Mucosal Immunization with a \u3cem\u3eStaphylococcus aureus\u3c/em\u3e IsdA-Cholera Toxin A\u3csub\u3e2\u3c/sub\u3e/B Chimera Induces Antigen-Specific Th2-Type Responses in Mice

Staphylococcus aureus is a leading cause of opportunistic infection worldwide and a significant public health threat. The iron-regulated surface determinant A (IsdA) adhesin is essential for S. aureus colonization on human nasal epithelial cells and plays an important role in iron acquisition and resistance to human skin defenses. Here we investigated the murine immune response to intranasal administration of a cholera toxin (CT) A2/B chimera containing IsdA. Plasmids were constructed to express the IsdA-CTA2/B chimera and control proteins in E. coli. Proper construction of the chimera was verified by SDS-PAGE, western blot, GM1 ELISA, and confocal microscopy. Groups of female BALB/c mice were immunized with IsdA-CTA2/B, IsdA mixed with CTA2/B, IsdA alone, or mock, followed by one booster immunization 10 days post-priming. Analysis of serum IgG and nasal, intestinal, and vaginal IgA suggested that mucosal immunization with IsdA-CTA2/B induces significant IsdA-specific humoral immunity. Functional in vitro assays revealed that α-IsdA immune serum significantly blocks the adherence of S. aureus to human epithelial cells. Splenocytes from mice immunized with IsdA-CTA2/B showed specific cellular proliferation and production of IL-4 after in vitro stimulation. Immunization with IsdA-CTA2/B drove isotype switching to IgG1, indicative of a Th2-type response. Our results suggest that the immunogenicity of the S. aureus IsdA-CTA2/B chimera merits further investigation as a potential mucosal vaccine candidate

α-Hemolysin as a Candidate for a Vaccine for \u3cem\u3eStaphylococcus aureus\u3c/em\u3e in Bovine Mastitis

Staphylococcus aureus is a Gram-positive bacteria responsible for many types of infections. It is abundant in nature, even present on our own skin, usually harmless. However, it is the leading cause of infection in humans. S. aureus also harms animals, and in dairy cows, causes Bovine mastitis. This disease results in a decreased quality and quantity of milk, inflammation of the mammary glands, and can even be transmitted to humans.(1) Because of this, there are massive economic ramifications estimated at $629 million annually.(2) This study focuses on a virulent factor known as a-hemolysin (Hla) and cloning this into S. aureus bacteria to make a vaccine to treat bovine mastitis. This is a protein present on the cell membrane of S. aureus, known for its cytotoxic properties. To harm eukaryotic cells, research suggests that Hla has a close relationship with a eukaryotic cell receptor known as ADAM10. Normally, this receptor has a role in the development of the nervous system, and in precursor formation of the amyloid protein. When S. aureus is exposed to these cell receptors, a bridge is formed between the Hla protein of the bacteria and the surface receptor ADAM10. After the link is formed, the Hla protein drills a pore into the eukaryotic cell causing it to lyse. (3) This makes the Hla protein a great candidate for a vaccine, as if this interaction could be prevented, then harm would be reduced in the host cell

Sle1 (\u3cem\u3eaaa\u3c/em\u3e) and Alpha Hemolysin (\u3cem\u3ehla\u3c/em\u3e) \u3cem\u3eStaphylococcus aureus\u3c/em\u3e Antigens as a Potential Vaccine for Cows

Knowledge of Staphylococcus aureus is essential to understanding how this pathogen causes different types of infections in humans. It can cause skin superficial infections and gastroenteritis. It can also cause infections in the joints and wounds, therefore causing humans to be severely ill by causing sepsis or infection in the blood. It is also very commonly antibiotic-resistant. In the lab, we are working with this priority pathogen because of antibiotic resistance. Cows can get S. aureus from humans, and it causes mastitis. This affects the dairy industry which is very important in the United States, but also specifically important in Idaho. We are making our vaccine through what\u27s called a chimera, we are fusing antigen proteins from S. aureus to cholera toxin (CT). The antigens we are using is Aaa/Sle1; a peptidoglycan hydrolase and adhesin, and Hla, a hemolytic pore protein. We have performed PCR and cloned this gene into a vector for chimera expression. The importance of this research is to improve productivity and quality of life by preventing S. aureus in cows. Everything that we learn about developing a vaccine for a cow can also translate to developing a vaccine for humans

Cellular Activity of \u3ci\u3eSalmonella\u3c/i\u3e Typhimurium ArtAB Toxin and Its Receptor-Binding Subunit

Salmonellosis is among the most reported foodborne illnesses in the United States. The Salmonella enterica Typhimurium DT104 phage type, which is associated with multidrug-resistant disease in humans and animals, possesses an ADP-ribosylating toxin called ArtAB. Full-length artAB has been found on a number of broad-host-range non-typhoidal Salmonella species and serovars. ArtAB is also homologous to many AB5 toxins from diverse Gram-negative pathogens, including cholera toxin (CT) and pertussis toxin (PT), and may be involved in Salmonella pathogenesis, however, in vitro cellular toxicity of ArtAB has not been characterized. artAB was cloned into E. coli and initially isolated using a histidine tag (ArtABHIS) and nickel chromatography. ArtABHIS was found to bind to African green monkey kidney epithelial (Vero) cells using confocal microscopy and to interact with glycans present on fetuin and monosialotetrahexosylganglioside (GM1) using ELISA. Untagged, or native, holotoxin (ArtAB), and the pentameric receptor-binding subunit (ArtB) were purified from E. coli using fetuin and D-galactose affinity chromatography. ArtAB and ArtB metabolic and cytotoxic activities were determined using Vero and Chinese hamster ovary (CHO) epithelial cells. Vero cells were more sensitive to ArtAB, however, incubation with both cell types revealed only partial cytotoxicity over 72 h, similar to that induced by CT. ArtAB induced a distinctive clustering phenotype on CHO cells over 72 h, similar to PT, and an elongated phenotype on Vero cells, similar to CT. The ArtB binding subunit alone also had a cytotoxic effect on CHO cells and induced morphological rounding. Results indicate that this toxin induces distinctive cellular outcomes. Continued biological characterization of ArtAB will advance efforts to prevent disease caused by non-typhoidal Salmonella

Burrowing owls, Pulex irritans and plague

Western burrowing owls (Athene cunicularia hypugaea) are small, ground-dwelling owls ofwestern North America that frequent prairie dog (Cynomys spp.) towns and other grasslands.As they rely on rodent prey and occupy burrows once or concurrently inhabited by fossorialmammals, the owls often harbor fleas. We examined the potential role of fleas found onburrowing owls in plague dynamics by evaluating prevalence of Yersinia pestis in fleas and inowl blood. During 2012-2013 fleas and blood were collected from burrowing owls in portionsof five states with endemic plague: Idaho, Oregon, Washington, Colorado, and South Dakota.Fleas were enumerated, taxonomically identified, pooled by nest and assayed for Y. pestis usingculturing and molecular (PCR) approaches. Owl blood underwent serological analysis for plagueantibodies and nested PCR for detection of Y. pestis. Of \u3e4750 fleas collected from owls, Pulexirritans, a known plague vector in portions of its range, comprised more than 99.4%. However,diagnostic tests for Y. pestis of flea pools (culturing and PCR) and owl blood (PCR and serology)were negative. Thus, despite that fleas were prevalent on burrowing owls, and the potentialfor a relationship with burrowing owls as a phoretic host of infected fleas exists, we found noevidence of Y. pestis in sampled fleas or in owls that harbored them. We suggest that studiessimilar to those reported here during plague epizootics will be especially useful for confirmingthese results

Immunogenicity of a West Nile Virus DIII-Cholera Toxin A\u3csub\u3e2\u3c/sub\u3e/B Chimera After Intranasal Delivery

West Nile virus (WNV) causes potentially fatal neuroinvasive disease and persists at endemic levels in many parts of the world. Despite advances in our understanding of WNV pathogenesis, there remains a significant need for a human vaccine. The domain III (DIII) region of the WNV envelope protein contains epitopes that are the target of neutralizing antibodies. We have constructed a chimeric fusion of the non-toxic cholera toxin (CT) CTA2/B domains to DIII for investigation as a novel mucosally-delivered WNV vaccine. Purification and assembly of the chimera, as well as receptor-binding and antigen delivery, were verified by western blot, GM1 ELISA and confocal microscopy. Groups of BALB/c mice were immunized intranasally with DIII-CTA2/B, DIII, DIII mixed with CTA2/B, or CTA2/B control, and boosted at 10 days. Analysis of serum IgG after 14 and 45 days revealed that mucosal immunization with DIII-CTA2/B induced significant DIII-specific humoral immunity and drove isotype switching to IgG2a. The DIII-CTA2/B chimera also induced antigen-specific IgM and IgA responses. Bactericidal assays indicate that the DIII-CTA2/B immunized mice produced DIII-specific antibodies that can trigger complement-mediated killing. A dose escalation resulted in increased DIII-specific serum IgG titers on day 45. DIII antigen alone, in the absence of adjuvant, also induced significant systemic responses after intranasal delivery. Our results indicate that the DIII-CTA2/B chimera is immunogenic after intranasal delivery and merits further investigation as a novel WNV vaccine candidate

Risk vs. Reward?: The Truth About Vaccines

Vaccines have had a profoundly positive impact on human health, but the practice of vaccination also has its detractors. In this podcast, Dr. Tinker explores the causes of the current loss of confidence in vaccine safety, and the impacts this loss has had on the incidence of disease. She also presents rationale regarding why vaccine research is needed and analyzes the pipeline for continued development of new and improved methods of immunization. Dr. Tinker examines the historical benefits of vaccines that have been used to fight smallpox, polio, rubella, and many other infectious diseases. Her current research regarding the search for a vaccine to fight staph infections is also chronicled.https://scholarworks.boisestate.edu/beyond_the_blue_podcasts/1020/thumbnail.jp

Immunogenicity of a West Nile Virus DIII-Cholera Toxin A2/B Chimera after Intranasal Delivery

West Nile virus (WNV) causes potentially fatal neuroinvasive disease and persists at endemic levels in many parts of the world. Despite advances in our understanding of WNV pathogenesis, there remains a significant need for a human vaccine. The domain III (DIII) region of the WNV envelope protein contains epitopes that are the target of neutralizing antibodies. We have constructed a chimeric fusion of the non-toxic cholera toxin (CT) CTA2/B domains to DIII for investigation as a novel mucosally-delivered WNV vaccine. Purification and assembly of the chimera, as well as receptor-binding and antigen delivery, were verified by western blot, GM1 ELISA and confocal microscopy. Groups of BALB/c mice were immunized intranasally with DIII-CTA2/B, DIII, DIII mixed with CTA2/B, or CTA2/B control, and boosted at 10 days. Analysis of serum IgG after 14 and 45 days revealed that mucosal immunization with DIII-CTA2/B induced significant DIII-specific humoral immunity and drove isotype switching to IgG2a. The DIII-CTA2/B chimera also induced antigen-specific IgM and IgA responses. Bactericidal assays indicate that the DIII-CTA2/B immunized mice produced DIII-specific antibodies that can trigger complement-mediated killing. A dose escalation resulted in increased DIII-specific serum IgG titers on day 45. DIII antigen alone, in the absence of adjuvant, also induced significant systemic responses after intranasal delivery. Our results indicate that the DIII-CTA2/B chimera is immunogenic after intranasal delivery and merits further investigation as a novel WNV vaccine candidate

Identification of Novel ADPRTs in \u3cem\u3eSalmonella infantis\u3c/em\u3e

Salmonella infantis, a serovar of Salmonella enterica, is an emerging pathogen that’s closely related to S. typhi, which causes typhoid fever. It has long been hypothesized that S. typhi does this through the action of its ADP ribosylating toxin (ADPRT), a type of toxin also found in Vibrio cholerae and Bordetella pertussis. As a bacteria which has recently been increasing in virulence and prevalence, it stands to reason that S. infantis may also possess such a toxin. Identification of a novel S. infantis toxin would lend insight into the mechanism of disease from S. infantis, as well as providing a target for vaccination and other treatment. Recent advances in analytical software and increases in the breadth and depth of online databases enable toxin identification to occur almost completely in silico; use of methods like DELTA-BLAST and Clustal Omega yielded forty-seven potential toxins from S. infantis, all labeled “pertussis toxin”. Further organization of these toxins into nine groups and careful alignment with known ADPRTs revealed that first, there was a high degree of variability among these toxins, and second, that several have 50% or less similarity to known ADPRTs like ArtA, which seems to be the most similar known toxin. Further analysis and stringent elimination of more similar groups leaves a single group with 30% similarity to ArtA as a probable example of a novel S. infantis toxin. Analysis of S. infantis genomes reveals that the toxins are almost certainly carried in prophage regions, but the original phage cannot definitively be identified. Further cloning and isolation will reveal whether this group is representative of an active toxin with APDRT capabilities, but certain structural characteristics seem to indicate that it is