ASU Scholarship Showcase

Streptococcus pneumoniae still causes severe morbidity and mortality worldwide, especially in young children and the elderly. Much effort has been dedicated to developing protein-based universal vaccines to conquer the current shortcomings of capsular vaccines and capsular conjugate vaccines, such as serotype replacement, limited coverage and high costs. A recombinant live vector vaccine delivering protective antigens is a promising way to achieve this goal. In this review, we discuss the researches using live recombinant vaccines, mainly live attenuated Salmonella and lactic acid bacteria, to deliver pneumococcal antigens. We also discuss both the limitations and the future of these vaccines.

Outer membrane vesicles (OMVs) isolated from Salmonella Typhimurium are potentially useful for developing subunit vaccines because of high immunogenicity and protective efficacy. However, flagella might remain in OMV pellets following OMV purification, resulting in non-essential immune responses and counteraction of bacterial protective immune responses when developing a vaccine against infection of multiple serotypes Salmonella. In this study, a flagellin-deficient S. Typhimurium mutant was constructed. Lipopolysaccharide profiles, protein profiles and cryo-electron microscopy revealed that there were no significant differences between the wild-type and mutant OMVs, with the exception of a large amount of flagellin in the wild-type OMVs. Neither the wild-type OMVs nor the non-flagellin OMVs were toxic to macrophages. Mice immunized with the non-flagellin OMVs produced high concentrations of IgG. The non-flagellin OMVs elicited strong mucosal antibody responses in mice when administered via the intranasal route in addition to provoking higher cross-reactive immune responses against OMPs isolated from S. Choleraesuis and S. Enteritidis. Both intranasal and intraperitoneal immunization with the non-flagellin OMVs provided efficient protection against heterologous S. Choleraesuis and S. Enteritidis challenge. Our results indicate that the flagellin-deficient OMVs may represent a new vaccine platform that could be exploited to facilitate the production of a broadly protective vaccine.

Bacterial lipopolysaccharides (LPS) are structural components of the outer membranes of Gram-negative bacteria and also are potent inducers of inflammation in mammals. Higher vertebrates are extremely sensitive to LPS, but lower vertebrates, like fish, are resistant to their systemic toxic effects. However, the effects of LPS on the fish intestinal mucosa remain unknown. Edwardsiella ictaluri is a primitive member of the Enterobacteriaceae family that causes enteric septicemia in channel catfish (Ictalurus punctatus). E. ictaluri infects and colonizes deep lymphoid tissues upon oral or immersion infection. Both gut and olfactory organs are the primary sites of invasion. At the systemic level, E. ictaluri pathogenesis is relatively well characterized, but our knowledge about E. ictaluri intestinal interaction is limited. Recently, we observed that E. ictaluri oligo-polysaccharide (O-PS) LPS mutants have differential effects on the intestinal epithelia of orally inoculated catfish. Here we evaluate the effects of E. ictaluri O-PS LPS mutants by using a novel catfish intestinal loop model and compare it to the rabbit ileal loop model inoculated with Salmonella enterica serovar Typhimurium LPS. We found evident differences in rabbit ileal loop and catfish ileal loop responses to E. ictaluri and S. Typhimurium LPS. We determined that catfish respond to E. ictaluri LPS but not to S. Typhimurium LPS. We also determined that E. ictaluri inhibits cytokine production and induces disruption of the intestinal fish epithelia in an O-PS-dependent fashion. The E. ictaluri wild type and ΔwibT LPS mutant caused intestinal tissue damage and inhibited proinflammatory cytokine synthesis, in contrast to E. ictaluri Δgne and Δugd LPS mutants. We concluded that the E. ictaluri O-PS subunits play a major role during pathogenesis, since they influence the recognition of the LPS by the intestinal mucosal immune system of the catfish. The LPS structure of E. ictaluri mutants is needed to understand the mechanism of interaction.

Antibiotic-resistant bacterial infections are difficult to treat, producing a burden on healthcare and the economy. Extraintestinal pathogenic Escherichia coli (ExPEC) strains frequently carry antibiotic resistance genes, cause infections outside of the intestine, and are causative agents of hospital-acquired infections. Developing a prevention strategy against this pathogen is challenging due to its antibiotic resistance and antigenic diversity. E. coli common pilus (ECP) is frequently found in ExPEC strains and may serve as a common antigen to induce protection against several ExPEC serotypes. In addition, live recombinant attenuated Salmonella vaccine (RASV) strains have been used to prevent Salmonella infection and can also be modified to deliver foreign antigens. Thus, the objective of this study was to design a RASV to produce ECP on its surface and assess its ability to provide protection against ExPEC infections. To constitutively display ECP in a RASV strain, we genetically engineered a vector (pYA4428) containing aspartate-β-semialdehyde dehydrogenase and E. coli ecp genes and introduced it into RASV χ9558. RASV χ9558 containing an empty vector (pYA3337) was used as a control to assess protection conferred by the RASV strain without ECP.

We assessed vaccine efficacy in in vitro bacterial inhibition assays and mouse models of ExPEC-associated human infections. We found that RASV χ9558(pYA4428) synthesized the major pilin (EcpA) and tip pilus adhesin (EcpD) on the bacterial surface. Mice orally vaccinated with RASV χ9558(pYA3337) without ECP or χ9558(pYA4428) with ECP, produced anti-Salmonella LPS and anti-E. coli EcpA and EcpD IgG and IgA antibodies. RASV strains showed protective potential against some E. coli and Salmonella strains as assessed using in vitro assays. In mouse sepsis and urinary tract infection challenge models, both vaccines had significant protection in some internal organs. Overall, this work showed that RASVs can elicit an immune response to E. coli and Salmonella antigens in some mice, provide significant protection in some internal organs during ExPEC challenge, and thus this study is a promising initial step toward developing a vaccine for prevention of ExPEC infections. Future studies should optimize the ExPEC antigens displayed by the RASV strain for a more robust immune response and enhanced protection against ExPEC infection.

Honey bees as other insects rely on the innate immune system for protection against diseases. The innate immune system includes the circulating hemocytes (immune cells) that clear pathogens from hemolymph (blood) by phagocytosis, nodulation or encapsulation. Honey bee hemocyte numbers have been linked to hemolymph levels of vitellogenin. Vitellogenin is a multifunctional protein with immune-supportive functions identified in a range of species, including the honey bee. Hemocyte numbers can increase via mitosis, and this recruitment process can be important for immune system function and maintenance. Here, we tested if hemocyte mediated phagocytosis differs among the physiologically different honey bee worker castes (nurses, foragers and winter bees), and study possible interactions with vitellogenin and hemocyte recruitment. To this end, we adapted phagocytosis assays, which—together with confocal microscopy and flow cytometry—allow qualitative and quantitative assessment of hemocyte performance. We found that nurses are more efficient in phagocytic uptake than both foragers and winter bees. We detected vitellogenin within the hemocytes, and found that winter bees have the highest numbers of vitellogenin-positive hemocytes. Connections between phagocytosis, hemocyte-vitellogenin and mitosis were worker caste dependent. Our results demonstrate that the phagocytic performance of immune cells differs significantly between honey bee worker castes, and support increased immune competence in nurses as compared to forager bees. Our data, moreover, provides support for roles of vitellogenin in hemocyte activity.

Salmonella enterica serovar Typhimurium genome encodes 13 fimbrial operons. Most of the fimbriae encoded by these operons are not produced under laboratory conditions but are likely to be synthesized in vivo. We used an in vivo expression technology (IVET) strategy to identify four fimbrial operons, agf, saf, sti, and stc that are expressed in the spleen. When any three of these operons were deleted, the strain retained wild-type virulence. However, when all four operons were deleted, the resulting strain was completely attenuated, indicating that these four fimbriae play functionally redundant roles critical for virulence. In mice, oral doses of as low as 1 × 10[superscript 5] CFU of the strain with four fimbrial operons deleted provided 100% protection against challenge with 1 × 10[superscript 9] CFU of wild-type S. Typhimurium. We also examined the possible effect of these fimbriae on the ability of a Salmonella vaccine strain to deliver a guest antigen. We modified one of our established attenuated vaccine strains, χ9088, to delete three fimbrial operons while the fourth operon was constitutively expressed. Each derivative was modified to express the Streptococcus pneumoniae antigen PspA. Strains that constitutively expressed saf or stc elicited a strong Th1 response with significantly greater levels of anti-PspA serum IgG and greater protective efficacy than strains carrying saf or stc deletions. The isogenic strain in which all four operons were deleted generated the lowest anti-PspA levels and did not protect against challenge with virulent S. pneumoniae. Our results indicate that these fimbriae play important roles, as yet not understood, in Salmonella virulence and immunogenicity.

Epigenetic changes enable genomes to respond to changes in the environment, such as altered nutrition, activity, or social setting. Epigenetic modifications, thereby, provide a source of phenotypic plasticity in many species. The honey bee (Apis mellifera) uses nutritionally sensitive epigenetic control mechanisms in the development of the royal caste (queens) and the workers. The workers are functionally sterile females that can take on a range of distinct physiological and/or behavioral phenotypes in response to environmental changes. Honey bees have a wide repertoire of epigenetic mechanisms which, as in mammals, include cytosine methylation, hydroxymethylated cytosines, together with the enzymatic machinery responsible for these cytosine modifications. Current data suggests that honey bees provide an excellent system for studying the “social repertoire” of the epigenome. In this review, we elucidate what is known so far about the honey bee epigenome and its mechanisms. Our discussion includes what may distinguish honey bees from other model animals, how the epigenome can influence worker behavioral task separation, and how future studies can answer central questions about the role of the epigenome in social behavior.

Extraintestinal pathogenic Escherichia coli (ExPEC) strains are important pathogens that cause diverse diseases in humans and poultry. Some E. coli isolates from chicken feces contain ExPEC-associated virulence genes, so appear potentially pathogenic; they conceivably could be transmitted to humans through handling and/or consumption of contaminated meat. However, the actual extraintestinal virulence potential of chicken-source fecal E. coli is poorly understood. Here, we assessed whether fecal E. coli isolates from healthy production chickens could cause diseases in a chicken model of avian colibacillosis and three rodent models of ExPEC-associated human infections. From 304 E. coli isolates from chicken fecal samples, 175 E. coli isolates were screened by PCR for virulence genes associated with human-source ExPEC or avian pathogenic E. coli (APEC), an ExPEC subset that causes extraintestinal infections in poultry. Selected isolates genetically identified as ExPEC and non-ExPEC isolates were assessed in vitro for virulence-associated phenotypes, and in vivo for disease-causing ability in animal models of colibacillosis, sepsis, meningitis, and urinary tract infection. Among the study isolates, 13% (40/304) were identified as ExPEC; the majority of these were classified as APEC and uropathogenic E. coli, but none as neonatal meningitis E. coli. Multiple chicken-source fecal ExPEC isolates resembled avian and human clinical ExPEC isolates in causing one or more ExPEC-associated illnesses in experimental animal infection models. Additionally, some isolates that were classified as non-ExPEC were able to cause ExPEC-associated illnesses in animal models, and thus future studies are needed to elucidate their mechanisms of virulence. These findings show that E. coli isolates from chicken feces contain ExPEC-associated genes, exhibit ExPEC-associated in vitro phenotypes, and can cause ExPEC-associated infections in animal models, and thus may pose a health threat to poultry and consumers.

Honey bees (Apis mellifera) provide a system for studying social and food-related behavior. A caste of workers performs age-related tasks: young bees (nurses) usually feed the brood and other adult bees inside the nest, while older bees (foragers) forage outside for pollen, a protein/lipid source, or nectar, a carbohydrate source. The workers' transition from nursing to foraging and their foraging preferences correlate with differences in gustatory perception, metabolic gene expression, and endocrine physiology including the endocrine factors vitellogenin (Vg) and juvenile hormone (JH). However, the understanding of connections among social behavior, energy metabolism, and endocrine factors is incomplete. We used RNA interference (RNAi) to perturb the gene network of Vg and JH to learn more about these connections through effects on gustation, gene transcripts, and physiology. The RNAi perturbation was achieved by single and double knockdown of the genes ultraspiracle (usp) and vg, which encode a putative JH receptor and Vg, respectively. The double knockdown enhanced gustatory perception and elevated hemolymph glucose, trehalose, and JH. We also observed transcriptional responses in insulin like peptide 1 (ilp1), the adipokinetic hormone receptor (AKHR), and cGMP-dependent protein kinase (PKG, or “foraging gene” Amfor). Our study demonstrates that the Vg–JH regulatory module controls changes in carbohydrate metabolism, but not lipid metabolism, when worker bees shift from nursing to foraging. The module is also placed upstream of ilp1, AKHR, and PKG for the first time. As insulin, adipokinetic hormone (AKH), and PKG pathways influence metabolism and gustation in many animals, we propose that honey bees have conserved pathways in carbohydrate metabolism and conserved connections between energy metabolism and gustatory perception. Thus, perhaps the bee can make general contributions to the understanding of food-related behavior and metabolic disorders.

Contemporary vaccine development relies less on empirical methods of vaccine construction, and now employs a powerful array of precise engineering strategies to construct immunogenic live vaccines. In this review, we will survey various engineering techniques used to create attenuated vaccines, with an emphasis on recent advances and insights. We will further explore the adaptation of attenuated strains to create multivalent vaccine platforms for immunization against multiple unrelated pathogens. These carrier vaccines are engineered to deliver sufficient levels of protective antigens to appropriate lymphoid inductive sites to elicit both carrier-specific and foreign antigen-specific immunity. Although many of these technologies were originally developed for use in Salmonella vaccines, application of the essential logic of these approaches will be extended to development of other enteric vaccines where possible. A central theme driving our discussion will stress that the ultimate success of an engineered vaccine rests on achieving the proper balance between attenuation and immunogenicity. Achieving this balance will avoid over-activation of inflammatory responses, which results in unacceptable reactogenicity, but will retain sufficient metabolic fitness to enable the live vaccine to reach deep tissue inductive sites and trigger protective immunity. The breadth of examples presented herein will clearly demonstrate that genetic engineering offers the potential for rapidly propelling vaccine development forward into novel applications and therapies which will significantly expand the role of vaccines in public health.