Development of a Multivalent Subunit Vaccine against Tularemia Using Tobacco Mosaic Virus (TMV) Based Delivery System

Francisella tularensisis a facultative intracellular pathogen, and is the causative agent of a fatal human disease known as tularemia. F. tularensis is classified as a Category A Biothreat agent by the CDC based on its use in bioweapon programs by several countries in the past and its potential to be used as an agent of bioterrorism. No licensed vaccine is currently available for prevention of tularemia. In this study, we used a novel approach for development of a multivalent subunit vaccine against tularemia by using an efficient tobacco mosaic virus (TMV) based delivery platform. The multivalent subunit vaccine was formulated to contain a combination of F. tularensis protective antigens: OmpA-like protein (OmpA), chaperone protein DnaK and lipoprotein Tul4 from the highly virulent F. tularensisSchuS4 strain. Two different vaccine formulations and immunization schedules were used. The immunized mice were challenged with lethal (10xLD100) doses of F. tularensisLVS on day 28 of the primary immunization and observed daily for morbidity and mortality. Results from this study demonstrate that TMV can be used as a carrier for effective delivery of multiple F. tularensisantigens. TMV-conjugate vaccine formulations are safe and multiple doses can be administered without causing any adverse reactions in immunized mice. Immunization with TMV-conjugated F. tularensisproteins induced a strong humoral immune response and protected mice against respiratory challenges with very high doses of F. tularensis LVS. This study provides a proof-of-concept that TMV can serve as a suitable platform for simultaneous delivery of multiple protective antigens of F. tularensis. Refinement of vaccine formulations coupled with TMV-targeting strategies developed in this study will provide a platform for development of an effective tularemia subunit vaccine as well as a vaccination approach that may broadly be applicable to many other bacterial pathogens

An Improved Tobacco Mosaic Virus (TMV)-Conjugated Multiantigen Subunit Vaccine Against Respiratory Tularemia

Francisella tularensis, the causative agent of the fatal human disease known as tularemia is classified as a Category A Select Agent by the Centers for Disease Control. No licensed vaccine is currently available for prevention of tularemia in the United States. Previously, we published that a tri-antigen tobacco mosaic virus (TMV) vaccine confers 50% protection in immunized mice against respiratory tularemia caused by F. tularensis. In this study, we refined the TMV-vaccine formulation to improve the level of protection in immunized C57BL/6 mice against respiratory tularemia. We developed a tetra-antigen vaccine by conjugating OmpA, DnaK, Tul4, and SucB proteins of Francisella to TMV. CpG was also included in the vaccine formulation as an adjuvant. Primary intranasal (i.n.) immunization followed by two booster immunizations with the tetra-antigen TMV vaccine protected 100% mice against i.n. 10LD100 challenges dose of F. tularensis live vaccine strain (LVS). Mice receiving three immunization doses of tetra-antigen TMV vaccine showed only transient body weight loss, cleared the infection rapidly, and showed minimal histopathological lesions in lungs, liver, and spleen following a lethal respiratory challenge with F. tularensis LVS. Mice immunized with the tetra-antigen TMV vaccine also induced strong ex vivo recall responses and were protected against a lethal challenge as late as 163 days post-primary immunization. Three immunization with the tetra-antigen TMV vaccine also induced a stronger humoral immune response predominated by IgG1, IgG2b, and IgG2c antibodies than mice receiving only a single or two immunizations. Remarkably, a single dose protected 40% of mice, while two doses protected 80% of mice from lethal pathogen challenge. Immunization of Interferongamma (IFN-g)-deficient mice with the tetra-antigen TMV vaccine demonstrated an absolute requirement of IFN-g for the generation of protective immune response against a lethal respiratory challenge with F. tularensis LVS. Collectively, this study further demonstrates the feasibility of TMV as an efficient platform for the delivery of multiple F. tularensis antigens and that tetra-antigen TMV vaccine formulation provides complete protection, and induces long-lasting protective and memory immune responses against respiratory tularemia caused by F. tularensis LVS

An O-Antigen glycoconjugate vaccine produced using protein glycan coupling technology is protective in an inhalational rat model of tularemia

There is a requirement for an efficacious vaccine to protect people against infection from Francisella tularensis, the etiological agent of tularemia. The lipopolysaccharide (LPS) of F. tularensis is suboptimally protective against a parenteral lethal challenge in mice. To develop a more efficacious subunit vaccine, we have used a novel biosynthetic technique of protein glycan coupling technology (PGCT) that exploits bacterial N-linked glycosylation to recombinantly conjugate F. tularensis O-antigen glycans to the immunogenic carrier protein Pseudomonas aeruginosa exoprotein A (ExoA). Previously, we demonstrated that an ExoA glycoconjugate with two glycosylation sequons was capable of providing significant protection to mice against a challenge with a low-virulence strain of F. tularensis. Here, we have generated a more heavily glycosylated conjugate vaccine and evaluated its efficacy in a Fischer 344 rat model of tularemia. We demonstrate that this glycoconjugate vaccine protected rats against disease and the lethality of an inhalational challenge with F. tularensis Schu S4. Our data highlights the potential of this biosynthetic approach for the creation of next-generation tularemia subunit vaccines

Intranasal Administration of a Two-Dose Adjuvanted Multi-Antigen TMV-Subunit Conjugate Vaccine Fully Protects Mice Against Francisella Tularensis LVS Challenge

Tularemia is a fatal human disease caused by Francisella tularensis, a Gram-negative encapsulated coccobacillus bacterium. Due to its low infectious dose, ease of aerosolized transmission, and lethal effects, the CDC lists F. tularensis as a Category A pathogen, the highest level for a potential biothreat agent. Previous vaccine studies have been conducted with live attenuated, inactivated, and subunit vaccines, which have achieved partial or full protection from F. tularensis live vaccine strain (LVS) challenge, but no vaccine has been approved for human use. We demonstrate the improved efficacy of a multi-antigen subunit vaccine by using Tobacco Mosaic virus (TMV) as an antigen carrier for the F. tularensis SchuS4 proteins DnaK, OmpA, SucB and Tul4 (DOST). The magnitude and quality of immune responses were compared after mice were immunized by subcutaneous or intranasal routes of administration with a TMV-DOST mixture, with or without four different adjuvants. Immune responses varied in magnitude and isotype profile, by antigen, by route of administration, and by protection in an F. tularensis LVS challenge model of disease. Interestingly, our analysis demonstrates an overwhelming IgG2 response to SucB after intranasal dosing, as well as a robust cellular response, which may account for the improved two-dose survival imparted by the tetravalent vaccine, compared to a previous study that tested efficacy of TMV-DOT. Our study provides evidence that potent humoral, cellular and mucosal immunity can be achieved by optimal antigen combination, delivery, adjuvant and appropriate route of administration, to improve vaccine potency and provide protection from pathogen challenge

Immunoproteomics Analysis of the Murine Antibody Response to Vaccination with an Improved Francisella tularensis Live Vaccine Strain (LVS)

Background: Francisella tularensis subspecies tularensis is the causative agent of a spectrum of diseases collectively known as tularemia. An attenuated live vaccine strain (LVS) has been shown to be efficacious in humans, but safety concerns have prevented its licensure by the FDA. Recently, F. tularensis LVS has been produced under Current Good Manufacturing Practice (CGMP guidelines). Little is known about the immunogenicity of this new vaccine preparation in comparison with extensive studies conducted with laboratory passaged strains of LVS. Thus, the aim of the current work was to evaluate the repertoire of antibodies produced in mouse strains vaccinated with the new LVS vaccine preparation. Methodology/Principal Findings: In the current study, we used an immunoproteomics approach to examine the repertoire of antibodies induced following successful immunization of BALB/c versus unsuccessful vaccination of C57BL/6 mice with the new preparation of F. tularensis LVS. Successful vaccination of BALB/c mice elicited antibodies to nine identified proteins that were not recognized by antisera from vaccinated but unprotected C57BL/6 mice. In addition, the CGMP formulation of LVS stimulated a greater repertoire of antibodies following vaccination compared to vaccination with laboratory passaged ATCC LVS strain. A total of 15 immunoreactive proteins were identified in both studies, however, 16 immunoreactive proteins were uniquely reactive with sera from the new formulation of LVS. Conclusions/Significance: This is the first report characterising the antibody based immune response of the new formulation of LVS in the widely used murine model of tularemia. Using two mouse strains, we show that successfully vaccinated mice can be distinguished from unsuccessfully vaccinated mice based upon the repertoire of antibodies generated. This opens the door towards downselection of antigens for incorporation into tularemia subunit vaccines. In addition, this work also highlights differences in the humoral immune response to vaccination with the commonly used laboratory LVS strain and the new vaccine formulation of LVS.Peer reviewed: YesNRC publication: Ye

Mining the Francisella tularensis proteome for vaccine candidates

2012 Summer.Includes bibliographical references.Based on methodologies developed for the identification of T cell antigen of other intracellular bacterial pathogens, a proteomic approach was applied for the elucidation of T cell antigens of Francisella tularensis (Covert, 2001). Specifically, subcellular components (membrane and soluble) of F. tularensis LVS were resolved by size using preparative SDS-PAGE and fractions collected using a whole gel elution technique. A total of 16 soluble and 19 membrane-sized fractions were produced, each of which were assessed for antigen reactivity based on the ability to elicit IFN-γ; from splenocytes of F. tularensis LVS-infected mice. Of these 35 preparative SDS-PAGE fractions, seven yielded a dominant T cell response. These seven fractions were further investigated using tandem mass spectrometry (MS/MS) to identify individual proteins in each immunodominant fraction. A total of 40 and 31 proteins were identified with greater than 95% confidence from the immunodominant membrane and soluble fractions, respectively. Further, MS/MS analysis of different protein quantities (2.5 μg to 10 μg) allowed for identification of the most abundant proteins in each fraction, thus focusing the number of possible proteins to nine proteins of interest. These data provide the basis for production of recombinant proteins and further immunological evaluations to select suitable candidates for inclusion in a subunit vaccine against tularemia

A humoral immune response study of concoction of recombinant FopA, DnaK and GroEL in mouse model as vaccine candidate against Francisella tularensis

16-21Tularemia, commonly prevalent in various regions of the northern side of the equator, is a zoonotic illness caused by Francisella tularensis. Among various Francisella tularensis species, subspecies tularensis is most pathogenic to human and included as category a biowarfare agent which can cause infection through aerosol, cut, injuries in skin and contact with tainted creatures. Right now, there is no approved vaccine is available for this very infectious intracellular pathogen. In this study, three Francisella proteins viz. FopA, GroEL and DnaK were cloned and expressed. The recombinant proteins were purified and utilized as a cocktail for immunization in mice. The ELISA results demonstrated that this cocktail elicited high antibody titres in sera of vaccinated animals. Among the IgG subtypes, IgG1 response was prevalent in immunized animals took after by IgG2. This indicated the induction of Th2 type immune responses against this cocktail a vaccine candidate. The investigation demonstrated that the blend of these three proteins has the potential as vaccine candidate for tularemia

The Francisella Tularensis Proteome and its Recognition by Antibodies

Francisella tularensis is the causative agent of a spectrum of diseases collectively known as tularemia. The extreme virulence of the pathogen in humans, combined with the low infectious dose and the ease of dissemination by aerosol have led to concerns about its abuse as a bioweapon. Until recently, nothing was known about the virulence mechanisms and even now, there is still a relatively poor understanding of pathogen virulence. Completion of increasing numbers of Francisella genome sequences, combined with comparative genomics and proteomics studies, are contributing to the knowledge in this area. Tularemia may be treated with antibiotics, but there is currently no licensed vaccine. An attenuated strain, the Live Vaccine Strain (LVS) has been used to vaccinate military and at risk laboratory personnel, but safety concerns mean that it is unlikely to be licensed by the FDA for general use. Little is known about the protective immunity induced by vaccination with LVS, in humans or animal models. Immunoproteomics studies with sera from infected humans or vaccinated mouse strains, are being used in gel-based or proteome microarray approaches to give insight into the humoral immune response. In addition, these data have the potential to be exploited in the identification of new diagnostic or protective antigens, the design of next generation live vaccine strains, and the development of subunit vaccines. Herein, we briefly review the current knowledge from Francisella comparative proteomics studies and then focus upon the findings from immunoproteomics approaches

Epitope-based vaccination against pneumonic tularemia

Francisella tularensis, the etiological agent of tularemia, is one of the most infectious bacterial pathogens known. No vaccine is currently approved for public use. Previously, we identified epitopes recognized specifically by T cells obtained from individuals following infection with F. tularensis. Here, we report that a subunit vaccine constructed based upon these epitopes elicited protective immunity in “humanized” HLA class II (DRB1*0401) transgenic mice. Vaccinated mice challenged intratracheally with a lethal dose of F. tularensis (Live Vaccine Strain) exhibited a rapid increase in pro-inflammatory cytokine production and diminished number of organisms in the lungs, and a concurrent increased rate of survival. These results demonstrate the efficacy of an epitope-based tularemia vaccine and suggest that such an approach might be widely applicable to the development of vaccines specific for intracellular bacterial pathogens

Francisella–Arthropod Vector Interaction and its Role in Patho-Adaptation to Infect Mammals

Francisella tularensis is a Gram-negative, intracellular, zoonotic bacterium, and is the causative agent of tularemia with a broad host range. Arthropods such as ticks, mosquitoes, and flies maintain F. tularensis in nature by transmitting the bacteria among small mammals. While the tick is largely believed to be a biological vector of F. tularensis, transmission by mosquitoes and flies is largely believed to be mechanical on the mouthpart through interrupted feedings. However, the mechanism of infection of the vectors by F. tularensis is not well understood. Since F. tularensis has not been localized in the salivary gland of the primary human biting ticks, it is thought that bacterial transmission by ticks is through mechanical inoculation of tick feces containing F. tularensis into the skin wound. Drosophila melanogaster is an established good arthropod model for arthropod vectors of tularemia, where F. tularensis infects hemocytes, and is found in hemolymph, as seen in ticks. In addition, phagosome biogenesis and robust intracellular proliferation of F. tularensis in arthropod-derived cells are similar to that in mammalian macrophages. Furthermore, bacterial factors required for infectivity of mammals are often required for infectivity of the fly by F. tularensis. Several host factors that contribute to F. tularensis intracellular pathogenesis in D. melanogaster have been identified, and F. tularensis targets some of the evolutionarily conserved eukaryotic processes to enable intracellular survival and proliferation in evolutionarily distant hosts