Antibody-Mediated Targeting of Antigens to Intestinal Aminopeptidase N Elicits Gut IgA Responses in Pigs.

Many pathogens enter the host via the gut, causing disease in animals and humans. A robust intestinal immune response is necessary to protect the host from these gut pathogens. Despite being best suited for eliciting intestinal immunity, oral vaccination remains a challenge due to the gastrointestinal environment, a poor uptake of vaccine antigens by the intestinal epithelium and the tolerogenic environment pervading the gut. To improve uptake, efforts have focused on targeting antigens towards the gut mucosa. An interesting target is aminopeptidase N (APN), a conserved membrane protein present on small intestinal epithelial cells shown to mediate epithelial transcytosis. Here, we aimed to further optimize this oral vaccination strategy in a large animal model. Porcine APN-specific monoclonal antibodies were generated and the most promising candidate in terms of epithelial transcytosis was selected to generate antibody fusion constructs, comprising a murine IgG1 or porcine IgA backbone and a low immunogenic antigen: the F18-fimbriated E. coli tip adhesin FedF. Upon oral delivery of these recombinant antibodies in piglets, both mucosal and systemic immune responses were elicited. The presence of the FedF antigen however appeared to reduce these immune responses. Further analysis showed that F18 fimbriae were able to disrupt the antigen presenting capacity of intestinal antigen presenting cells, implying potential tolerogenic effects of FedF. Altogether, these findings show that targeted delivery of molecules to epithelial aminopeptidase N results in their transcytosis and delivery to the gut immune systems. The results provide a solid foundation for the development of oral subunit vaccines to protect against gut pathogens.

Aspherical and Spherical InvA497-Functionalized Nanocarriers for Intracellular Delivery of Anti-Infective Agents.

PURPOSE: The objective of this work was to evaluate the potential of polymeric spherical and aspherical invasive nanocarriers, loaded with antibiotic, to access and treat intracellular bacterial infections. METHODS: Aspherical nanocarriers were prepared by stretching of spherical precursors, and both aspherical and spherical nanocarriers were surface-functionalized with the invasive protein InvA497. The relative uptake of nanocarriers into HEp-2 epithelial cells was then assessed. Nanocarriers were subsequently loaded with a preparation of the non-permeable antibiotic gentamicin, and tested for their ability to treat HEp-2 cells infected with the enteroinvasive bacterium Shigella flexneri. RESULTS: InvA497-functionalized nanocarriers of both spherical and aspherical shape showed a significantly improved rate and extent of uptake into HEp-2 cells in comparison to non-functionalized nanocarriers. Functionalized and antibiotic-loaded nanocarriers demonstrated a dose dependent killing of intracellular S. flexneri. A slight but significant enhancement of intracellular bacterial killing was also observed with aspherical as compared to spherical functionalized nanocarriers at the highest tested concentration. CONCLUSIONS: InvA497-functionalized, polymer-based nanocarriers were able to efficiently deliver a non-permeable antibiotic across host cell membranes to affect killing of intracellular bacteria. Functionalized nanocarriers with an aspherical shape showed an interesting future potential for intracellular infection therapy.

Regional immune response to immunization with Escherichia coli O157:H7-derived intimin in cattle.

Escherichia coli O157:H7 is an enteric pathogen of animals and humans that can result in deadly sequelae. Cattle are asymptomatic carriers and shedders of the bacteria and serve as an important reservoir of human infection. E. coli O157:H7 colonizes the gastrointestinal tract, most frequently at the rectoanal junction mucosa in cattle. Vaccination is a potentially highly effective means of decreasing cattle colonization and shedding and thereby decreasing human infections. Currently available vaccines are administered subcutaneously or intramuscularly, and immune responses have been evaluated solely by systemic immunoglobulin responses. This study evaluated local and systemic lymphoproliferative responses in addition to immunoglobulin responses following subcutaneous or mucosal (rectal) immunization with E. coli O157:H7 outer membrane protein intimin over three trials. In all three trials, significant local and systemic lymphoproliferative responses (P < 0.05) occurred following immunization in the majority of animals, as well as significant immunoglobulin responses (P < 0.001) in all animals. Surprisingly, local responses in the mesorectal lymph nodes were very similar between the subcutaneous and mucosal immunization groups. Moreover, the responses in mesorectal lymph nodes appeared targeted rather than generalized, as minimal or no significant responses were observed in the associated prescapular lymph nodes of subcutaneously immunized animals. The results indicate that both subcutaneous and mucosal immunizations are effective methods of inducing immune responses against E. coli O157:H7 in cattle.

[Expression of integrin α5 and ß1 in osteoblast in the process of gingipains-induced apoptosis].

OBJECTIVE: To investigate the regulatory mechanisms of integrin α5 and ß1 in osteoblast in the process of gingipains-induced apoptosis. METHODS: Gingipains were isolated and purified from supernatants of Porphyromonas gingivalis W83 which was cultured under standard anaerobic conditions. MC3T3-E1 was challenged with or without 8.3480 U/L gingipains for 48 h and apoptosis was examined by transferase-mediated deoxyuridine triphosphate-biotin nick end labeling-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (TUNEL-DAPI) staining. The expression of integrin α5 and ß1 was analyzed by Western blotting after MC3T3-E1 was treated under different conditions. RESULTS: Arginine-specific proteinases(Rgp) activity was (41.74 ± 2.11) U/L and lysine-specific proteinase(Kgp) was (1.02 ± 0.25) U/L.Gingipains induced MC3T3-E1 cells apoptosis after 48 h. Compared with control group, expression of integrin α5 and ß1 was down-regulated by gingipains in a time-dependent manner within short periods ( ≤ 72 h), integrin α5 and ß1 relative expression was (0.485 ± 0.039),(0.504 ± 0.002) at 48 h,(0.398 ± 0.058),(0.179 ± 0.001) at 72 h respectively (P < 0.05). After 72 h, integrin α5 expression in MC3T3-E1 cells was stable compared with control group while integrin ß1 was still lower(control group:1.000 ± 0.000, 96 h:0.604 ± 0.003, 120 h: 0.357 ± 0.002) (P < 0.05). Proteinase inhibitor tosyl- L- lysine-chloromethyl-ketone(TLCK) effectively blocked the activity of gingipain and inhibited down-regulation of integrin α5 and ß1 induced by gingipains from (0.398 ± 0.058,0.179 ± 0.001 ) to (0.781 ± 0.012, 0.857 ± 0.060) (P < 0.05). TLCK alone did not have any effect on integrin α5 and ß1(P > 0.05). Gingipains also decreased integrin α5 and ß1 in a dose-dependent manner.When cells were treated with 20.8700 U/L gingipains, integrin α5 and ß1 relative expression reached to the lowest(0.105 ± 0.004,0.020 ± 0.000) (P < 0.05). CONCLUSIONS: Gingipains inhibited the expression of integrin α5 and ß1 in a time- and dose- dependent manner in osteoblasts in the process of apoptosis, which may not be mediated by direct proteolytic effect.

In vivo transfection study of chitosan-DNA-FAP-B nanoparticles as a new non viral vector for gene delivery to the lung.

Gene therapy targeted at the respiratory epithelium holds therapeutic potential for diseases such as cystic fibrosis and lung cancer. We recently reported that Chitosan-DNA-FAP-B nanoparticles are good candidates for targeted gene delivery to fibronectin molecules (FAP-B receptors) of lung epithelial cell membrane. In this study Chitosan-DNA-FAP-B nanoparticles were nebulized to mice using air jet nebulizer. The effect of nebulization on size, zeta potential and DNA binding ability of nanoparticles were studied. The level of gene expression in the mice lungs was evaluated. Nebulization did not affect the physicochemical properties of nanoparticles. Aerosol delivery of Chitosan-DNA-FAP-B nanoparticles resulted in 16-fold increase of gene expression in the mice lungs compared with Chitosan-DNA nanoparticles. This study suggested that Chitosan-FAP-B nanoparticle can be a promising carrier for targeted gene delivery to the lung.

HGP44 induces protection against Porphyromonas gingivalis-Induced alveolar bone loss in mice.

The protective effect of DNA vaccines expressing the Arg-gingipain A domain against bone loss induced by Porphyromonas gingivalis infection was investigated in a murine model. phgp44, which expresses the 44-kDa adhesion/hemagglutinin domain of Arg-gingipain A, prevented P. gingivalis-induced alveolar bone loss. The results indicate that phgp44 could be a candidate antigen for a vaccine against P. gingivalis infection.

Immunogenicity of orally administrated recombinant Lactobacillus casei Zhang expressing Cryptosporidium parvum surface adhesion protein P23 in mice.

Cryptosporidium parvum, an intestinal apicomplexan parasite, is a significant cause of diarrheal diseases in both humans and animals. What is more, there is no promising strategy for controlling cryptosporidiosis. In this study, the P23 immunodominant surface protein of C. parvum sporozoites was stably expressed in the Lactobacillus casei Zhang strain and its immunogenicity was evaluated in a mouse model. The molecular weight (23 kDa) and immunogenicity of p23 gene expressed by L. casei Zhang were similar to that of the native P23 protein. Oral immunization with control L. casei Zhang and recombinant L. casei Zhang-p23 activated the mucosal immune system to elicit serum immunoglobulin G (IgG) and mucosal IgA in mice. Furthermore, the expression of cytokines such as IL-4, IL-6, and IFN-γ in splenocytes of mice was detected by real-time PCR after oral immunization. P23-specific immunocyte activation was also verified. These findings indicate that the live L. casei Zhang vector may be a new tool for the production of mucosal vaccines against cryptosporidiosis in animals.

[Recombinant fragments of conservative proteins of group B streptococci as a basis of specific vaccine].

AIM: The study devoted to problem of using of recombinant fragments of group B streptococci (GBS) conservative proteins for induction of immune response against streptococcal infections. Two recombinant polypeptides (ScaAB and-ScpB1) corresponding to immunogenic epitopes of two surface GBS proteins ScaAB and C5a-peptidase, which are presented in other streptococcal species, were studied. The objective of the study was to assess specificity and protective activity of mentioned polypeptides against homologous and heterologous strains of pathogenic streptococci from different groups. MATERIALS AND METHODS: Strains of Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus agalactiae were used in the study. Array of used methods included opsonophagocytic test as well as active and passive protection of experimental animals against streptococcal infection. RESULTS: It was shown that antibodies specific to studied polypeptides opsonized several strains of group A and B streptococci as well as pneumococci. Immunization of mice with ScpB1 polypeptide resulted in more rapid recovery of animals from challenge systemic group B streptococcal infection. Antisera specific to both polypeptides provided passive protection of animals from infection caused either GBS or GAS. CONCLUSION: Obtained data confirm the feasibility to use recombinant fragments of several GBS conservative proteins in vaccine for induction of protection against infections caused by different species of pathogenic streptococci.

Immune responses to a DNA/protein vaccination strategy against Staphylococcus aureus induced mastitis in dairy cows.

The fibronectin binding protein (FnBP) and clumping factor A (ClfA) of Staphylococcus aureus are important proteins involved in the pathogenesis of staphylococcal bovine mastitis. These antigens were the targets of a DNA and protein vaccination strategy against S. aureus induced mastitis in dairy cows. The DNA vaccine comprised the bicistronic plasmid (pCI-D(1)D(3)-IRES-ClfA) that encoded the fusion of two sequences, (D1(21-34); D3(20-33)) from the fibronectin-binding motifs of FnBP and a fragment from ClfA (aa 221-550) of S. aureus 8325-4 separated by an Internal Ribosomal Entry Site (IRES) sequence. In addition, the vaccine contained the plasmid encoding the bovine granulocyte-macrophage-colony stimulatory factor gene (pCI-bGM-CSF). Four, 7-month pregnant heifers were immunized twice with the DNA vaccine and boosted once with recombinant D(1)D(3) and ClfA proteins while four others were not immunized. The immunization induced lymphoproliferative responses and functional antibodies against D(1)D(3) and ClfA antigens. Three weeks after calving, three mammary quarters of each vaccinated and non-vaccinated cow were challenged with 900 CFU/each of S. aureus Newbould 305. The fourth quarter received saline only. Serum haptoglobin levels, cardiac rhythm and the body temperature of vaccinated cows during the 24-72 h post-challenge were lower than in non-vaccinated animals. At 21 days post-challenge, bacteria were present in 5 of the vaccinated and 11 of the control challenged quarters. The bacteria averaged 1.4 and 3.3 log(10) CFU/ml of milk from vaccinated and control cows respectively. In summary, DNA-protein vaccination against FnBP and ClfA of S. aureus caused both lymphoproliferative and humoral immune responses that provided partial protection of mammary gland from staphylococcal mastitis and better post-challenge conditions in vaccinated cows.

Proteolysis of ICAM-1 on human oral epithelial cells by gingipains.

Cysteine proteinases (gingipains) from Porphyromonas gingivalis are considered key virulence factors of severe periodontitis and host immune evasion. Since expression of intercellular adhesion molecule-1 (ICAM-1) on gingival epithelium is indispensable in polymorphonuclear leukocyte (PMN) migration at the site of periodontitis, we examined the effects of gingipains on the expression of ICAM-1 on human oral epithelial cell lines (KB and HSC-2) by flow cytometry and Western blotting. We found that three purified forms of gingipains efficiently reduced ICAM-1 expression on the cells in a time- and dose-dependent manner. Gingipains reduced the expression on fixed cells and degraded the ICAM-1 in the cell membranes, indicating that the reduction resulted from direct proteolysis. They then disturbed the ICAM-1-dependent adhesion of PMNs to the cells. These results indicate that gingipains cleave ICAM-1 on oral epithelial cells, consequently disrupting PMN-oral epithelial cell interaction, and are involved in immune evasion by the bacterium in periodontal tissues.

Vaccination with FimH adhesin protects cynomolgus monkeys from colonization and infection by uropathogenic Escherichia coli.

Escherichia coli FimH adhesin mediates binding to the bladder mucosa. In mice, a FimH vaccine protects against bacterial challenge. In this study, 4 monkeys were inoculated with 100 microgram of FimCH adhesin-chaperone complex mixed with MF59 adjuvant, and 4 monkeys were given adjuvant only intramuscularly. After 2 doses (day 0 and week 4), a booster at 48 weeks elicited a strong IgG antibody response to FimH in the vaccinated monkeys. All 8 monkeys were challenged with 1 mL of 108 E. coli cystitis isolate NU14. Three of the 4 vaccinated monkeys were protected from bacteruria and pyuria; all control monkeys were infected. These findings suggest that a vaccine based on the FimH adhesin of E. coli type 1 pili may have utility in preventing cystitis in humans.