Intranasal vaccination of neonatal mice with polysaccharide conjugate vaccine for protection against pneumococcal otitis media

Streptococcus pneumoniae is the leading bacterial cause of acute otitis media (OM) in young children, and can often produce invasive disease. Typical intramuscular routes of vaccination are poorly protective against development of OM. In the present study, we intranasally (i.n.) inoculated neonatal 1-week-old mice with pneumococcal polysaccharide conjugate vaccine using IL-12 as a mucosal adjuvant. The protective efficacy of this treatment was tested by challenging immunized infant (3-week-old) mice with bacteria to induce OM and invasive disease. i.n. vaccination was found to enhance levels of specific antibodies in middle ear (ME) washes and sera from wild-type (but not IFN-γ^−/−) mice. Immunization in the presence of IL-12 resulted in enhanced clearance of S. pneumoniae from the ME. Opsonization of bacteria with ME wash fluids or sera from immunized mice caused increased bacterial clearance from the ME of naïve mice. In addition, immunized mice demonstrated 89% survival after OM-induced invasive pneumococcal infection, compared to 22% survival in unvaccinated mice. These results indicate that i.n. vaccination of neonatal mice in the presence of IL-12 is able to enhance IFN-γ dependent ME mucosal and systemic immune responses to pneumococci and efficiently protect against both OM and invasive infection.     

Protective immunity against Toxoplasma challenge in mice by coadministration of T. gondii antigens and Eimeria profilin-like protein as an adjuvant

Toll-like receptor (TLR) ligands are attractive adjuvant candidates in vaccine development. Eimeria tenella profilin-like protein has recently been shown to be a potent agonist of the innate immune system through its recognition by Toll-like receptor-11. In this report, we studied the systemic and mucosal adjuvant activity of Eimeria profilin-like protein within a vaccinal strategy against Toxoplasma gondii in mice. Using intraperitoneal (i.p.) immunization, we observed that coadministration of the recombinant Eimeria antigen (rEA) with T. gondii antigen (TAg) effectively elevates plasma levels of IL-12p70 and consequently induced both enhanced specific humoral and Th1 cellular immune responses. The co-administration of TAg plus rEA by i.p route significantly enhanced the protection against T. gondii infection (62% brain cyst reduction) in comparison with control mice and with mice immunized with TAg alone (only 36% brain cyst reduction). After intranasal immunization, humoral and cellular responses were weak. However mice immunized nasally with TAg plus rEA were significantly protected with 50% of brain cyst reduction, conversely TAg immunized mice did not present any brain cyst reduction.These results indicate that Eimeria profilin-like protein would serve as an efficacious systemic and mucosal adjuvant inducing protective immune response against chronical stage of T. gondii infection through TLR11 activation.     

Intradermal vaccination with a Pseudomonas aeruginosa vaccine adjuvanted with a mutant bacterial ADP-ribosylating enterotoxin protects against acute pneumonia

Respiratory infections are a leading cause of morbidity and mortality globally. This is partially due to a lack of effective vaccines and a clear understanding of how vaccination route and formulation influence protective immunity in mucosal tissues such as the lung. Pseudomonas aeruginosa is an opportunistic pathogen capable of causing acute pulmonary infections and is a leading cause of hospital-acquired and ventilator-associated pneumonia. With multidrug-resistant P. aeruginosa infections on the rise, the need for a vaccine against this pathogen is critical. Growing evidence suggests that a successful P. aeruginosa vaccine may require mucosal antibody and Th1- and Th17-type CD4⁺ T cells to prevent pulmonary infection. Intradermal immunization with adjuvants, such as the bacterial ADP-Ribosylating Enterotoxin Adjuvant (BARE) double mutant of E. coli heat-labile toxin (dmLT), can direct protective immune responses to mucosal tissues, including the lungs. We reasoned that intradermal immunization with P. aeruginosa outer membrane proteins (OMPs) adjuvanted with dmLT could drive neutralizing antibodies and migration of CD4⁺ T cells to the lungs and protect against P. aeruginosa pneumonia in a murine model. Here we show that mice immunized with OMPs and dmLT had significantly more antigen-specific IgG and Th1- and Th17-type CD4⁺ memory T cells in the pulmonary environment compared to control groups of mice. Furthermore, OMPs and dmLT immunized mice were significantly protected against an otherwise lethal lung infection. Protection was associated with early IFN-γ and IL-17 production in the lungs of immunized mice. These results indicate that intradermal immunization with dmLT can drive protective immunity to the lung mucosa and may be a viable vaccination strategy for a multitude of respiratory pathogens.     

Mice immunized with bone marrow-derived dendritic cells stimulated with recombinant Coxiella burnetii Com1 and Mip demonstrate enhanced bacterial clearance in association with a Th1 immune response

The recombinant membrane-associated proteins of Coxiella burnetii, Com1, Mip and GroEL, were used in vitro to stimulate BALB/c mouse bone marrow-derived dendritic cells (BMDCs). The antigen-activated BMDCs were transferred into naïve BALB/c mice. Seven days after challenge of C. burnetii, the bacterial loads of mice receiving BMDCs activated with Com1 or Mip, but not GroEL, were significantly lower than that of mice receiving BMDCs pulsed with TrxA (Esherichia coli thioredoxin) in a quantitative polymerase chain reaction assay. After in vitro interaction with cognate antigen-pulsed BMDCs, the percentages of CD69-positive cells and TNF-α-positive cells in CD4⁺ and CD8⁺ T cells isolated from the spleens of mice receiving Com1-, Mip-, or GroEL-pulsed BMDCs were significantly higher than that of mice receiving mock-pulsed BMDCs in flow cytometric analysis. The percentages of IFN-γ-positive cells in CD4⁺ and CD8⁺ T cells from mice receiving Com1- or Mip-pulsed BMDCs were significantly greater than that of mice receiving GroEL-pulsed BMDCs. However, the percentage of IL-4-positive cells in CD4⁺ T cells of mice receiving GroEL-pulsed BMDCs was obviously higher than that of mice receiving Com1- or Mip-pulsed BMDCs. Our results demonstrate that Com1 and Mip are protective antigens and strongly indicate that they favor to induce IFN-γ-producing Th1 and Tc1 cells, whereas the non-protective antigen GroEL is biased to induce a Th2 response. Therefore, Com1 and Mip are key antigens to induce a protective immune response against C. burnetii infection.     

Protective immunity against mouse upper genital tract pathology correlates with high IFNγ but low IL-17 T cell and anti-secretion protein antibody responses induced by replicating chlamydial organisms in the airway

To search for optimal immunization conditions for inducing protective immunity against upper genital tract pathologies caused by chlamydial intravaginal infection, we compared protection efficacy in mice immunized intranasally or intramuscularly with live or inactivated Chlamydia muridarum organisms. Mice immunized intranasally with live organisms developed strong protection against both vaginal shedding of infectious organisms and upper genital tract pathologies. The protection correlated with a robust antigen-specific T cell response with high IFNγ but low IL-17. Although a significant level of IL-5 was also detected, these mice maintained an overall Th1-dorminant immunity following immunization and challenge infection. On the contrary, mice immunized intranasally with inactivated organisms or intramuscularly with live or inactivated organisms produced high levels of IL-17 and still developed significant upper genital tract pathologies. High titers of antibodies against chlamydial secretion antigens were detected only in mice immunized intranasally with live organisms but not mice in other groups, suggesting that the intranasally inoculated live organisms were able to undergo replication and immune responses to the chlamydial secretion proteins may contribute to protective immunity. These observations have provided important information on how to develop subunit vaccines for inducing protective immunity against urogenital infection with Chlamydia trachomatis organisms.     

Long-term control of Coxiellosis in sheep by annual primary vaccination of gimmers

Coxiella (C.) burnetii, a Gram-negative intracellular bacterium, causes Q fever in humans and Coxiellosis in animals. Ruminants are a primary source of human infection with C. burnetii. In 2013, vaccination was implemented in a sheep flock with 650 ewes associated with two outbreaks of Q fever in humans in 2008 and 2012. Only gimmers (yearlings) received two doses of a commercial C. burnetii phase I whole cell vaccine three weeks apart (primary vaccination) without any revaccination. Vaginal and nasal swabs collected shortly after lambing were tested by qPCR. Additionally, a group of non-vaccinated sentinels was serologically monitored for phase I (PhI), II (PhII) antibodies and for Interferon γ (IFN-γ) after stimulation of whole blood cells with PhII-antigen with and without an IL-10-neutralizing monoclonal antibody. In 2021, 679 sera collected in 2014–2021 were retested retrospectively with three commercial ELISA kits and one batch of an in-house PhI/PhII-ELISA.A low-level shedding of C. burnetii (<10³ mean C. burnetii/swab) was observed until 2014. In 2021 C. burnetii was detected in two animals (<10^3.1 C. burnetii/swab), but vaginal swabs collected at two subsequent lambing seasons remained negative. Seroconversion of sentinels was detected until 2017. However, the retrospective analysis of sentinels in 2021 revealed additional single seropositive animals from 2018 to 2021. IFN-γ reactivity was observed during the whole study period; it peaked in 2014 and in 2018 and decreased thereafter.The sporadic detection of C. burnetii and the immune responses of sentinels suggested that a subliminal infection persisted despite vaccination. Nevertheless, vaccination of gimmers prevented the development of a major outbreak, it controlled the infection and reduced the risk of human infection.     

Induction of protection against vaginal shedding and infertility by a recombinant Chlamydia vaccine

A vaccine formulated with the Chlamydia muridarum recombinant major outer membrane protein, plus the adjuvants CpG and Montanide, was tested for its ability to protect BALB/c mice against a vaginal challenge. Mice were immunized by mucosal [intravaginal (i.vag.) plus colonic (col.), or intranasal (i.n.) plus sublingual (s.l.)], or systemic [intramuscular (i.m.) plus subcutaneous (s.c.)] routes, and a combination of mucosal priming and systemic boosting routes. A negative control group was vaccinated with the Neisseria gonorrhoeae porin B (Ng-rPorB) and a positive control group was inoculated in the nares with live Chlamydia. The strongest Chlamydia-specific humoral and cell-mediated immune responses were observed in the groups immunized by a combination of mucosal and systemic routes. Following the vaginal challenge, groups immunized using mucosal priming followed by systemic immunization had a significant decrease in the number of mice with positive vaginal cultures. For example, of the mice immunized i.n./s.l. + i.m./s.c., 24% had positive cultures during the six weeks of the experiment versus 69% for the negative control group immunized with Ng-rPorB (P < 0.05). Similarly, the groups of mice primed by the mucosal routes and boosted by the systemic routes had significantly less IFU in the vaginal cultures when compared to the Ng-rPorB animals (P < 0.05). These combination groups were also protected against infertility. The two groups had fertility rates of 100% (i.n./s.l. + i.m./s.c.) and 81% (i.vag./col. + i.m./s.c.) equivalent to the positive-control group immunized with live Chlamydia (100% fertility; P > 0.05). These results show the importance of the schedule and routes of vaccination and represent the first study to show protection against infertility by a Chlamydia recombinant subunit vaccine.     

Intranasal immunization protects against Acinetobacter baumannii-associated pneumonia in mice

Multidrug-resistant Acinetobacter baumannii has become an important causative agent of healthcare associated infections. Hospital- and community-acquired pneumonia is the most common clinical manifestation of A. baumannii infection worldwide and is often associated with high mortality. Most experimental vaccine studies to date have evaluated vaccines against systemic A. baumannii infections following systemic immunization. We recently demonstrated that a mouse model of respiratory A. baumannii infection using the strain LAC-4 results in disease progression that is similar to that observed in humans. Here we used this model in conjunction with an inactivated whole cell vaccine to evaluate the feasibility of developing protective mucosal vaccines against respiratory A. baumannii infection and to investigate the potential mechanism of protection of such vaccines. Our results showed that intranasal immunization with formalin-killed whole cells of the LAC-4 strain elicited mucosal and systemic antigen-specific immune responses, and protected mice against lethal intranasal or intraperitoneal challenges. Compared to naïve mice, immunized mice had significantly fewer bacteria in their lungs, and the pathogen was barely detectable in blood and spleens at 24 h post challenge, indicating the ability of immunized mice to control extrapulmonary dissemination of the pathogen. Mechanistic studies using gene-deficient mice, neutropenic mice, or passive immunization showed that B cells and neutrophils, but not FcRγ, played crucial roles in the protection against respiratory A. baumannii challenge of intranasally immunized mice whereas passive transfer of hyperimmune sera only prolonged the survival time of challenged mice by 48 h. These results provide immunological insights for the rational design of novel mucosal vaccines to protect against respiratory A. baumannii infection and demonstrate the feasibility to develop such vaccines.     

Development of recombinant vaccine candidate molecule against Shigella infection

Shigellosis is an acute bacillary diarrheal disease caused by the gram negative bacillus Shigella. The existence of multiple Shigella serotypes and their growing resistance to antibiotics stress the urgent need for the development of vaccine that is protective across all serotypes. Shigella’s IpaB antigen is involved in translocon pore formation, promotes bacterial invasion and induces apoptosis in macrophages. S. Typhi GroEL (Hsp 60) is the immunodominant antigen inducing both arms of immunity and has been explored as adjuvant in this study. The present study evaluates the immunogenicity and protective efficacy of recombinant IpaB domain-GroEL fusion protein in mice against lethal Shigella infection. The IpaB domain and GroEL genes were fused using overlap extension PCR and cloned in pRSETA expression vector. Fused gene was expressed in Escherichia coli BL-21 cells and the resulting 90 KDa fusion protein was purified by affinity chromatography. Intranasal (i.n.) immunization of mice with fusion protein increased the IgG and IgA antibody titers as compared to the group immunized with IpaB and GroEL and control PBS immunized group. Also IgG1 and IgG2a antibodies induced in fusion protein immunized mice were higher than co-immunized group. Significant increase in lymphocyte proliferation and cytokine levels (IFN-γ, IL-4 and IL-10), indicates induction of both Th1 and Th2 immune responses in both immunized groups. Immunization with fusion protein protected 90–95% of mice whereas 80–85% survivability was observed in co-immunized group against lethal challenge with S. flexneri, S. boydii and S. sonnei. Passive immunization conferred 60–70% protection in mice against all these Shigella species. Organ burden and histopathology studies also revealed significant decrease in lung infection as compared to the co-immunized group. Since IpaB is the conserved dominant molecule in all Shigella species, this study will lead to an ideal platform for the development of safe, efficacious and cost-effective recombinant vaccine against Shigella serotypes.     

Immunisation with the glycolytic enzyme enolase confers effective protection against Candida albicans infection in mice

Candida albicans is an opportunistic human fungal pathogen that continues to be a leading cause of candidal infections in immunocompromised hosts. Enolase, an important glycolytic enzyme located on the cell wall of C. albicans, was cloned, purified, and characterized by molecular cloning, affinity chromatography and Western blotting. C57BL/6J mice were immunized with recombinant enolase subcutaneously every two weeks, and the protective effect against systemic challenge evaluated by fungal burdens in target organs, titres of specific antibodies to enolase, and by levels of Th1/2 cytokines in serum. After challenge with C. albicans strains SC5314 and 3630, fungal burdens in the liver, kidney, brain, spleen and lung were significantly decreased in immunized mice. Histopathological assessment demonstrated that enolase protected the tissue structure, and decreased the infiltration of inflammatory cells. The titres of enolase-specific IgG1 and IgG2a in the immune serum reached up to 1:51200. Furthermore, opsonization with immune serum resulted in enhanced killing of both 3630 and SC5314 by murine neutrophils. Levels of IL-12 and IL-8 in the immune serum increased, whereas the concentration of the Th2 cytokine, IL-10, was significantly higher in immunized mice compared to the control group. It was concluded that recombinant enolase effectively protected mice against disseminated candidiasis, and may be a promising target for vaccination against different strains of C. albicans.     

Oral vaccination with Ts87 DNA vaccine delivered by attenuated Salmonella typhimurium elicits a protective immune response against Trichinella spiralis larval challenge

We have previously reported that Ts87 is an immunodominant antigen that induces protective immunity against Trichinella spiralis larval challenge. In this study, the Ts87 gene was cloned into an expression plasmid, pVAX1, and the recombinant Ts87 DNA was transformed into attenuated Salmonella typhimurium strain SL7207. Oral immunization of mice with Ts87 DNA delivered in S. typhimurium elicited a significant local mucosal IgA response and a systemic Th1/Th2 immune response. Cytokine profiling also showed a significant increase in the Th1 (IFN-γ) and Th2 (IL-5, 6, 10) responses in splenocytes of immunized mice upon stimulation with Ts87 antigen. An immunofluorescence assay performed with antisera revealed that the recombinant Ts87 protein was expressed in mesenteric lymph nodes of immunized mice. The mice immunized with Salmonella-delivered Ts87 DNA displayed a statistically significant 29.8% reduction in adult worm burden and a 34.2% reduction in muscle larvae following challenge with T. spiralis larvae, compared with mice immunized with empty Salmonella or a PBS control. Our results demonstrate that Ts87 DNA delivered by attenuated live S. typhimurium elicits a local IgA response and a balanced Th1/Th2 immune response and produces partial protection against T. spiralis infection in mice.     

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.     

Intranasal immunization with Leish-111f induces IFN-γ production and protects mice from Leishmania major infection

The mucosal vaccination is a non-invasive alternative approach for not only mucosal pathogens but also parenteral pathogens, since it induces both mucosal and systemic immunoreactions. The purpose of this study was to evaluate the application of intranasal (i.n.) immunization with a recombinant leishmanial protein against Leishmania infection. BALB/c mice were i.n. administered 1–3 times with Leish-111f plus cholera toxin (CT) adjuvant (Leish-111f/CT). Splenocytes from i.n. immunized mice produced high level of IFN-γ but not IL-4 in response to Leish-111f. When infected with 1 × 10⁶ of Leishmania major promastigotes 2 weeks after the final administration, lesion development was completely controlled in all mice i.n. administered with Leish-111f/CT. Mice i.n. administered with Leish-111f alone showed neither cytokine productions nor lesion control even after 6 administrations, suggesting the importance of CT adjuvant. This report demonstrated for the first time that i.n. administration of a recombinant leishmanial protein induces Th1 type immunity and protects mice from Leishmania infection.     

Immunization with PCEP microparticles containing pertussis toxoid, CpG ODN and a synthetic innate defense regulator peptide induces protective immunity against pertussis

We investigated the efficacy of a novel microparticle (MP) based vaccine formulation consisting of pertussis toxoid (PTd), polyphosphazene (PCEP), CpG ODN 10101 and synthetic cationic innate defense regulator peptide 1002 (IDR) against Bordetella pertussis in mice. We studied whether encapsulation of these IDR-CpG ODN complexes into polyphosphazene-based microparticles further enhanced their immunomodulatory activity compared to soluble formulations containing PCEP (SOL), or without PCEP (AQ). In vitro stimulation of murine macrophages showed MP induced significantly higher levels of pro-inflammatory cytokines. When assessed in a B. pertussis infection challenge model, a single immunization with MP formulation led to significantly lower bacterial loads compared to other formulations and non-vaccinated animals. ELISPOT of splenocytes showed that MP group mice had significantly higher number of antigen-specific IL-17 secreting cells. The cytokine profile in lung homogenates of MP group mice after challenge showed significantly higher amounts of MCP-1, TNF-α, IFN-γ, IL-12 and IL-17 and significantly lowered IL-10 levels suggesting a strong Th1 shift. Protection was observed against challenge infection with B. pertussis. On the other hand protective immune responses elicited in Quadracel^® immunized mice were Th2 skewed. Hence, we conclude that formulation of PTd, PCEP, CpG ODN and IDR into MP generates a protective immune response in mice against pertussis emphasizing the potential of MP as a delivery vehicle for the potential development of single-shot vaccines.     

Construction of a recombinant-attenuated Salmonella Enteritidis strain secreting Escherichia coli heat-labile enterotoxin B subunit protein and its immunogenicity and protection efficacy against salmonellosis in chickens

A live attenuated Salmonella Enteritidis (SE) strain secreting Escherichia coli heat-labile enterotoxin B subunit (LTB) protein was constructed as a new vaccine candidate. The comparative effect of this vaccine candidate was evaluated with a previously reported SE vaccine, JOL919. An asd+, p15A ori plasmid containing eltB-encoding LTB was introduced into a ΔlonΔcpxRΔasd SE strain, and designated as JOL1364. In a single immunization experiment, group A chickens were orally inoculated with phosphate-buffered saline as a control, group B chickens were orally immunized with JOL919, and group C chickens were orally immunized with JOL1364. The immunized groups B and C showed significantly higher systemic, mucosal and cellular immune responses as compared to those of the control group. In addition, the immunized group C showed significantly higher mucosal and cellular immune responses as compared to those of the immunized group B at the 1_st week post-immunization. In the examination of protection efficacy, the immunized groups B and C showed lower gross lesion scores in the liver and spleen, and lower bacterial counts of SE challenge strain in the liver, spleen, and caeca as compared to those of the control group. The number of SE-positive birds was significantly lower in the immunized group C as compared to that of the control group at the 14th day post-challenge. In addition, the number of birds carrying the challenge strain in the caeca was significantly lower in the immunized group C than those in the immunized group B and control group at the 7th and 14th day post-challenge. These results indicate that immunization with the JOL1364 vaccine candidate can induce higher mucosal and cellular immune responses than those of the JOL919 for efficient protection against salmonellosis.     

A lipopolysaccharide-free outer membrane vesicle vaccine protects against Acinetobacter baumannii infection

Outer membrane vesicles (OMVs) were isolated from an Acinetobacter strain deficient in lipopolysaccharide (LPS) due to a mutation in lpxD (IB010). Two immunizations with 10 µg of IB010 OMVs elicited total IgG, IgM, IgG1 and IgG2c titers similar to those observed after immunization with OMVs derived from the parental strain (ATCC 19606), and IB010 OMVs plus purified LPS. Immunization with IB010 OMVs resulted in significantly reduced post-infection spleen bacterial loads and serum IL-1β and IL-6 levels compared to control mice in a disseminated sepsis model. Mice immunized with 10 µg IB010 OMVs demonstrated significant, but partial, protection (75%) against infection, whereas mice immunized with ATCC 19606 OMVs or IB010 OMVs plus purified LPS were completely protected. Immunization of mice with 100 µg of IB010 OMVs completely protected mice from infection. This study demonstrates that LPS deficient A. baumannii produces OMVs, and that immunization with these OMVs elicits protective immunity against infection.     

Induction of Th2-related immune responses and production of systemic IgA in mice intranasally immunized with Brucella abortus malate dehydrogenase loaded chitosan nanoparticles

The aim of this study was to investigate the induction of mucosal immune responses by an important Brucella abortus antigen, malate dehydrogenase (Mdh), loaded in mucoadhesive chitosan nanoparticles (CNs) and immunized intranasally in a BALB/c mouse model. The production of cytokines was investigated in human leukemic monocyte cells (THP-1 cells) after stimulation with the nanoparticles. Mdh-loaded CNs (CNs-Mdh) induced higher interleukin (IL)-6 production than unloaded antigens and TF loaded CNs (CNs-TF). Using ELISpot to quantify cytokines and antibody-secreting cells in the intranasally immunized mice, IL-4 and IgG-secreting cells were found to be significantly increased at 4?weeks and 6?weeks post-immunization in the CNs-Mdh immunized group, respectively. Increases in Mdh-specific IgG, IgG1, and IgG2a antibodies were confirmed at 6?weeks after immunization, indicating a predominant IgG1 response. Analysis of the mucosal immune response in the intranasally immunized mice revealed, Mdh-specific IgA and total IgA in the nasal washes, genital secretions, fecal extracts and sera that were remarkably increased in the CNs-Mdh-immunized group compared to the CNs-TF-immunized group except total IgA of nasal wash. Therefore, the results indicated that the intranasal immunization of CNs-loaded B. abortus Mdh antigen effectively induced antigen-specific mucosal immune responses through the elicitation of Th2-related immune responses.     

Partial protective immunity against toxoplasmosis in mice elicited by recombinant Toxoplasma gondii malate dehydrogenase

Toxoplasma gondii can infect humans and wildlife, sometimes causing serious clinical presentations. Currently, no viable vaccine or effective drug strategies exist to prevent and control toxoplasmosis. T. gondii malate dehydrogenase (TgMDH) is a crucial enzyme in cellular redox reactions and has been shown to be an immunogenic compound that could be a potential vaccine candidate. Here, we investigate the protective efficacy of recombinant TgMDH (rTgMDH) against T. gondii infection in BALB/c mice. All mice were vaccinated via the nasal route. We determined the optimal vaccination dose by monitoring systemic and mucosal immune responses. The results showed that mice vaccinated with 30 μg of rTgMDH produced the highest antibody titers in serum, a strong lymphoproliferative response, marked increases in their levels of IL-2 and IFN-γ, and significantly greater levels of specific secretory IgA (sIgA) in mucosal washes. In addition, the vaccinated mice were orally challenged with tachyzoites of the virulent T. gondii RH strain 2 weeks after the final vaccination. Compared to the control group, we found that vaccination with rTgMDH increased the survival rate of infected mice by 47% and also significantly reduced the tachyzoite loads in their liver (by 58%) and brain (by 41%). Therefore, the rTgMDH protein triggers a strong systemic and mucosal immune response and provides partial protection against T. gondii infection.     

Intranasal immunization with recombinant PspA fused with a flagellin enhances cross-protective immunity against Streptococcus pneumoniae infection in mice

Streptococcus pneumoniae is a major respiratory pathogen that causes high levels of mortality and morbidity in infants and the elderly. Despite the use of antibiotics and vaccines, fatal pneumococcal disease remains prevalent. Pneumococcal surface protein A (PspA), a highly immunogenic surface protein produced by all strains of S. pneumoniae, can elicit protective immunity against fatal pneumococcal infection. We have previously demonstrated that the Vibrio vulnificus FlaB, a bacterial flagellin protein and agonist of TLR5, has strong mucosal adjuvant activity and induces protective immunity upon co-administration with tetanus toxoid. In this study, we have tested whether intranasal immunization with recombinant fusion proteins consisted of PspA and FlaB (PspA-FlaB and FlaB-PspA) is able to elicit more efficient protective mucosal immune responses against pneumococcal infection than immunization with PspA alone or with a stoichiometric mixture of PspA and FlaB. When mice were intranasally immunized with fusion proteins, significantly higher levels of anti-PspA IgG and IgA were induced in serum and mucosal secretions. The mice immunized intranasally with the FlaB-PspA fusion protein were the most protected from a lethal challenge with live S. pneumoniae, as compared to the mice immunized with PspA only, a mixture of PspA and FlaB, or the PspA-FlaB fusion protein. FlaB-PspA also induced a cross protection against heterologous capsular types. These results suggest that a FlaB-PspA fusion protein alone could be used as an anti-pneumococcal mucosal vaccine or as an effective partner protein for multivalent capsular polysaccharide conjugate vaccines.     

Evaluation of carbopol as an adjuvant on the effectiveness of progressive atrophic rhinitis vaccine

The Gram-negative pathogen toxigenic P. multocida causes progressive atrophic rhinitis (PAR) in swine throughout the world. Although some vaccines are being developed against PAR, their efficacy has not been evaluated using carbopol. In our study, a mixture of killed B. bronchiseptica and P. multocida bacteria, combined with recombinant proteins containing the C- and N-termini of PMT, was emulsified using two different adjuvants (ISA-15A and carbopol 971). The efficacy of these two vaccines was evaluated in a mouse model. Balb/C mice were immunized twice at a 14-day interval. Two weeks after the secondary immunization, blood samples were collected and the mice were challenged with toxigenic P. multocida. Thirty-five days later, the mice were euthanized, blood and tissue samples were collected. Compared with mice inoculated with vaccine emulsified with ISA-15A, higher titers of SN (1:64) and significantly increased levels of TNF-α, IL-6 and IL-17A were observed in mice inoculated with vaccine emulsified with the carbopol 971P. Especially, mice immunized with vaccine emulsified with the carbopol 971P had no detectable pathological changes in snouts or organs after challenge. The results demonstrated that carbopol adjuvanted vaccine provides good protection against PAR and P. multocida infection which can induce robust humoral and cell-mediated responses. We conclude that the carbopol adjuvanted vaccine is a good candidate for PAR prevention.