheal infections and almost 13,000 deaths per year in the United States. Pathogenic strains of C. difficile produce toxin A (TcdA) and toxin B (TcdB), which can directly kill cells and induce an inflammatory response in the colonic mucosa. Hirota et al. (S. A. Hirota et al., Infect Immun 80:4474-4484, 2012) first introduced the intrarectal instillation model of intoxication using TcdA and TcdB purified from VPI 10463 (VPI 10463 reference strain [ATCC 43255]) and 630 C. difficile strains. Here, we expand this technique by instilling purified, recombinant TcdA and TcdB, which allows for the interrogation of how specifically mutated toxins affect tissue. Mouse colons were processed and stained with hematoxylin and eosin for blinded evaluation and scoring by a board-certified gastrointestinal pathologist. The amount of TcdA or TcdB needed to produce damage was lower than previously reported in vivo and ex vivo Furthermore, TcdB mutants lacking either endosomal pore formation or glucosyltransferase activity resemble sham negative controls. Immunofluorescent staining revealed how TcdB initially damages colonic tissue by altering the epithelial architecture closest to the lumen. Tissue sections were also immunostained for markers of acute inflammatory infiltration. These staining patterns were compared to slides from a human C. difficile infection (CDI). The intrarectal instillation mouse model with purified recombinant TcdA and/or TcdB provides the flexibility needed to better understand structure/function relationships across different stages of CDI pathogenesis.

Macrophage migration inhibitory factor regulates innate γδ T-cell responses via IL-17 expression.

T cells expressing invariant γδ antigen receptors (γδ T cells) bridge innate and adaptive immunity and facilitate barrier responses to pathogens. Macrophage migration inhibitory factor (MIF) is an upstream mediator of host defense that up-regulates the expression of pattern recognition receptors and sustains inflammatory responses by inhibiting activation-induced apoptosis in monocytes and macrophages. Surprisingly, Mif-/- γδ T cells, when compared with wild type, were observed to produce >10-fold higher levels of the proinflammatory cytokine IL-17 after stimulation with gram-positive exotoxins. High-IL-17 expression was associated with the characteristic features of IL-17-producing γδ T (γδ17) cells, including expression of IL-23R, IL-1R1, and the transcription factors RORγt and Sox13. In the gram-positive model of shock mediated by toxic shock syndrome toxin (TSST-1), Mif-/- mice succumbed to death more quickly with increased pulmonary neutrophil accumulation and higher production of cytokines, including IL-1ß and IL-23. Mif-/- γδ T cells also produced high levels of IL-17 in response to Mycobacterium lipomannan, and depletion of γδ T cells improved survival from acutely lethal Mycobacterium infection or TSST-1 administration. These data indicate that MIF deficiency is associated with a compensatory amplification of γδ17 cell responses, with implications for innate immunity and IL-17-mediated pathology in situations such as gram-positive toxic shock or Mycobacterium infection.-Kim, H. K., Garcia, A. B., Siu, E., Tilstam, P., Das, R., Roberts, S., Leng, L., Bucala, R. Macrophage migration inhibitory factor regulates innate γδ T-cell responses via IL-17 expression.

Immunizations with Enterotoxigenic Escherichia coli Heat-Stable Toxin Conjugates Engender Toxin-Neutralizing Antibodies in Mice That Also Cross-React with Guanylin and Uroguanylin.

Infection with enterotoxigenic Escherichia coli (ETEC) is a common cause of childhood diarrhea in low- and middle-income countries, as well as of diarrhea among travelers to these countries. In children, ETEC strains secreting the heat-stable toxin (ST) are the most pathogenic, and there are ongoing efforts to develop vaccines that target ST. One important challenge for ST vaccine development is to construct immunogens that do not elicit antibodies that cross-react with guanylin and uroguanylin, which are endogenous peptides involved in regulating the activity of the guanylate cyclase-C (GC-C) receptor. We immunized mice with both human ST (STh) and porcine ST (STp) chemically coupled to bovine serum albumin, and the resulting sera neutralized the toxic activities of both STh and STp. This suggests that a vaccine based on either ST variant can confer cross-protection. However, several anti-STh and anti-STp sera cross-reacted with the endogenous peptides, suggesting that the ST sequence must be altered to reduce the risk of unwanted cross-reactivity. Epitope mapping of four monoclonal anti-STh and six anti-STp antibodies, all of which neutralized both STh and STp, revealed that most epitopes appear to have at least one amino acid residue shared with guanylin or uroguanylin. Despite this, only one monoclonal antibody displayed demonstrable cross-reactivity to the endogenous peptides, suggesting that targeted mutations of a limited number of ST residues may be sufficient to obtain a safe ST-based vaccine.

Interleukin-10 (IL-10) Produced by Mutant Toxic Shock Syndrome Toxin 1 Vaccine-Induced Memory T Cells Downregulates IL-17 Production and Abrogates the Protective Effect against Staphylococcus aureus Infection.

Development of long-term memory is crucial for vaccine-induced adaptive immunity against infectious diseases such as Staphylococcus aureus infection. Toxic shock syndrome toxin 1 (TSST-1), one of the superantigens produced by S. aureus, is a possible vaccine candidate against infectious diseases caused by this pathogen. We previously reported that vaccination with less toxic mutant TSST-1 (mTSST-1) induced T helper 17 (Th17) cells and elicited interleukin-17A (IL-17A)-mediated protection against S. aureus infection 1 week after vaccination. In the present study, we investigated the host immune response induced by mTSST-1 vaccination in the memory phase, 12 weeks after the final vaccination. The protective effect and IL-17A production after vaccination with mTSST-1 were eliminated because of IL-10 production. In the presence of IL-10-neutralizing monoclonal antibody (mAb), IL-17A production was restored in culture supernatants of CD4+ T cells and macrophages sorted from the spleens of vaccinated mice. Vaccinated mice treated with anti-IL-10 mAb were protected against systemic S. aureus infection in the memory phase. From these results, it was suggested that IL-10 produced in the memory phase suppresses the IL-17A-dependent vaccine effect through downregulation of IL-17A production.

Induced Proton Perturbation for Sensitive and Selective Detection of Tight Junction Breakdown.

Tight junctions (TJs) in the epithelial cell gap play primary roles in body defense and nutrient absorption in multicellular organisms. Standard in vitro assays lack sensitivity, selectivity, temporal resolution, and throughput for bioengineering applications. Prompted by the rigorous barrier functions of TJ, we developed a TJ assay that senses proton leaks in the cell gap using ion-sensitive field-effect transistors. Upon exposure of Madin-Darby canine kidney (MDCK) cells cultured on a gate dielectric to a calcium-chelator EGTA, ammonia-assisted pH perturbation was enhanced solely in TJ-forming cells. This was supported by simulations with increased ion permeability in the paracellular pathway. Following administration of Clostridium perfringens enterotoxin as a specific TJ inhibitor to the MDCK cells, our method detected TJ breakdown at a 13× lower concentration than a conventional trans-epithelial electrical resistance assay. Thus, the semiconductor-based active pH sensing could offer an alternative to current in vitro assays for TJs in pathological, toxicological, and pharmaceutical studies.

Different frequencies of memory B-cells induced by tetanus, botulinum, and heat-labile toxin binding domains.

Along with robust immunogenicity, an ideal vaccine candidate should be able to produce a long lasting protection. In this regard, the frequency of memory B-cells is possibly an important factor in memory B-cell persistency and duration of immunological memory. On this basis, binding domains of tetanus toxin (HcT), botulinum type A1 toxin (HcA), and heat-labile toxin (LTB) were selected as antigen models that induced long-term, midterm and short-term immune memory, respectively. In the present study, the frequency of total memory B-cells after immunization with HcT, HcA and LTB antigens after 90 and 180 days, and also after one booster, in 190 days, was evaluated. The results showed a significant correlation between frequency of total memory B-cells and duration of humoral immunity. Compared to other antigens, the HcT antibody titers and HcT total memory B-cell populations were greater and persistent even after 6 months. At 6 months after the final immunization, all HcT- and HcA-immunized mice survived against tetanus and botulinum toxins, and also LT toxin binding to GM1 ganglioside was blocked in LTB-immunized mice. We conclude the frequency of memory B-cells and their duration are likely a key factor for vaccine memory duration.

Significance of Enterotoxigenic Escherichia coli (ETEC) Heat-Labile Toxin (LT) Enzymatic Subunit Epitopes in LT Enterotoxicity and Immunogenicity.

Enterotoxigenic Escherichia coli (ETEC) strains producing heat-labile toxin (LT) and/or heat-stable toxin (STa) are a top cause of children's diarrhea and travelers' diarrhea. Holotoxin-structured GM1-binding LT is a strong immunogen and an effective adjuvant, and can serve a carrier or a platform for multivalent vaccine development. However, the significance of peptide domains or epitopes of LT particularly enzymatic LTA subunit in association with LT enterotoxicity and immunogenicity has not been characterized. In this study, we identified B-cell epitopes in silico from LTA subunit and examined epitopes for immunogenicity and association with LT enterotoxicity. Epitopes identified from LTA subunit were individually fused to a modified chicken ovalbumin carrier protein, and each epitope-ovalbumin fusion was used to immunize mice. Data showed all 11 LTA epitopes were immunogenic; epitope 7 (105SPHPYEQEVSA115) induced greater titers of anti-LT antibodies which neutralized LT enterotoxicity more effectively. To examine these epitopes for the significance in LT enterotoxicity, we constructed LT mutants by substituting each of 10 epitopes at the toxic A1 domain of LTA subunit with a foreign epitope and examined LT mutants for enterotoxicity and GM1-binding activity. Data showed that LT mutants exhibited no enterotoxicity but retained GM1-binding activity. The results from this study indicated that while not all immunodominant LTA epitopes were neutralizing, LT mutants with an individual epitope substituted lost enterotoxicity but retained GM1-binding activity. These results provided additional information to understand LT immunogenicity and enterotoxicity and suggested the potential application of LT platform for multivalent vaccines against ETEC diarrhea and other diseases.IMPORTANCE No vaccine is licensed for enterotoxigenic Escherichia coli (ETEC) strains, which remain a leading cause of diarrhea in children from developing countries and international travelers. GM1-binding heat-labile toxin (LT) which is a key virulence factor of ETEC diarrhea is a strong vaccine antigen and a self-adjuvant. LT can also serve a backbone or platform for MEFA (multiepitope fusion antigen), a newly developed structural vaccinology technology, to present heterogeneous epitopes (by replacing LT epitopes) and to mimic epitope antigenicity for development of broadly protective vaccines. Data from this study identified neutralizing LT epitopes and demonstrated that substitution of LT epitopes eliminated LT enterotoxicity without altering GM1-binding activity, suggesting LT is potentially a versatile MEFA platform to present heterogeneous epitopes for multivalent vaccines against ETEC and other pathogens.

TNF and CD28 Signaling Play Unique but Complementary Roles in the Systemic Recruitment of Innate Immune Cells after Staphylococcus aureus Enterotoxin A Inhalation.

Staphylococcus aureus enterotoxins cause debilitating systemic inflammatory responses, but how they spread systemically and trigger inflammatory cascade is unclear. In this study, we showed in mice that after inhalation, Staphylococcus aureus enterotoxin A rapidly entered the bloodstream and induced T cells to orchestrate systemic recruitment of inflammatory monocytes and neutrophils. To study the mechanism used by specific T cells that mediate this process, a systems approach revealed inducible and noninducible pathways as potential targets. It was found that TNF caused neutrophil entry into the peripheral blood, whereas CD28 signaling, but not TNF, was needed for chemotaxis of inflammatory monocytes into blood and lymphoid tissue. However, both pathways triggered local recruitment of neutrophils into lymph nodes. Thus, our findings revealed a dual mechanism of monocyte and neutrophil recruitment by T cells relying on overlapping and nonoverlapping roles for the noninducible costimulatory receptor CD28 and the inflammatory cytokine TNF. During sepsis, there might be clinical value in inhibiting CD28 signaling to decrease T cell-mediated inflammation and recruitment of innate cells while retaining bioactive TNF to foster neutrophil circulation.

Induction of mucosal immune responses against Helicobacter pylori infection after sublingual and intragastric route of immunization.

There is a current lack of effective mucosal vaccines against major gastroenteric pathogens and particularly against Helicobacter pylori, which causes a chronic infection that can lead to peptic ulcers and gastric cancer in a subpopulation of infected individuals. Mucosal CD4+ T-cell responses have been shown to be essential for vaccine-induced protection against H. pylori infection. The current study addresses the influence of the adjuvant and site of mucosal immunization on early CD4+ T-cell priming to H. pylori antigens. The vaccine formulation consisted of H. pylori lysate antigens and mucosal adjuvants, cholera toxin (CT) or a detoxified double-mutant heat-labile enterotoxin from Escherichia coli (dmLT), which were administered by either the sublingual or intragastric route. We report that in vitro, adjuvants CT and dmLT induce up-regulation of pro-inflammatory gene expression in purified dendritic cells and enhance the H. pylori-specific CD4+ T-cell response including interleukin-17A (IL-17A), interferon-γ (IFN-γ) and tumour necrosis factor-α (TNF-α) secretion. In vivo, sublingual immunization led to an increased frequency of IL-17A+ , IFN-γ+ and TNF-α+ secreting CD4+ T cells in the cervical lymph nodes compared with in the mesenteric lymph nodes after intragastric immunization. Subsequently, IL-17A+ cells were visualized in the stomach of sublingually immunized and challenged mice. In summary, our results suggest that addition of an adjuvant to the vaccine clearly activated dendritic cells, which in turn, enhanced CD4+ T-cell cytokines IL-17A, IFN-γ and TNF-α responses, particularly in the cervical lymph nodes after sublingual vaccination.

Therapeutic blockade of CD54 attenuates pulmonary barrier damage in T cell-induced acute lung injury.

Acute respiratory distress syndrome (ARDS) is a serious, often fatal condition without available pharmacotherapy. Although the role of innate cells in ARDS has been studied extensively, emerging evidence suggests that T cells may be involved in disease etiology. Staphylococcus aureus enterotoxins are potent T-cell mitogens capable of triggering life-threatening shock. We demonstrate that 2 days after inhalation of S. aureus enterotoxin A, mice developed T cell-mediated increases in vascular permeability, as well as expression of injury markers and caspases in the lung. Pulmonary endothelial cells underwent sequential phenotypic changes marked by rapid activation coinciding with inflammatory events secondary to T-cell priming, followed by reductions in endothelial cell number juxtaposing simultaneous T-cell expansion and cytotoxic differentiation. Although initial T-cell activation influenced the extent of lung injury, CD54 (ICAM-1) blocking antibody administered well after enterotoxin exposure substantially attenuated pulmonary barrier damage. Thus CD54-targeted therapy may be a promising candidate for further exploration into its potential utility in treating ARDS patients.

An engineered food-grade Lactococcus lactis strain for production and delivery of heat-labile enterotoxin B subunit to mucosal sites.

BACKGROUND: Recent researches have been focusing on mucosal immune adjuvants, which play the key roles in mucosal immunization and have become the limitation for non-injected vaccine development. Escherichia coli heat-labile enterotoxin B subunit (LTB) was regarded as a promising mucosal adjuvant for its nontoxicity and potent activity. LTB preparation issues have always been recurring, in part owing to that the recombinant LTB expressed by E. coli does not act as its native form. RESULTS: We constructed an engineered Lactococcus lactis strain using a food-grade expression system. The LTB secreted by the engineered strain was detected in the culture supernatant, constituting 10.3% of the supernatant proteins, and recognized by mouse anti-LTB antibodies. The engineered strain, co-administered orally to SPF BALB/c mice with a H. pylori vaccine candidate expressing Lpp20 antigen, could significantly enhance the Lpp20-induced mucosal SIgA antibody responses against H. pylori. CONCLUSIONS: This is the first report that LTB was efficiently produced and delivered via using a food-grade lactococcal expression system, which offers a novel production and utilization mode of this crucial mucosal adjuvant. The engineered L. lactis strain secreting LTB has considerable potential for oral vaccine formulation owing to its outstanding safety, adjuvant activity and high-level production.

Staphylococcal enterotoxin B administration in pregnant rats alters the splenic lymphocyte response in adult offspring rats.

BACKGROUND: Our previous study suggested that SEB exposure in pregnant rats could lead to the change of T cells subpopulation in both peripheral blood and thymus of the offspring rats. However, rarely is known about the influence of SEB exposure in pregnant rats on T cell subpopulation in the spleens of offspring rats. RESULTS: SEB was intravenously administered to the pregnant rats at gestational day 16 in this study. The percentages, in vivo and in vitro responses of CD4 and CD8 T cells were investigated with flow cytometry. The prenatal SEB exposure obviously increased splenic CD4 T cell percentages of both neonates and adult offspring rats, and obviously reduced splenic CD8 T cell percentages of both the fifth day neonates and adult offspring rats. After spleens in the adult offspring rats were re-stimulated with SEB in vivo or in vitro, in vivo SEB stimulation could lead to the marked decrease of splenic CD4 T cell percentage and the marked increase of splenic CD8 T cell percentage. While in vitro SEB stimulation to the cultured splenocytes markedly decreased the proliferation of the splenic lymphocytes and the CD4 T cell percentage, and had no influence on CD8 T cell percentage. CONCLUSION: The prenatal SEB exposure could alter the percentages of CD4/CD8 T cell subpopulation and the response of CD4 and CD8 T cells to the in vivo and in vitro secondary SEB stimulation in the splenocytes of adult offspring rats.

Immunogenicity of recombinant Lactobacillus casei-expressing F4 (K88) fimbrial adhesin FaeG in conjunction with a heat-labile enterotoxin A (LTAK63) and heat-labile enterotoxin B (LTB) of enterotoxigenic Escherichia coli as an oral adjuvant in mice.

AIMS: The aims of this study were to develop an effective oral vaccine against enterotoxigenic Escherichia coli (ETEC) infection and to design new and more versatile mucosal adjuvants. METHODS AND RESULTS: Genetically engineered Lactobacillus casei strains expressing F4 (K88) fimbrial adhesin FaeG (rLpPG-2-FaeG) and either co-expressing heat-labile enterotoxin A (LTA) subunit with an amino acid mutation associated with reduced virulence (LTAK63) and a heat-labile enterotoxin B (LTB) subunit of E. coli (rLpPG-2-LTAK63-co-LTB) or fused-expressing LTAK63 and LTB (rLpPG-2-LTAK63-fu-LTB) were constructed. The immunogenicity of rLpPG-2-FaeG in conjunction with rLpPG-2-LTAK63-co-LTB or rLpPG-2-LTAK63-fu-LTB as an orally administered mucosal adjuvant in mice was evaluated. Results showed that the levels of FaeG-specific serum IgG and mucosal sIgA, as well as the proliferation of lymphocytes, were significantly higher in mice orally co-administered rLpPG-2-FaeG and rLpPG-2-LTAK63-fu-LTB compared with those administered rLpPG-2-FaeG alone, and were lower than those co-administered rLpPG-2-FaeG and rLpPG-2-LTAK63-co-LTB. Moreover, effective protection was observed after challenge with F4+ ETEC strain CVCC 230 in mice co-administered rLpPG-2-FaeG and rLpPG-2-LTAK63-co-LTB or rLpPG-2-FaeG and rLpPG-2-LTAK63-fu-LTB group compared with those that received rLpPG-2-FaeG alone. CONCLUSIONS: rLpPG-2-FaeG showed greater immunogenicity in combination with LTAK63 and LTB as molecular adjuvants. SIGNIFICANCE AND IMPACT OF THE STUDY: Recombinant Lactobacillus provides a promising platform for the development of vaccines against F4+ ETEC infection.

Parenteral adjuvant potential of recombinant B subunit of Escherichia coli heat-labile enterotoxin.

BACKGROUND: The B subunit of Escherichia coli heat-labile enterotoxin (LTB) is a potent mucosal immune adjuvant. However, there is little information about LTB's potential as a parenteral adjuvant. OBJECTIVES: We aimed at evaluating and better understanding rLTB's potential as a parenteral adjuvant using the fused R1 repeat of Mycoplasma hyopneumoniae P97 adhesin as an antigen to characterise the humoral immune response induced by this construct and comparing it to that generated when aluminium hydroxide is used as adjuvant instead. METHODS: BALB/c mice were immunised intraperitoneally with either rLTBR1 or recombinant R1 adsorbed onto aluminium hydroxide. The levels of systemic anti-rR1 antibodies (total Ig, IgG1, IgG2a, and IgA) were assessed by enzyme-linked immunosorbent assay (ELISA). The ratio of IgG1 and IgG2a was used to characterise a Th1, Th2, or mixed Th1/Th2 immune response. FINDINGS: Western blot confirmed rR1, either alone or fused to LTB, remained antigenic; anti-cholera toxin ELISA confirmed that LTB retained its activity when expressed in a heterologous system. Mice immunised with the rLTBR1 fusion protein produced approximately twice as much anti-rR1 immunoglobulins as mice vaccinated with rR1 adsorbed onto aluminium hydroxide. Animals vaccinated with either rLTBR1 or rR1 adsorbed onto aluminium hydroxide presented a mixed Th1/Th2 immune response. We speculate this might be a result of rR1 immune modulation rather than adjuvant modulation. Mice immunised with rLTBR1 produced approximately 1.5-fold more serum IgA than animals immunised with rR1 and aluminium hydroxide. MAIN CONCLUSIONS: The results suggest that rLTB is a more powerful parenteral adjuvant than aluminium hydroxide when administered intraperitoneally as it induced higher antibody titres. Therefore, we recommend that rLTB be considered an alternative adjuvant, even if different administration routes are employed.

The role of purified Clostridium difficile glucosylating toxins in disease pathogenesis utilizing a murine cecum injection model.

Most pathogenic Clostridium difficile produce two major exotoxins TcdA and TcdB, in the absence of which the bacterium is non-pathogenic. While it is important to investigate the role of each toxin in the pathogenesis of C. difficile infection (CDI) using isogenic strains, it is impossible to precisely control the expression levels of individual toxins and exclude bacterial factors that may contribute to the toxins' effects during infection. In this study, we utilized an acute intestinal disease model by injecting purified toxins directly into mouse cecum after a midline laparotomy. We evaluated the physical condition of mice by clinical score and survival, and the intestinal tissue damage and inflammation by histology. Depending on the dose of the toxins, mice developed mild to severe colitis, experienced diarrhea or rapidly died. We found that both purified TcdA and TcdB were able to induce clinical disease, intestinal inflammation, and tissue damage that resembled CDI. TcdA was significantly faster in inducing intestinal inflammation and tissue damage, and was approximately five times more potent than TcdB in terms of inducing severe gut disease and death outcomes in mice. Moreover, we found that the two toxins had significant synergistic effects on disease induction. Comparison of the in vivo toxicity of TcdB from clinical strains revealed that TcdB from an epidemic RT 027 strain was more toxic than the others. Our study thus demonstrates that both TcdA and TcdB, independent of other factors from C. difficile bacterium, are able to cause disease that resembles CDI and highlights the importance of targeting both toxins for vaccines and therapeutics against the disease.

Positive Contribution of Adjuvanted Influenza Vaccines to the Resolution of Bacterial Superinfections.

BACKGROUND: Most preclinical studies assess vaccine effectiveness in single-pathogen infection models. This is unrealistic given that humans are continuously exposed to different commensals and pathogens in sequential and mixed infections. Accordingly, complications from secondary bacterial infection are a leading cause of influenza-associated morbidity and mortality. New vaccination strategies are needed to control infections on simultaneous fronts. METHODS: We compared different anti-influenza vaccines for their protective potential in a model of viral infection with bacterial superinfection. Mice were immunized with H1N1/A/California/7/2009 subunit vaccines, formulated with different adjuvants inducing either T-helper type 1 (Th1) (MF59 plus CpG)-, Th1/2 (MF59)-, or Th17 (LTK63)-prone immune responses and were sequentially challenged with mouse-adapted influenza virus H1N1/A/Puerto Rico/8/1934 and Staphylococcus aureus USA300, a clonotype emerging as a leading contributor in postinfluenza pneumonia in humans. RESULTS: Unadjuvanted vaccine controlled single viral infection, yet mice had considerable morbidity from viral disease and bacterial superinfection. In contrast, all adjuvanted vaccines efficiently protected mice in both conditions. Interestingly, the Th1-inducing formulation was superior to Th1/2 or Th17 inducers. CONCLUSIONS: Our studies should help us better understand how differential immunity to influenza skews immune responses toward coinfecting bacteria and discover novel modes to prevent bacterial superinfections in the lungs of persons with influenza.

Novel Strategy To Protect against Influenza Virus-Induced Pneumococcal Disease without Interfering with Commensal Colonization.

Streptococcus pneumoniae commonly inhabits the nasopharynx as a member of the commensal biofilm. Infection with respiratory viruses, such as influenza A virus, induces commensal S. pneumoniae to disseminate beyond the nasopharynx and to elicit severe infections of the middle ears, lungs, and blood that are associated with high rates of morbidity and mortality. Current preventive strategies, including the polysaccharide conjugate vaccines, aim to eliminate asymptomatic carriage with vaccine-type pneumococci. However, this has resulted in serotype replacement with, so far, less fit pneumococcal strains, which has changed the nasopharyngeal flora, opening the niche for entry of other virulent pathogens (e.g., Streptococcus pyogenes, Staphylococcus aureus, and potentially Haemophilus influenzae). The long-term effects of these changes are unknown. Here, we present an attractive, alternative preventive approach where we subvert virus-induced pneumococcal disease without interfering with commensal colonization, thus specifically targeting disease-causing organisms. In that regard, pneumococcal surface protein A (PspA), a major surface protein of pneumococci, is a promising vaccine target. Intradermal (i.d.) immunization of mice with recombinant PspA in combination with LT-IIb(T13I), a novel i.d. adjuvant of the type II heat-labile enterotoxin family, elicited strong systemic PspA-specific IgG responses without inducing mucosal anti-PspA IgA responses. This response protected mice from otitis media, pneumonia, and septicemia and averted the cytokine storm associated with septic infection but had no effect on asymptomatic colonization. Our results firmly demonstrated that this immunization strategy against virally induced pneumococcal disease can be conferred without disturbing the desirable preexisting commensal colonization of the nasopharynx.

Tagging staphylococcal enterotoxin B (SEB) with TGFaL3 for breast cancer therapy.

Recent research has attempted to direct superantigens towards tumors by means of tumor-targeted superantigen (TTS) strategy. In this study, we explored the antitumor property of TTS by fusing the third loop of transforming growth factor α (TGFαL3) to staphylococcal enterotoxin type B (SEB) and investigated the possibility of the therapeutic application of TGFαL3-SEB as a novel antitumor candidate in mice bearing breast cancer. Treatment was performed through intratumoral and intravenous injection of TGFαL3-SEB. Tumor size/volume, long-term survival, and cytokine secretion were assessed. In addition, the toxicity of each treatment on liver and kidneys was examined. Our results indicated that the relative tumor volume significantly increased in the mice receiving intratumoral TGFaL3-SEB (p < 0.05). Surprisingly, 5 out of the 14 mice were cleared from the tumor thoroughly in 10-25 days after intratumoral administration of TGFaL3-SEB. Quantification of cytokines clearly showed that the mice receiving intratumoral SEB significantly secreted higher interferon γ (IFN-γ) and tumor necrosis factor α (TNF-α) compared with the other groups (p < 0.05). The antitumor effect was followed by inhibition of cell proliferation (Ki-67) and micro vascularization (CD31). The highest and lowest levels of tumor necrosis were observed in the intratumoral administration of TGFαL3-SEB (85 %) and PBS (14 %), respectively. Intratumoral injection of TGFαL3-SEB increased the lifespan of the mice so 37.5 % of them could survive for more than 6 months (p < 0.05). Overall, our findings indicated that intratumoral administration of TGFαL3-SEB effectively inhibited the growth of breast tumors through induction of necrosis and suppressing proliferation and angiogenesis without systemic toxicity.

Induction of nasal polyps using house dust mite and Staphylococcal enterotoxin B in C57BL/6 mice.

BACKGROUND: The murine polyp model was developed previously using ovalbumin and Staphylococcus aureus enterotoxin B (SEB). Here, we established a model mimicking key aspects of chronic eosinophilic rhinosinusitis with nasal polyps using the house dust mite (HDM), a clinically relevant aeroallergen, co-administered with SEB. We assessed the inflammatory response and formation of nasal polypoid lesions in an experimental murine model using intranasal delivery of HDM and ovalbumin. METHODS: After induction of HDM-induced allergic rhinosinusitis in C57BL/6 mice, SEB (10ng) was instilled into the nasal cavity of mice for eight weeks. Phosphate-buffered saline-challenged mice served as control. Histopathological changes were evaluated using haematoxylin and eosin for overall inflammation, Sirius red for eosinophils, and periodic acid-Schiff stain for goblet cells. The distribution of mast cells in mouse nasal tissue was determined by immunohistochemistry. Serum total IgE was measured using enzyme-linked immunosorbent assay. RESULTS: Compared to mice treated with HDM only, the HDM+SEB-treated mice demonstrated nasal polypoid lesion formation and a significant increase in the number of secretory cells and eosinophilic infiltration. Moreover, mice challenged intranasally with HDM showed highly abundant mast cells in the nasal mucosa. In contrast, OVA+SEB-challenged mice showed a significantly lower degree of mast cell infiltration. CONCLUSION: We established an in vivo model of chronic allergic rhinosinusitis with nasal polypoid lesions using HDM aeroallergen. This study demonstrated that the HDM+SEB-induced murine polyp model could be utilised as a suitable model for nasal polyps, especially with both eosinophil and mast cell infiltration.