Induction Of Mucosal Immune Responses Research Articles

Intranasal nanoemulsion adjuvanted S-2P vaccine demonstrates protection in hamsters and induces systemic, cell-mediated and mucosal immunity in mice.

With the rapid progress made in the development of vaccines to fight the SARS-CoV-2 pandemic, almost >90% of vaccine candidates under development and a 100% of the licensed vaccines are delivered intramuscularly (IM). While these vaccines are highly efficacious against COVID-19 disease, their efficacy against SARS-CoV-2 infection of upper respiratory tract and transmission is at best temporary. Development of safe and efficacious vaccines that are able to induce robust mucosal and systemic immune responses are needed to control new variants. In this study, we have used our nanoemulsion adjuvant (NE01) to intranasally (IN) deliver stabilized spike protein (S-2P) to induce immunogenicity in mouse and hamster models. Data presented demonstrate the induction of robust immunity in mice resulting in 100% seroconversion and protection against SARS-CoV-2 in a hamster challenge model. There was a significant induction of mucosal immune responses as demonstrated by IgA- and IgG-producing memory B cells in the lungs of animals that received intranasal immunizations compared to an alum adjuvanted intramuscular vaccine. The efficacy of the S-2P/NE01 vaccine was also demonstrated in an intranasal hamster challenge model with SARS-CoV-2 and conferred significant protection against weight loss, lung pathology, and viral clearance from both upper and lower respiratory tract. Our findings demonstrate that intranasal NE01-adjuvanted vaccine promotes protective immunity against SARS-CoV-2 infection and disease through activation of three arms of immune system: humoral, cellular, and mucosal, suggesting that an intranasal SARS-CoV-2 vaccine may play a role in addressing a unique public health problem and unmet medical need.

Respiratory Vaccination with Hemagglutinin Nanoliposomes Protects Mice from Homologous and Heterologous Strains of Influenza Virus.

Intranasal vaccination offers the potential advantage of needle-free prevention of respiratory pathogens such as influenza viruses with induction of mucosal immune responses. Optimal design of adjuvants and antigen delivery vehicles for intranasal delivery has not yet been well established. Here, we report that an adjuvant-containing nanoliposome antigen display system that converts soluble influenza hemagglutinin antigens into nanoparticles is effective for intranasal immunization. Intranasal delivery of nanoliposomes in mice delivers the particles to resident immune cells in the respiratory tract, inducing a mucosal response in the respiratory system as evidenced by nasal and lung localized IgA antibody production, while also producing systemic IgG antibodies. Intranasal vaccination with nanoliposome particles decorated with nanogram doses of hemagglutinin protected mice from homologous and heterologous H3N2 and H1N1 influenza virus challenge. IMPORTANCE A self-assembling influenza virus vaccine platform that seamlessly converts soluble antigens into nanoparticles is demonstrated with various H1N1 and H3N2 influenza antigens to protect mice against influenza virus challenge following intranasal vaccination. Mucosal immune responses following liposome delivery to lung antigen-presenting cells are demonstrated.

Recombinant probiotic-Based Vaccines; Attractive vehicles for immunization via mucosal routes

Introduction: The importance of genetically modified microorganisms, including probiotics, now widely have considered as attractive vehicles for mucosal delivery of therapeutic molecules. Previous studies have indicated that mucosal administration of these molecules can effectively trigger humoral along with cellular immune responses. Therefore, development of induction of mucosal immune responses, utilizing of recombinant probiotics-based vaccines, as live delivery vectors with no side effects in humans, may contribute to effectiveness of control strategies against infection agents.

CFP10-loaded PLGA nanoparticles as a booster vaccine confer protective immunity against Mycobacterium bovis.

Introduction: The limited efficacy of BCG (bacillus Calmette-Guérin) urgently requires new effective vaccination approaches for the control of tuberculosis. Poly lactic-co-glycolic acid (PLGA) is a prevalent drug delivery system. However, the effect of PLGA-based nanoparticles (NPs) against tuberculosis for the induction of mucosal immune response is no fully elucidated. In this study, we hypothesized that intranasal immunization with culture filtrate protein-10 (CFP10)-loaded PLGA NPs (CFP10-NPs) could boost the protective immunity of BCG against Mycobacterium bovis in mice. Methods: The recombinant protein CFP10 was encapsulated with PLGA NPs to prepare CFP10-NPs by the classical water-oil-water solvent-evaporation method. Then, the immunoregulatory effects of CFP10-NPs on macrophages in vitro and on BCG-immunized mice in vivo were investigated. Results: We used spherical CFP10-NPs with a negatively charged surface (zeta-potential -28.5 ± 1.7 mV) having a particle size of 281.7 ± 28.5 nm in diameter. Notably, CFP10-NPs significantly enhanced the secretion of tumor necrosis factor α (TNF-α) and interleukin (IL)-1β in J774A.1 macrophages. Moreover, mucosal immunization with CFP10-NPs significantly increased TNF-α and IL-1β production in serum, and immunoglobulin A (IgA) secretion in bronchoalveolar lavage fluid (BALF), and promoted the secretion of CFP10-specific interferon-γ (IFN-γ) in splenocytes of mice. Furthermore, CFP10-NPs immunization significantly reduced the inflammatory area and bacterial load in lung tissues at 3-week post-M. bovis challenge. Conclusion: CFP10-NPs markedly improve the immunogenicity and protective efficacy of BCG. Our findings explore the potential of the airway mucosal vaccine based on PLGA NPs as a vehicle for targeted lung delivery.

A single intranasal dose of human parainfluenza virus type 3-vectored vaccine induces effective antibody and memory T cell response in the lungs and protects hamsters against SARS-CoV-2

Respiratory tract vaccination has an advantage of needle-free delivery and induction of mucosal immune response in the portal of SARS-CoV-2 entry. We utilized human parainfluenza virus type 3 vector to generate constructs expressing the full spike (S) protein of SARS-CoV-2, its S1 subunit, or the receptor-binding domain, and tested them in hamsters as single-dose intranasal vaccines. The construct bearing full-length S induced high titers of neutralizing antibodies specific to S protein domains critical to the protein functions. Robust memory T cell responses in the lungs were also induced, which represent an additional barrier to infection and should be less sensitive than the antibody responses to mutations present in SARS-CoV-2 variants. Following SARS-CoV-2 challenge, animals were protected from the disease and detectable viral replication. Vaccination prevented induction of gene pathways associated with inflammation. These results indicate advantages of respiratory vaccination against COVID-19 and inform the design of mucosal SARS-CoV-2 vaccines.

Staphylococcus aureus-specific IgA antibody in milk suppresses the multiplication of S. aureus in infected bovine udder

BackgroundBovine mastitis caused by Staphylococcus aureus (S. aureus) is extremely difficult to control and new methods for its prevention and management are required. Nasal vaccines may prevent initial bovine mastitis infection caused by S. aureus. However, limited information is available regarding induction of mucosal immune response through nasal immunization with antigen and its suppression of S. aureus multiplication during bovine mastitis. This study sought to investigate whether induction of immunoglobulin A (IgA) in milk by nasal immunization could suppress multiplication of S. aureus in the bovine udder.ResultsNasal immunization with formalin-killed S. aureus conjugated with a cationic cholesteryl-group-bearing pullulan-nanogel was performed. Anti-S. aureus-specific IgA antibodies were significantly more abundant in the milk of immunized cows than in non-immunized animals (P < 0.05). S. aureus counts in the quarter were negative in both non-immunized and nasal-immunized cows 1 week after mock infusion. In S. aureus-infused quarters, S. aureus multiplication was significantly suppressed in immunized compared with non-immunized cows (P < 0.05). Furthermore, a significant negative correlation was found between S. aureus-specific IgA antibodies and S. aureus counts in infused quarters of both non-immunized and nasal-immunized cows (r = − 0.811, P < 0.01).ConclusionIn conclusion, the present study demonstrates that S. aureus-specific IgA antibodies in milk successfully suppressed the multiplication of S. aureus in infected bovine udders. Although the exact mechanism explaining such suppressive effect remains to be elucidated, nasal vaccines that can induce humoral immunity may help prevent initial infection with S. aureus and the onset of bovine mastitis.

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.

Optimization of human papillomavirus (HPV) type 16 E7-expressing lactobacillus-based vaccine for induction of mucosal E7-specific IFNγ-producing cells

Therapeutic HPV vaccine is an agent to induce E7-specific Th1 immune responses to treat cervical neoplasia (CIN2-3). Our previous clinical trial has demonstrated that oral administration of HPV16 E7-expressing Lactobacillus casei (L. casei), GLBL101c, resulted in the regression of HPV16-related CIN3. Here we examined optimization of the E7-expressing L. casei for induction of the mucosal immune responses to E7. Various doses of HPV16 E7 molecule were displayed on the L. casei. Immunization with E7-bound L. casei showed the induction of E7-specific mucosal IFNγ-producing cells was dependent on displayed E7-doses but saturated beyond 0.3 μg/108 cells. A new agent, L. casei with endogenous expression of E7 (IGMKK16E7), showed the optimal amount of displayed-E7. Immunization with IGMKK16E7 demonstrated 4-fold higher induction of E7-specific mucosal IFNγ-producing cells when compared with the former one. Our new system provided the optimal E7-expressing L. casei for displayed-E7 amount and induction of mucosal Th1 immune response.

Nanoemulsion adjuvant augments retinaldehyde dehydrogenase activity in dendritic cells and regulates expression of gut homing receptors on T cells

Abstract Mucosal surfaces are the primary point of entry for most infectious agents. Therefore, induction of mucosal immune responses serves as the primary defense by preventing and clearing pathogens. In order to mount an effective immune response, secretory IgA and T cell homing towards mucosal surfaces are two very important factors. Conventional vaccines induce strong systemic immunity, but often fail to produce mucosal immune responses. Our group has developed an oil-in-water nanoemulsion which has been shown to provide protection against mucosal pathogens when administered as an intranasal vaccine adjuvant. In the present study, we demonstrate that intranasal immunization with nanoemulsion indirectly activates the retinaldehyde dehydrogenase in dendritic cells, leading to an increase in secretory IgA production as well as expression of α4β7 and CCR9 gut homing receptors on the mesenteric lymph node cells. Ex vivo stimulation of splenocytes from NE immunized animals established increase in Th1/Th17 cytokines while suppressing Th2, suggesting RALDH increase does not suppress Th1/Th17 response. Our nanoemulsion based adjuvant activates retinaldehyde dehydrogenase without possessing any active ligand and imprints T cells for gut homing without altering systemic immunity. These results provide new insights into the mechanism of our nanoemulsion adjuvant and have significant implications for the design of effective mucosal vaccines.

The Mouse Model of Infection with Citrobacter rodentium.

Citrobacter rodentium is a murine mucosal pathogen used as a model to elucidate the molecular and cellular pathogenesis of infection with two clinically important human gastrointestinal pathogens, enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC). C. rodentium infection provides an excellent model to study different aspects of host-pathogen interaction in the gut, including intestinal inflammatory responses during bacteria-induced colitis, mucosal healing and epithelial repair, the induction of mucosal immune responses, and the role of the intestinal microbiota in mediating resistance to colonization by enteric pathogens. This unit provides detailed protocols for growing this bacterium, infecting mice by intragastric inoculation, measuring bacterial loads in feces and organs, and monitoring intestinal pathology induced by infection. Additional protocols describe steps needed to create frozen stocks, establish a growth curve, perform ex vivo organ cultures, isolate immune cells from the large intestine, and measure immune response by flow cytometry. © 2017 by John Wiley & Sons, Inc.

Induction of antibody response in the oral cavity of dogs following intraocular (eye drop) immunization with Porphyromonas gingivalis cell lysate incorporated in pH-sensitive fusogenic polymer-modified liposomes.

Induction of mucosal immune responses against Porphyromonas gingivalis within the oral cavity of dogs was studied by immunizing with pH-sensitive fusogenic polymer (MGluPG)-modified liposome-associated cell lysate. Dogs immunized with P. gingivalis cell lysate-containing MGluPG-modified liposomes by intraocular (eye drop) route displayed significant levels of P. gingivalis cell lysate-specific serum IgG and IgA as well as mucosal IgA antibodies in saliva secretion. Serum and salivary antibodies generated by intraocularly immunized with MGluPG-modified liposome-associated P. gingivalis cell lysate revealed a significant aggregation activity against P. gingivalis, whereas serum and saliva from dogs receiving MGluPG-modified liposomes unentrapping P. gingivalis cell lysate did not show the aggregation activity against P. gingivalis. Furthermore, P. gingivalis-specific antibodies in saliva of immunized dogs inhibited the adherence of P. gingivalis to cultured HeLa cells. More importantly, salivary antibodies induced by intraocular immunization with P. gingivalis cell lysate-containing MGluPG-modified liposomes significantly inhibited the coaggregation of P. gingivalis with Actinomyces naeslundii and the cell damage activity of P. gingivalis against FaDu cells, an oral epithelial cell. These results suggest that intraocularly administered P. gingivalis cell lysate-containing MGluPG-modified liposomes should be an effective mucosal vaccine against P. gingivalis infection in dogs and may be an important tool for the prevention of periodontitis.

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.

Marked Differences in Mucosal Immune Responses Induced in Ileal versus Jejunal Peyer's Patches to Mycobacterium avium subsp. paratuberculosis Secreted Proteins following Targeted Enteric Infection in Young Calves.

In cattle, Mycobacterium avium subsp. paratuberculosis infection is primarily mediated through M cells overlying Peyer’s patches (PP) in the ileum. The capacity of M. avium subsp. paratuberculosis to invade ileal PP (IPP) versus discrete PP in the jejunum (JPP) and subsequent differences in mucosal immune responses were investigated. Intestinal segments were surgically prepared in both mid-jejunum, containing two JPPs, and in terminal small intestine containing continuous IPP. M. avium subsp. paratuberculosis (109 CFU) was injected into the lumen of half of each intestinal segment when calves were 10–14 days-old and infection confirmed 1–2 months later by PCR and immunohistochemistry. Thirteen recombinant M. avium subsp. paratuberculosis proteins, previously identified as immunogenic, were used to analyze pathogen-specific B- and T-cell responses in PP and mesenteric lymph nodes. IgA plasma cell responses to 9 of 13 recombinant proteins were detected in JPP but not in IPP. Secretory IgA reacting in ELISA with 9 of the 13 recombinant proteins was detected in luminal contents from both jejunal and ileal segments. These observations support the conclusion that pathogen-specific IgA B cells were induced in JPP but not IPP early after a primary infection. The presence of secretory IgA in intestinal contents is consistent with dissemination of IgA plasma cells from the identified mucosa-associated immune induction sites. This is the first direct evidence for M. avium subsp. paratuberculosis uptake by bovine JPP and for local induction of pathogen-specific IgA plasma cell responses after enteric infection. We also provide evidence that bacterial invasion of IPP, a primary B lymphoid tissue, provides a novel strategy to evade induction of mucosal immune responses. Over 60% of PPs in the newborn calf small intestine is primary lymphoid tissue, which has significant implications when designing oral vaccines or diagnostic tests to detect early M. avium subsp. paratuberculosis infections.

Parenteral Vaccination Can Be an Effective Means of Inducing Protective Mucosal Responses.

The current paradigm in vaccine development is that nonreplicating vaccines delivered parenterally fail to induce immune responses in mucosal tissues. However, both clinical and experimental data have challenged this concept, and numerous studies have shown that induction of mucosal immune responses after parenteral vaccination is not a rare occurrence and might, in fact, significantly contribute to the protection against mucosal infections afforded by parenteral vaccines. While the mechanisms underlying this phenomenon are not well understood, the realization that parenteral vaccination can be an effective means of inducing protective mucosal responses is paradigm-shifting and has potential to transform the way vaccines are designed and delivered.

RANKL regulates differentiation of microfold cells in mouse nasopharynx-associated lymphoid tissue (NALT)

Murine nasopharynx-associated lymphoid tissue (NALT), located at the base of the nasal cavity, serves as a major site for the induction of mucosal immune responses against airway antigens. The follicle-associated epithelium (FAE) covering the luminal surface of NALT is characterized by the presence of microfold cells (M cells), which take up and transport luminal antigens to lymphocytes. Glycoprotein 2 (GP2) has recently been identified as a reliable marker for M cells in Peyer's patches of the intestine. However, the expression of GP2 and other functional molecules in the M cells of NALT has not yet been examined. We have immunohistochemically detected GP2-expressing cells in the FAE of NALT and the simultaneous expression of other intestinal M-cell markers, namely Tnfaip2, CCL9, and Spi-B. These cells have been further identified as M cells because of their higher uptake capacity of luminal microbeads. Electron microscopic observations have shown that GP2-expressing cells on the FAE display morphological features typical of M cells: they possess short microvilli and microfolds on the luminal surface and are closely associated with intraepithelial lymphocytes. We have also found that the receptor activator of nuclear factor kappa-B ligand (RANKL) is expressed by stromal cells underneath the FAE, which provides its receptor RANK. The administration of RANKL markedly increases the number of GP2(+)Tnfaip2(+) cells on the NALT FAE and that of intestinal M cells. These results suggest that GP2(+)Tnfaip2(+) cells in NALT are equivalent to intestinal M cells, and that RANKL-RANK signaling induces their differentiation.

Distribution patterns of mucosally applied particles and characterization of the antigen presenting cells

Mucosal application is the most common route of vaccination to prevent outbreaks of infectious diseases like Newcastle disease virus (NDV). To gain more knowledge about distribution and uptake of a vaccine after mucosal vaccination, we studied the distribution pattern of antigens after different mucosal routes of administration. Chickens were intranasally (i.n.), intratracheally (i.t.) or intraocularly (i.o.) inoculated with fluorescent beads and presence of beads in nasal-associated lymphoid tissue (NALT), Harderian gland (HG), conjunctiva-associated lymphoid tissue (CALT), trachea, lungs, air sacs, oesophagus and blood was characterized. The distribution patterns differed significantly between the three inoculation routes. After i.t. inoculation, the beads were mainly retrieved from trachea, NALT and lung. I.n. inoculation resulted in beads found mainly in NALT but detectable in all organs sampled. Finally, after i.o. inoculation, the beads were detected in NALT, CALT, HG and trachea. The highest number of beads was retrieved after i.n. inoculation. Development of novel vaccines requires a comprehensive knowledge of the mucosal immune system in birds in order to target vaccines appropriately and to provide efficient adjuvants. The NALT is likely important for the induction of mucosal immune responses. We therefore studied the phenotype of antigen-presenting cells isolated from NALT after i.n. inoculation with uncoated beads or with NDV-coated beads. Both types of beads were efficiently taken up and low numbers of bead+ cells were detected in all organs sampled. Inoculation with NDV-coated beads resulted in a preferential uptake by NALT antigen-presenting cells as indicated by high percentages of KUL01+-, MHC II+ and CD40+ bead+ cells.

Induction of mucosal immune responses and protection of cattle against direct-contact challenge by intranasal delivery with foot-and-mouth disease virus antigen mediated by nanoparticles.

The aim of this study was to enhance specific mucosal, systemic, and cell-mediated immunity and to induce earlier onset of protection against direct-contact challenge in cattle by intranasal delivery of a nanoparticle-based nasal vaccine against type A foot-and-mouth disease (FMD). In this study, two kinds of nanoparticle-based nasal vaccines against type A FMD were designed: (1) chitosan-coated poly(lactic-co-glycolic acid) (PLGA) loaded with plasmid DNA (Chi-PLGA-DNA) and (2) chitosan-trehalose and inactivated foot-and-mouth disease virus (FMDV) (Chi-Tre-Inactivated). Cattle were immunized by an intranasal route with nanoparticles and then challenged for 48 hours by direct contact with two infected donor cattle per pen. Donors were inoculated intradermally in the tongue 48 hours before challenge, with 0.2 mL cattle-passaged FMDV. Serological and mucosal antibody responses were evaluated, and virus excretion and the number of contact infections were quantified. FMDV-specific secretory immunoglobulin (Ig)A (sIgA) antibodies in nasal washes were initially detected at 4 days postvaccination (dpv) with two kinds of nanoparticles. The highest levels of sIgA expression were observed in nasal washes, at 10 dpv, from animals with Chi-PLGA-DNA nanoparticles, followed by animals immunized once by intranasal route with a double dose of Chi-Tre-Inactivated nanoparticles and animals immunized by intranasal route three times with Chi-Tre-Inactivated nanoparticles (P<0.05). FMDV-specific IgA antibodies in serum showed a similar pattern. All animals immunized by intranasal route developed low levels of detectable IgG in serum at 10 dpv. Following stimulation with FMDV, the highest levels of proliferation were observed in splenocytes harvested from Chi-PLGA-DNA-immunized animals, followed by proliferation of cells harvested from Chi-Tre-Inactivated nanoparticle-immunized animals (P<0.05). Higher protection rates were associated with the highest sIgA antibody responses induced in the Chi-PLGA-DNA nanoparticle-immunized group. Only one animal was clinically affected with mild signs after 7 days of contact challenge, after a delay of 2–3 days compared with the clinically affected negative-control group. Of the five animals directly challenged that were vaccinated by intranasal route with a double dose of Chi-Tre-Inactivated, four were clinically infected; however, the degree of severity of disease in this group was lower than in control cattle. The number of viral RNA copies in nasal swabs from the vaccinated, severely infected group was significantly higher than in swabs from the vaccinated, clinically protected group. These data suggested that intranasal delivery of Chi-PLGA-DNA nanoparticles resulted in higher levels of mucosal, systemic, and cell-mediated immunity than did of Chi-Tre-Inactivated nanoparticles. In conclusion, although intranasal delivery with FMDV antigen mediated by nanoparticles did not provide complete clinical protection, it reduced disease severity and virus excretion and delayed clinical symptoms. Chi-PLGA-DNA nanoparticle vaccines have potential as a nasal delivery system for vaccines.

Lactococcus lactis IBB477 presenting adhesive and muco-adhesive properties as a candidate carrier strain for oral vaccination against influenza virus.

In the gastrointestinal tract (GIT), adhesion is a prerequisite for bacterial colonization. Lactococci can be used in functional food (probiotics) and health-related applications (mucosal vaccines, therapeutic drug delivery), both potentially involving adhesive properties. A candidate lactic acid bacterium for influenza antigen delivery through the GIT should display the ability to adhere. The present work probes the interactions between Lactococcus lactis and mucins using pig gastric mucin (PGM) as a model. Two strains were used for the optimization of the screening method for adhesion: L. lactis subsp. cremoris IBB477 persistent in the GIT of germ-free rats, and the low-adhering control strain MG1820. High adhesion to bare and mucin-coated polystyrene of IBB477 in comparison with MG1820 was observed. We searched for genetic determinants potentially involved in the adhesion/muco-adhesion of IBB477, identifying two such genes: prtP and a gene coding for a protein with MUB and MucBP domains. Based on its persistence in the GIT and adhesive properties, L. lactis IBB477 is a candidate carrier strain for expression of influenza haemagglutinin (HA) protein for induction of mucosal immune response.

Mucosal immunization of calves with recombinant bovine adenovirus-3 coexpressing truncated form of bovine herpesvirus-1 gD and bovine IL-6

Previous studies have suggested an important role of the cytokine adjuvant IL-6 in the induction of mucosal immune responses in animals, including mice. Here, we report the in vivo ability of bovine adenovirus (BAdV)-3 expressing bovine (Bo) IL-6, to influence the systemic and mucosal immune responses against bovine herpesvirus (BHV)-1 gDt in calves. To co-express both antigen and cytokine, we first constructed a recombinant BAdV-3 expressing chimeric gDt.BoIL-6 protein (BAV326). Secondly, we constructed another recombinant BAdV-3 simultaneously expressing gDt and BoIL-6 using IRES containing a bicistronic cassette gDt-IRES.IL-6, (BAV327). Recombinant proteins expressed by BAV326 and BAV327 retained antigenicity (gDt) and biological activity (BoIL-6). Intranasal immunization of calves with recombinant BAV326, BAV327 or BAV308 (gDt alone) resulted in demonstrable levels of gDt-specific IgG responses in sera and IgA response in nasal secretions, in all animals. In addition, all calves developed complement-independent neutralizing antibody responses against BHV-1. However, no significant difference could be observed in the induction of systemic or mucosal immune response in animals immunized with recombinant BAV326 or BAV327 co-expressing BoIL-6. Moreover, there was no difference in the protection against BHV-1 challenge particularly in the amount of virus excretion in the nasal cavity in calves immunized with BAV326, BAV327 or BAV308. These data suggest that the BoIL-6 had no modulating effect on the induction of gDt specific mucosal and systemic immune responses in calves.

Recent progress in mucosal immunology and vaccine development

Recent progress in mucosal immunology and vaccine development