• Characterization of Live, Attenuated Shigella flexneri Vaccine Candidates

      DeLaine-Elias, BreOnna; Barry, Eileen M.; 0000-0002-4217-6309 (2016)
      Shigella flexneri is one of the leading causes of diarrheal disease in children under five in developing countries, yet currently there is no licensed vaccine to prevent disease. The primary aim of this study was to assess two new vaccine candidates in combination with CVD 1208S as a multi-component broad-spectrum vaccine. Additionally, the development of model systems for studying Shigella pathogenesis and profiling of host and bacterial responses served to advance our capacity to characterize the vaccine candidates. Host responses to the vaccines were measured using macrophage cytotoxicity assays, epithelial cell invasion assays, qRT PCR for transcriptional activity and enzyme-linked immunosorbent assays for cytokine secretion. The CVD 1208S vaccine was further evaluated for its impact on host gene expression using RNA sequencing. In addition to in vitro experiments, in vivo guinea pig and infant mouse studies were performed to assess Shigella specific antibody responses and protection against wild type challenge following immunization with each vaccine individually and as a trivalent formulation. In vitro studies demonstrated the new vaccine strains have a similar profile as the well-studied CVD 1208S vaccine. Guinea pig immunization studies revealed a robust induction of Shigella serotype specific IgA and IgG antibodies following immunization with each individual vaccine strain and a combined inoculum of all three. Challenge using the guinea pig Sereny test demonstrated protection against wild type S. flexneri serotypes 2a, 3a and 6 following immunizations with a mixed immunization including CVD 1208S, CVD 1213 and CVD 1215. Furthermore, the vaccines were safe and immunogenic in infant mice and experiments are ongoing to assess protection against wild type challenge. CVD 1208S was also used to evaluate three-dimensional and ex vivo models for Shigella pathogenesis. These data indicate that CVD 1213 and CVD 1215 are viable candidates for the creation of a multivalent vaccine that could confer broad-spectrum protection against S. flexneri infections. Moreover, global transcriptional analysis provides a broader view of host response to the vaccines as well as the effects of vaccine attenuation on bacterial gene expression. This data contributes to a broader profile of Shigella pathogenesis for future vaccine development.
    • Confirmation of vaccine candidate expression in multiple Staphylococcus aureus strains

      Muench, Anna Rachael; Shirtliff, Mark (2012)
      Staphylococcus aureus has emerged as an important pathogen due to its ability to form persistent infections through the biofilm mode of growth. S. aureus is responsible for thousands infections per year, which include a vast array of diseases and is rapidly developing the ability to become antibiotic resistant resulting in failure of our current methods of treatment. This antibiotic resistance is increased during biofilm formation when the bacteria form a polysaccharide matrix, which prevents infection clearance through antimicrobial means or by the host immune response. This project analyzed 14 Staphylococcus aureus strains and one Staphylococcus epidermidis strain obtained from NARSA (Network on Antimicrobial Resistance in Staphylococcus aureus) detecting the genomic presence and expression of three previously identified immunogenic proteins - glucosaminidase, SA0486 (a hypothetical lipoprotein), and SA0688 (an ABC transporter lipoprotein) - which have been incorporated into a vaccine described by Brady et al 2011. The strains were analyzed for the presence of the three specific genes through PCR and gel electrophoresis and for the expression of these proteins under biofilm growth conditions through western blot analysis. All strains contain the genetic code for these antigens and express at least 2 of the 3 antigens under biofilm growth conditions. Two strains were further analyzed for expression of these antigens under planktonic growth at 2.5hrs, 5hrs, and 24hrs comparing them to biofilm growth utilizing western blot analysis. These strains showed varying expression of the antigens under planktonic conditions with SA0688 expressed under all growth conditions in both strains, glucosaminidase with a gradual increase in expression as growth time increase in both strains, and SA0486 showing different expression patterns in the two strains but with the highest level at 5hrs and 24hrs. To truly understand the expression patterns under planktonic growth, further analysis will be required. The data generated provided the evidence of the universal nature of the vaccine previously developed in the Shirtliff laboratory described in Brady et al 2011.
    • Development and Testing of a Five-Subunit Biofilm Vaccine for the Prevention of Pulmonary Tuberculosis

      Kerns, Patrick W.; Shirtliff, Mark (2014)
      Mycobacterium tuberculosis (MTB) is the causative agent of tuberculosis, a disease that kills 1.4 million people and infects 8.7 million people worldwide every year. This bacterium has traditionally been studied using in vitroshaking cultures including detergents, which force the bacteria into an artificial style of planktonic growth. We utilized a static model of MTB growth that allows the bacteria to naturally grow as a biofilm pellicle at the air-liquid interface and utilized this model to identify antigens common to both shaking and biofilm cultures through a process of two-dimensional gel electrophoresis combined with western blotting. Having identified proteins that were both immunogenic and produced at three biofilm time points (three, five, and seven weeks) as well as in our control shaking culture, we proceeded to recombinantly express five in their full length forms. Three proteins were purified in their native forms and two proteins first denatured during the purification process and then refolded. We vaccinated female BALB/c and C3HeB/FeJ mice using 25 micrograms of each of the five antigens combined with either cyclic-di-GMP or DDA/MPL as an adjuvant over the course of three vaccine trials. While all vaccine formulations elicited a strong immune humoral response with high IgG1 and IgG2a titers against the vaccine antigens, there were no significant reductions in the number of CFU/g tissue in spleens or lungs of vaccinated animals compared to unvaccinated controls. Overall lung burden in BALB/c mice was significantly reduced in BCG vaccinated animals (p < 0.05) while there was a trend for bacterial burden reduction in animals vaccinated with the antigen subunits and DDA/MPL adjuvant (p = 0.055). In addition, the difference in the immune responses of the difference strains of mice was apparent when the T-helper responses of BALB/c and C3HEB/FeJ mice were evaluated. In the case of testing the DDA/MPL vaccine, BALB/c mice produced a strong antigen-specific IFN-gamma; response from vaccinated mouse splenocytes compared to the C3HeB/FeJ mice that produced significantly more IL-10 relative to IFN-gamma;. This indicated the fundamental differences in mouse strain responses to vaccination with BALB/c mice skewing towards a Th1 response relative to C3HeB/FeJ mice that had a more Th2/Treg skewed immune response. In addition, there were fundamental differences in the properties of the adjuvant-dependent immune responses. While DDA/MPL adjuvant showed a trend towards challenge protection, we found that cyclic-di-GMP-treated animals did significantly worse than control animals with an accompanying elevation in lung IFN-gamma; levels. These results call into question the use of cyclic-di-GMP adjuvant for tuberculosis vaccines, since it may be counterproductive to a protective immune response to MTB. Furthermore, the recombinant proteins 35kdag, CeoB, Mkl, and TB31.7 warrant further investigation as potential diagnostic markers for active or latent tuberculosis.
    • The Effect of Live-attenuated and Wild-type Shigella Strains on the Gastrointestinal Microbiota in Cynomolgus Macaques

      Seekatz, Anna Maria; Fraser, Claire M. (2013)
      Little is known about the role of the gastrointestinal microbiota in susceptibility to infection with enteric pathogens and response to live oral vaccines. This study examined the effect of immunization with an oral live-attenuated Shigella dysenteriae 1 vaccine and challenge with wild-type S. dysenteriae 1 on the gastrointestinal microbiota of cynomolgus macaques using 16S rRNA analysis. Multi-dimensional cluster analysis identified distinct bacterial community types within healthy macaques. The microbiota found in association with Mauritian macaques is distinct from and characterized by significantly higher diversity than the microbiota found in macaques from other geographic origins. Mauritian macaques also contain genetically distinct microsatellites in loci spanning the major histocompatibility complex (MHC) region, providing a possible link between the MHC repertoire and the intestinal microbiota. The intestinal microbiota in distinct macaque populations responds differently to immunization and subsequent challenge with wild-type Shigella, and is altered in fecal samples collected post-immunization and post-challenge in macaques from Indonesia, Indochina and the Philippines, but not from Mauritius. Specifically, Shigella exposure results in the appearance of a community type that is dominated by Enterococcus, a genus typically present at low abundance. While both Mauritian and non-Mauritian macaques exhibit anti-Shigella antibody responses upon immunization and challenge, clinical symptoms of shigellosis post-challenge are only observed in non-Mauritian macaques. These studies highlight the importance of further investigation into the possible protective role of the microbiota against enteric pathogens and the importance of host genetic backgrounds in conducting vaccine studies.
    • Evaluation of Lassa virus vaccine immunogenicity in a CBA/J-ML29 mouse model

      Goicochea, Marco Aurelio; Lukashevich, Igor S. (2011)
      Lassa virus (LASV) is widely spread in West Africa and can cause fatal Lassa fever. In addition to the significant public health problem in endemic regions there have been numerous imported cases to non-endemic countries. Due to limitations in treatment options and difficulties posed by reservoir control vaccination remains the most logical method of disease control; however we have no vaccine approved for human use. LASV is a pathogen that requires the highest level of biocontainment for study and as such vaccine development is both difficult and costly. ML29 is a reassortant virus containing the replication machinery of the nonpathogenic Mopeia virus and major immunogens of LASV. It displays an attenuated phenotype both in vitro and in vivo as compared to wild type LASV and therefore ML29 offers a safer immunogenic surrogate of LASV for vaccine research outside of BSL-4 facilities. We have established a small animal model for the evaluation of immunogenicity of LASV vaccine candidates based on unique phenotypic characteristics of ML29 in CBA/J mice. A single intraperitoneal immunization with the reassortant virus ML29 into CBA/J mice is non-pathogenic and sufficient to protect animals against a lethal homologous intracerebral challenge. Immunized mice display negligible levels of ML29-specific antibody titers, but LASV antigen-specific cell mediated immune (CMI) responses are detectable early and peak around day 8-10 after immunization. ML29 immune splenocytes display high numbers of IFN-? producing cells by ELISPOT and robust numbers of IFN-?+ and TNF-?+ CD4 and CD8 T lymphocytes by flow cytometry. In vivo CTL experiments show a correlation between Ag-specific cytotoxicity and the timing of protection induced by a single immunization with ML29. Furthermore, splenocyte transfers using donor cells from ML29 immunized mice display a similar kinetics of protection. Finally, mice that received splenocytes from ML29 immunized mice depleted of CD8+ cells all succumbed to a lethal challenge, further demonstrating the critical role of CD8 T cells in protection. This model has proven a useful immunological tool for the preliminary evaluation of immunogenicity and efficacy for other heterologous vaccine candidates against LASV outside of BSL-4 containment facilities necessitated by LASV.
    • Role of Vaccine-Induced IgG in Protection Against Bordetella Pertussis

      Masterson, Mary; Pasetti, Marcela F. (2019)
      Bordetella pertussis is a highly infectious respiratory pathogen that can induce severe bronchopneumonia and respiratory failure in infants (whooping cough). Vaccine formulations consisting of Diphtheria toxoid, Tetanus toxoid, and Acellular Pertussis (aP) components (DTaP and Tdap) protect against disease. It remains unclear how a parenteral vaccine, which primarily elicits systemic IgG, contributes to protection against a respiratory pathogen. The goal of this study was to investigate mechanisms by which vaccine-induced IgG reaches the respiratory mucosa and contributes to protection against B. pertussis infection. We hypothesized that pertussis-specific systemic IgG is transported from circulation into the airways via the neonatal Fc receptor (FcRn). To test this hypothesis, wild type mice and mice lacking FcRn (FcRn-/-) were immunized with DTaP or passively transferred DTaP-immune serum and challenged with B. pertussis. Post-challenge readouts included kinetics of Pertussis Toxin (PT) IgG in serum and bronchoalveolar lavage fluid (BALf), bacterial load quantification, and histopathology of lung tissues. WT vaccinated mice were able to clear the infection, whereas FcRn-/- vaccinated mice had residual bacterial counts and increased lung inflammation. Passive administration of DTaP-immune sera reduced lung colonization in both WT and FcRn-/- mice. However, FcRn-/- recipients exhibited moderate bronchopneumonia (absent in WT mice). The lower bacterial clearance and exacerbated tissue damage observed in actively and passively immunized FcRn-/- mice was not due to the absence of PT-IgG (or differences in IgG isotype) in BALf. Rather, WT and FcRn-/- mice had similar PT-IgG levels in serum and BALf, suggesting that FcRn-independent mechanisms mediate IgG transport across the lung. PT-IgG progressively increased in BALf of passively immunized FcRn-/- mice post-challenge (along with lung inflammation,) suggesting IgG also diffuses through damaged lung epithelium. We observed that neutrophils from FcRn-/- mice had lower B. pertussis opsonophagocytic capacity as compared to WT. This impairment in IgG-mediated antimicrobial function in the absence of FcRn could explain the increased inflammation in FcRn-/- mice. In conclusion, we have shown that pertussis-specific IgG translocation into the airways appears to be FcRn-independent, and that IgG-mediated B. pertussis neutrophil phagocytosis may contribute to bacterial clearance and tissue preservation post-infection through FcRn interactions.
    • Whole-genome analysis of Plasmodium falciparum isolates to understand allele-specific immunity to malaria

      Shah, Zalak; Takala-Harrison, Shannon (2020)
      After repeated P. falciparum infections, individuals in high-transmission areas acquire clinical immunity to malaria. However, the genes important in determining allele-specific immunity are not entirely known. Previous genome-wide approaches explored signatures of selection in the parasite genome to identify targets of clinical immunity; however, these approaches did not account for individual level allele-specific immunity. Here we take a whole-genome approach to identify genes that may be involved in acquisition of allele-specific immunity to malaria by analyzing parasite genomes collected from infected individuals in Malawi. However, obtaining whole genome sequence data from clinical samples is one of the major hurdles in the field of malaria genomics. In order to obtain whole genome sequence data from non-leukocyte depleted, low parasitemia samples, we optimized a selective-whole genome amplification (sWGA) by filtering the DNA prior to sWGA, to generate high coverage, whole genome sequence data from P. falciparum clinical samples with low amounts of parasite DNA. Using this optimized approach, we successfully performed whole-genome sequencing on 202 parasite isolates. We compared parasite genomes from individuals with varying levels of clinical immunity, defined using an individual’s proportion of symptomatic infections during the course of the study, hypothesizing that individuals with higher immunity become symptomatically ill due to infection with parasites with less common alleles. Using FST, we identified 161 SNPs to be genetically differentiated between the two groups and the median allele frequency was significantly lower at these sites in individuals in higher immunity group compared to the lower immunity group. We also examined pairs of parasites collected at different time points from the same individuals and identified 225 loci in 174 genes that vary within same individuals more often than expected by chance. Using both of these approaches, we identified 25 genes that encode likely targets of immunity, including a known antigen, CLAG8. Further analysis of clag8 global diversity showed evidence of immune selection in the C-terminal region, supporting the use of this approach in identification of new vaccine targets. Identifying and further analyzing these genomic regions will provide insights into mechanisms involved in allele-specific acquired immunity.