Poliovirus replicons encoding the B subunit of Helicobacter pylori urease protect mice against H. pylori infection

We developed a novel vaccine for Helicobacter pylori based on a poliovirus vector in which capsid genes were replaced with the gene for the B subunit of H. pylori urease (UreB). Mice were vaccinated with UreB or control (L1) replicon and challenged with H. pylori. Twenty percent of mice vaccinated prophylactically with UreB, but 80% vaccinated with L1, and then challenged with H. pylori became infected (P = 0.003). Seventy-three percent of mice with established H. pylori infection vaccinated therapeutically with UreB replicon cleared their infection compared to 33% vaccinated with L1 (P = 0.067). In therapeutically vaccinated mice with residual infection, UreB-vaccinated animals had fewer H. pylori than L1-vaccinated mice (P < 0.05). Anti-urease antibody titres in prophylactically, but not therapeutically, vaccinated mice were markedly higher in animals that received UreB versus L1 replicon (P = 0.01). Vaccination with poliovirus vector containing the gene for the B subunit of H. pylori urease provides significant prophylactic and strong therapeutic protection against H. pylori in mice.     

Poliovirus replicons encoding the B subunit of Helicobacter pylori urease elicit a Th1 associated immune response.

The development of a vaccine for Helicobacter pylori is a key strategy for reducing the worldwide prevalence of H. pylori infection. Although immunization with recombinant B subunit of H. pylori urease (ureB) has yielded promising results, for the most part, these studies relied on the use of strong adjuvant, cholera toxin, precluding the use in humans. Thus, the development of new vaccine strategies for H. pylori is essential. Previous studies from our laboratory have described a vaccine vector based on poliovirus in which foreign genes are substituted for the poliovirus capsid genes. The genomes encoding foreign proteins (replicons) are encapsidated into authentic poliovirions by providing the capsids in trans. To test the utility of replicons as a vaccine vector for H. pylori, a replicon was constructed which encodes ureB. Expression of ureB in cells from the replicon was demonstrated by metabolic labeling followed by immunoprecipitation with anti-urease antibodies. To investigate the immunogenicity of the replicons, mice containing the transgene for the receptor for poliovirus were immunized via the intramuscular route. Mice given three doses of replicons did not develop substantial antibodies to ureB as determined by Western blot analysis using lysates from H. pylori. In contrast, mice given two doses of replicon followed by a single injection of recombinant ureB developed serum antibodies to ureB which were predominately IgG2a. Splenic lymphocytes from mice immunized with replicons alone, or replicons plus recombinant ureB produced abundant interferon-gamma and no detectable interleukin-4 upon stimulation with recombinant ureB. These results establish that poliovirus replicons encoding H. pylori ureB are immunogenic and induce primarily a T helper 1 associated immune response.     

Transcutaneous immunization by merely prolonging the duration of antigen presence on the skin of mice induces a potent antigen-specific antibody response even in the absence of an adjuvant

Transcutaneous immunization (TCI) is a promising needle-free technique for vaccination. In this method, strong adjuvants, such as the cholera toxin, are generally crucial to elicit a robust immune response. Here, we showed that prolonged antigen presence on the skin of mice during TCI could effectively enhance the immune response. Substantial antigen-specific antibodies were produced in the sera of mice even after non-adjuvanted TCI when the antigen presence was for longer than 16 h. This non-adjuvanted TCI method was applied using the tetanus toxoid, and potent tetanus toxoid-specific antibodies were successfully induced in the sera of mice; they survived a lethal tetanus toxin challenge with no clinical signs. Thus, non-adjuvanted approach might be a possible option for TCI, and this method might improve the safety and practicality of transcutaneous vaccination.     

Expression of Helicobacter pylori urease subunit B gene in Lactococcus lactis MG1363 and its use as a vaccine delivery system against H. pylori infection in mice

The use of Lactococcus lactis as an antigen delivery vehicle for mucosal immunisation has been proposed. To determine whether L. lactis could effectively deliver Helicobacter pylori antigens to the immune system, a recombinant L. lactis expressing H. pylori urease subunit B (UreB) was constructed. Constitutive expression of UreB by a pTREX1 vector resulted in the intracellular accumulation of UreB to approximately 6.25% of soluble cellular protein. Five different oral regimens were used to vaccinate C57BL/6 mice and the immune response measured. One regimen, which consisted of four weekly doses of 10(10) bacteria, followed after an interval of approximately 4 weeks by three successive daily doses, was able to elicit a systemic antibody response to UreB in the mice, although subsequently, a similar regimen produced a significant antibody response in only one out of six mice. The other three regimes, in which mice were vaccinated with two or three sets of three consecutive daily doses of recombinant bacteria over 30 days, failed to elicit significant anti-UreB serum antibody responses. In three regimens, the immunised mice were then challenged by H. pylori strain SS1 and no protective effect was observed. These findings suggest that any adjuvant effects of L. lactis are unlikely to be sufficient to produce an effective immune response and to protect against H. pylori challenge, when used to deliver a weak immunogen, such as UreB.     

Short term renourishment of severely malnourished mice induces an enhanced rebound of the immune response after oral immunization

Mice fed a protein deficient diet containing 2% ovalbumin were renourished with a diet containing 20% ovalbumin for the final 4 or 8 days of a 6 week diet feeding period. The effect of this short term renourishment on the IgM and IgA immune response after oral immunization with sheep red blood cells was determined as the number of plaque forming cells (PFC) in the spleen. Both renourishment intervals resulted in a significant increase in body weight. Mice renourished for 4 days had a significantly enhanced IgA PFC response as compared to mice fed the low protein diet only. Mice renourished for 8 days had an even greater IgA PFC response and also had an enhanced number of IgM PFC. These responses were also significantly higher than previously reported values for the IgA and IgM PFC of mice fed the control diet for 6 weeks. These results indicate that the immune response to a mucosally encountered antigen recovers very quickly with renourishment, even to the extent of allowing an enhanced rebound of the PFC response.     

Synergistic effect of anti-Helicobacter pylori urease immunoglobulin Y from egg yolk of immunized hens and Lactobacillus johnsonii No.1088 to inhibit the growth of Helicobacter pylori in vitro and in vivo

Helicobacter pylori is a pathogenic bacterium that infects the stomach, causing chronic gastritis; and it is also considered to be related to the occurrence of gastric cancers. Although some eradication regimens including multiple antibiotics have been developed, the emergence of resistance to antibiotics becomes problematic. Therefore, other approaches to compensate or augment the effects of standard regimens are needed. In this study, we examined the possible synergistic effects of anti-H. pylori urease IgY and Lactobacillus johnsonii No.1088 (LJ88) both in vitro and in vivo. Anti-H. pylori urease IgY was purified from egg yolks laid by the hens immunized with urease purified from H. pylori. LJ88 is a unique strain of lactic acid bacterium isolated from human gastric juice, and it has been reported to inhibit H. pylori both in vitro and in vivo. The in vitro mixed culture study showed that anti-H. pylori urease IgY augmented the anti-H. pylori activity of LJ88 against both clarithromycin-sensitive and -resistant H. pylori strains. In a germ-free mice infection model, combined administration of daily anti-H. pylori urease IgY and weekly living LJ88 significantly reduced H. pylori infections, whereas either monotherapy did not. In an in vivo human gut microbiota-associated mice model, not only daily administration of living LJ88 but also heat-killed one significantly reduced an H. pylori infection in the stomach when combined with anti-H. pylori urease IgY. The extent of reduction of the stomach H. pylori by such a combination therapy was larger than that reported for LJ88 monotherapy. These results taken together revealed a synergistic effect of anti-H. pylori urease IgY and living or heat-killed LJ88, thus suggesting that such a combination might be a promising therapy to possibly compensate and/or augment standard anti-H. pylori regimens.     

Safety and immunogenicity of live recombinant Salmonella enterica serovar Typhi Ty21a expressing urease A and B from Helicobacter pylori in human volunteers.

Helicobacter pylori urease was expressed in the common live typhoid vaccine Ty21a yielding Ty21a(pDB1). Nine volunteers received Ty21a(pDB1) and three control volunteers received Ty21a. No serious adverse effects were observed in any of the volunteers. Ten out of 12 volunteers developed humoral immune responses to the Salmonella carrier as detected by antigen-specific antibody-secreting cells but only two volunteers seroconverted. A total of five volunteers showed responses in one or two out of three assays for cellular responses to the carrier (proliferation, IFN-gamma-secretion, IFN-gamma-ELISPOT). Three of the volunteers that had received Ty21a(pDB1) showed a weak but significant T-cell response to Helicobacter urease, while no volunteer had detectable humoral responses to urease. Ty21a(pDB1) is a suitable prototype to optimize Salmonella-based vaccination for efficient cellular responses that could mediate protective immunity against Helicobacter.     

A truncated variant of the hepatitis C virus core induces a slow but potent immune response in mice following DNA immunization

Vaccination of BALB/c mice with pIDKCo, a plasmid containing the coding sequence for the first 176 amino acids of the hepatitis C virus (HCV) core protein, induced both humoral and cellular specific immune responses. Particularly, the level of anti-core antibodies increased slowly with time up to a mean value above 1:8000 that was generally superior than that found in anti-HCV positive individuals. Six out of nine anti-HCV positive human sera were able to inhibit at different extent the binding of mouse anti-core sera to a recombinant capsid protein. Our results show that it is possible to elicit a potent humoral and cellular immune response against the HCV core antigen in mice following DNA immunization.     

Impact of vector-priming on the immunogenicity of a live recombinant Salmonella enterica serovar typhi Ty21a vaccine expressing urease A and B from Helicobacter pylori in human volunteers

Orally administered recombinant Salmonella vaccines represent an attractive option for mass vaccination programmes against various infectious diseases. Therefore, it is crucial to gather knowledge about the possible impact of preexisiting immunity to carrier antigens on the immunogenicity of recombinant vaccines. Thirteen volunteers were preimmunized with Salmonella typhi Ty21a in order to evaluate the effects of prior immunization with the carrier strain. Then, they received three doses of 1-2 x 10(10) viable organisms of either the vaccine strain S. typhi Ty21a (pDB1) expressing subunits A and B of recombinant Helicobacter pylori urease (n = 9), or placebo strain S. typhi Ty21a (n = 4). Four volunteers were preimmunized and boosted with the vaccine strain S. typhi Ty21a (pDB1). No serious adverse effects were observed in any of the volunteers. Whereas none of the volunteers primed and boosted with the vaccine strain responded to the recombinant antigen, five of the nine volunteers preimmunized with the carrier strain showed cellular immune responses to H. pylori urease (56%). This supports the results of a previous study in non-preimmunized volunteers where 56% (five of nine) of the volunteers showed a cellular immune response to urease after immunisation with S. typhi Ty21a (pDB1).     

Comparison of the patterns of antibody recall responses to HIV-1 gp120 and hepatitis B surface antigen in immunized mice

To date, we still lack an ideal strategy for designing envelope glycoprotein (Env) vaccines to elicit potent protective antibodies against HIV-1 infection. Since the human hepatitis B virus surface antigen (HBsAg) is representative of effective vaccines that can induce ideal humoral immune responses, knowledge of how it elicits antibody responses and T helper cells would be an useful reference for HIV vaccine development. We compared the characteristics of the HIV-1 Env gp120 trimer and HBsAg in antibody elicitation and induction of T follicular helper (Tfh) and memory B cells in immunized Balb/c mice. Using the strategy of protein prime-protein boost, we found that HIV-1 gp120 induced slower recall antibody responses but redundant non-specific IgG responses at early time after boosting compared to HBsAg. The higher frequency of PD-1^hiCD4⁺ T cells and Tfh cells that appeared at the early time point after gp120 boosting is likely to limit the development of memory B cells, memory T cells, and specific antibody recall responses. These findings regarding the different features of HIV envelope and HBsAg in T helper cell responses may provide a direction to improve HIV envelope immunogenicity.     

Repeated daily exposure to 2 ppm nitrogen dioxide upregulates the expression of IL-5, IL-10, IL-13, and ICAM-1 in the bronchial epithelium of healthy human airways

Background: Repeated daily exposure of healthy human subjects to NO₂ induces an acute airway inflammatory response characterised by neutrophil influx in the bronchial mucosa

Aims: To assess the expression of NF-κB, cytokines, and ICAM-1 in the bronchial epithelium.

Methods: Twelve healthy, young non-smoking volunteers were exposed to 2 ppm of NO₂/filtered air (four hours/day) for four successive days on separate occasions. Fibreoptic bronchoscopy was performed one hour after air and final NO₂ exposures. Bronchial biopsy specimens were immunostained for NF-κB, TNF-α, eotaxin, Gro-α, GM-CSF, IL-5, -6, -8, -10, -13, and ICAM-1 and their expression was quantified using computerised image analysis.

Results: Expression of IL-5, IL-10, IL-13, and ICAM-1 increased following NO₂ exposure.

Conclusion: Upregulation of the Th2 cytokines suggests that repeated exposure to NO₂ has the potential to exert a "pro-allergic" effect on the bronchial epithelium. Upregulation of ICAM-1 highlights an underlying mechanism for leucocyte influx, and could also explain the predisposition to respiratory tract viral infections following NO₂ exposure since ICAM-1 is a major receptor for rhino and respiratory syncytial viruses.

     

Induction of systemic and mucosal antibody responses in mice immunized intranasally with aluminium-non-adsorbed diphtheria toxoid together with recombinant cholera toxin B subunit as an adjuvant

Nasal mucosal immunization is very attractive for vaccination to prevent various bacterial and viral infectious diseases because of induction of systemic and mucosal immune responses. The aim of the present study was to investigate the possibility of changing the immunization procedure of diphtheria toxoid (Dt) from intramuscular or subcutaneous injection to intranasal administration. Intranasal immunization with aluminium-non-adsorbed diphtheria toxoid (nDt) together with recombinant cholera toxin B subunit (rCTB, 10 μg) induced, at a concentration of 5 Lf, high levels of serum Dt-specific IgG antibody responses and high or moderate levels of the specific IgA antibody responses in all mice and only a slight level of the specific IgE antibody responses in some mice. Furthermore, sufficiently high diphtheria antitoxin titres more than 0.1 international units (IU) ml⁻¹ were obtained from mice which showed high levels of serum Dt-specific IgG antibody responses. Under the same experimental conditions, induction of significant levels of mucosal Dt-specific IgA antibody responses occurred in the nasal cavity, the lung, the saliva and vaginal secretions and the small and large intestines of all mice, although there were different titres between individual mice. Similar results were also obtained with rCTB-specific serum IgG and IgA and mucosal IgA antibody responses; serum rCTB-specific IgE antibody titres were not detected. These results show that intranasal administration of nDt with rCTB must be a very useful means for vaccination against diphtheria.     

Nasal immunization of mice with AFCo1 or AFPL1 plus capsular polysaccharide Vi from Salmonella typhi induces cellular response and memory B and T cell responses

The response to infection against Salmonella involves both B and T cell mediated immunity. An effective immunization can activate an adequate immune response capable to control the primary infection and protect against a secondary infection. Mucosal vaccination, by inducing local pathogen-specific immune responses, has the potential to counter mucosally transmitted pathogens at the portal of entry, thereby increasing the efficacy of vaccines. The aim of this work was to explore the efficacy of AFCo1 or AFPL1, as mucosal adjuvants to stimulate cell immunity and memory responses against Vi polysaccharide antigen of Salmonella typhi (PsVi). Mice immunized with 3 intranasal doses exhibited high levels of PsVi-specific IgG (p < 0.05), IgG2a and IgG2c subclasses. Also, an amplified recall response after a booster immunization with a plain polysaccharide vaccine was induced. Avidities index were higher in mice immunized with adjuvanted formulations at different chaotropic concentrations. Furthermore, IL-12 and IFN-γ levels in nasally vaccinated mice with both adjuvants were induced. Moreover, priming with 3 doses followed by booster immunization with VaxTyVi^® resulted in high levels of anti-Vi specific IgG, IgG subclasses and antibody avidity. Long lived plasma cells in bone marrow, memory B cells and long-term memory T cells after booster dose were induced. The combined formulation of Vi polysaccharide with mucosal adjuvants provides an improved immunogenicity, in particular with regard to cellular responses and long lasting cells responses.     

Construction and evaluation of the eukaryotic expression plasmid encoding two copies of somatostatin genes fused with hepatitis B surface antigen gene S

The aim of current study was to evaluate the prospects of somatostatin DNA vaccine. Two copies of somatostatin (SS) genes were fused with the hepatitis B surface antigen (HBsAg) S gene using genetic engineering methods, the identified recombinant plasmid designated as pcS/2SS was transfected into HeLa cells to detect expression and antigenicity of target fusion protein, and its immunoreaction as well as safety was evaluated with animal experiments. The expressed target protein had a specific reaction with somatostatin antibody and showed a single strip result. A single injection of this vector stimulated long-term antigen-specific antibody responses in rats, and peak antibody levels occurred at the 2nd week of the initial injection. Additionally, the 50 microg immunized group resulted in a 13.5% increase in growth rate as compared with control group (111.7 g vs. 98.4 g). The genomic DNA was assayed for integrated plasmid using a sensitive PCR method, and the risk of mutation due to integration of pcS/2SS plasmid following intramuscular injection in mice was negligible. The successful construction of pcS/2SS DNA vaccine with good immunogenicity and safety has prospects to promote growth of animals.     

A validation of the construct and reliability of an emotional intelligence scale applied to nursing students

OBJECTIVE:

The current study aimed to validate the construct and reliability of an emotional intelligence scale.

METHOD:

The Trait Meta-Mood Scale-24 was applied to 349 nursing students. The process included content validation, which involved expert reviews, pilot testing, measurements of reliability using Cronbach''s alpha, and factor analysis to corroborate the validity of the theoretical model''s construct.

RESULTS:

Adequate Cronbach coefficients were obtained for all three dimensions, and factor analysis confirmed the scale''s dimensions (perception, comprehension, and regulation).

CONCLUSION:

The Trait Meta-Mood Scale is a reliable and valid tool to measure the emotional intelligence of nursing students. Its use allows for accurate determinations of individuals'' abilities to interpret and manage emotions. At the same time, this new construct is of potential importance for measurements in nursing leadership; educational, organizational, and personal improvements; and the establishment of effective relationships with patients.     

Characterization of an inactivated whole-virus bivalent vaccine that induces balanced protective immunity against coxsackievirus A6 and A10 in mice

Coxsackievirus A6 (CVA6) and CVA10 are two of the major pathogens associated with hand, foot and mouth disease (HFMD) in children. The majority of CVA6 and CVA10 infections result in mild, self-limiting episodes (fever and herpangina) in pediatric populations; however, in some cases, can proceed to severe neurological disease and death. Efforts to mitigate viral transmission to decrease the morbidity and mortality associated with infection would be greatly strengthened by the availability of an efficacious CVA6 and CVA10 bivalent vaccine. Here we report the immunogenicity and protective efficacy of a bivalent combination vaccine comprised of formaldehyde-inactivated, whole-virus CVA6 and CVA10. We demonstrate that subcutaneous delivery of the bivalent vaccine can induce antigen-specific systemic immune responses, particularly the induction of polyfunctional T cells, which elicit active immunization to achieve a protection rate of >80% in the infected neonatal mice. Furthermore, passive transfer of the antisera from vaccinated mice potently protected recipient mice against CVA6 and CVA10 challenge. Importantly, the bivalent vaccine could induce high levels of IgG and neutralizing antibodies in adult female mice and the maternal antibody transmitted to the recipient mice played an important role in controlling homotypic and heterotypic CVA6 and CVA10 infections and viral replication in vivo. Collectively, these findings indicate that there is no immunological interference between the two antigens with respect to their ability to induce virus-specific immune responses, and thus provides proof-of-concept for further development of multivalent vaccines for broad protection against HFMD.     

The role of IL-10 and IgG1 in the protection and granulomatous response in Schistosoma mansoni P24-immunized mice

Previous work by our laboratory identified a fraction of Schistosoma mansoni soluble adult worm antigenic preparation, designated PIII, able to elicit significant in vitro cell proliferation, and lower in vitro and in vivo granuloma formation. In the present work, we investigated some biological activities of P24, an antigenic component of PIII. Immunization of mice with this antigen induced a significant protection degree against challenge infection and significant decrease in the hepatic granuloma formation. Pre-incubation of spleen cells from P24-immunized mice with S. mansoni antigens induced a significant increase of interleukin (IL)-10 levels, but not interferon-gamma, in the cell supernatants. In addition, mice immunized with different S. mansoni antigens and P24 displayed indistinguishable levels of IgG2a in response to anti-S. mansoni antigens, while IgG1 levels were significantly increased. Collectively, our results indicate that P24 might mediate protective anti-parasite immunity and downregulate granulomatous hypersensitivity to S. mansoni eggs in part by its ability to induce a higher production of IgG1 and IL-10.     

Boosting systemic and secreted antibody responses in mice orally immunized with recombinant Bacillus subtilis strains following parenteral priming with a DNA vaccine encoding the enterotoxigenic Escherichia coli (ETEC) CFA/I fimbriae B subunit

Recombinant Bacillus subtilis strains, either spores or vegetative cells, may be employed as safe and low cost orally delivered live vaccine vehicles. In this study, we report the use of an orally delivered B. subtilis vaccine strain to boost systemic and secreted antibody responses in mice i.m. primed with a DNA vaccine encoding the structural subunit (CfaB) of the CFA/I fimbriae encoded by enterotoxigenic Escherichia coli (ETEC), an important etiological agent of diarrhea among travelers and children living in endemic regions. DBA/2 female mice submitted to the prime-boost immunization regimen developed synergic serum (IgG) and mucosal (IgA) antibody responses to the target CfaB antigen. Moreover, in contrast to mice immunized only with one vaccine formulation, sera harvested from prime-boosted vaccinated individuals inhibited adhesion of ETEC cells to human red blood cells. Additionally, vaccinated dams conferred full passive protection to suckling newborn mice challenged with a virulent ETEC strain. Taken together the present results further demonstrate the potential use of recombinant B. subtilis strains as an alternative live vaccine vehicle.     

Oral immunization with attenuated Salmonella carrying a co-expression plasmid encoding the core and E2 proteins of hepatitis C virus capable of inducing cellular immune responses and neutralizing antibodies in mice

Hepatitis C virus (HCV) core protein has long been considered an attractive candidate for inclusion in a protective vaccine. However, this protein may hamper the development of systemic immune responses because of its immune suppressive properties. We previously reported that immune responses to HCV core protein could be efficiently induced by attenuated Salmonella carrying the HCV core protein, but not the HCV core DNA vaccine. To optimize the combination of the core protein and envelope protein 2 (E2) into a vaccine formulation to induce cellular immune responses and neutralizing antibodies, we constructed a plasmid containing two expression cassettes. One expression cassette was included to regulate the expression of HCV core protein by an inducible in vivo-activated Salmonella promoter, the other was included to regulate the expression of HCV E2 protein by the cytomegalovirus enhancer/promoter. Oral immunization of BALB/c mice with the attenuated Salmonella strain SL7207 carrying this plasmid efficiently induced HCV core and E2-specific cellular immune responses and antibodies. IgG purified from immunized mice could neutralize the infectivity of HCV pseudoparticles (HCVpp) of both the autologous Con 1 isolate and the heterologous H77 isolate, and cell culture produced HCV (HCVcc) of Con1-JFH1 chimera. These results indicated that this vaccine strategy can effectively deliver core and E2 protein to the immune system and provide a promising approach for the development of prophylactic and therapeutic vaccines against HCV infection.     

Nasal or intramuscular immunization of mice with influenza subunit antigen and the B subunit of Escherichia coli heat-labile toxin induces IgA- or IgG-mediated protective mucosal immunity

Local mucosal IgA antibodies play a central role in protection of the respiratory tract against influenza virus infection. Therefore, new-generation influenza vaccines should aim at stimulating not only systemic, but also local antibody responses. Previously, we demonstrated that the recombinant B subunit of the Escherichia coli heat-labile toxin (LTB) is a potent adjuvant towards nasally administered influenza subunit antigen. Here, we investigated the protection conferred by LTB-supplemented influenza subunit antigen given intranasally (i.n.) or intramuscularly (i.m.) to mice. Both i.n. and i.m. immunization with subunit antigen and LTB completely protected the animals against viral infection. Protection upon i.n. immunization was associated with the induction of antigen-specific serum IgG and mucosal IgA, whereas protection upon i.m. immunization correlated with strong serum and mucosal IgG, but not IgA responses. We conclude that LTB-supplemented influenza subunit antigen, given either i.n. or i.m, induces protective antibody-mediated mucosal immunity and thus represents a promising novel flu vaccine candidate.