Francisella tularensis is 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. tularensis SchuS4 strain. Two different vaccine formulations and immunization schedules were used. The immunized mice were challenged with lethal (10xLD100) doses of F. tularensis LVS 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. tularensis antigens. 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. tularensis proteins 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.
Chemical conjugation of CTL peptides to tobacco mosaic virus (TMV) has shown promise as a molecular adjuvant scaffold for augmentation of cellular immune responses to peptide vaccines. This study demonstrates the ease of generating complex multipeptide vaccine formulations using chemical conjugation to TMV for improved vaccine efficacy. We have tested a model foreign antigen target-the chicken ovalbumin-derived CTL peptide (Ova peptide), as well as mouse melanoma-associated CTL epitopes p15e and tyrosinase-related protein 2 (Trp2) peptides that are self-antigen targets. Ova peptide fusions to TMV, as bivalent formulations with peptides encoding additional T-help or cellular uptake via the integrin-receptor binding RGD peptide, showed improved vaccine potency evidenced by significantly enhanced numbers of antigen-reactive T cells measured by in vitro IFNgamma cellular analysis. We measured the biologically relevant outcome of vaccination in protection of mice from EG.7-Ova tumor challenge, which was achieved with only two doses of vaccine ( approximately 600 ng peptide) given without adjuvant. The p15e peptide alone or Trp2 peptide alone, or as a bivalent formulation with T-help or RGD uptake epitopes, was unable to stimulate effective tumor protection. However, a vaccine with both CTL peptides fused together onto TMV generated significantly improved survival. Interestingly, different bivalent vaccine formulations were required to improve vaccine efficacy for Ova or melanoma tumor model systems.
Therapeutic monoclonal antibodies continue to achieve clinical success for the treatment of many different diseases, particularly cancer. However, the production and purification of antibodies continues to be a time and labor-intensive process with considerable technical challenges. Gene-based delivery of antibodies may address this, via direct production within the host that achieves therapeutic levels. In this report, we validate the feasibility that gene-based delivery is a viable approach for efficacious delivery of antibodies in the preclinical and, presumably, clinical setting. We demonstrate high and sustained in vivo expression of the murine antihuman epidermal growth factor receptor antibody 14E1 following intramuscular delivery by adeno-associated virus (AAV) 2/1. Incorporating the Furin/2A technology for monocistronic expression of both heavy and light chains, we achieved sustained serum levels of full-length 14E1 peaking over 1 mg ml À1 in athymic nude mice. In the A431 xenograft tumor model, 14E1was capable of significantly inhibiting tumor growth and prolonging survival when AAV was administered prior to tumor challenge. Furthermore, 14E1 demonstrated significant antitumor efficacy against well-established tumors (B400 mm 3 ) when AAV was administered up to 20 days after tumor challenge. Here we demonstrate for the first time growth inhibition of a well-established tumor by a full-length antibody following delivery by AAV.
We have used an RNA nanoparticle system to test the hypothesis that co-expression of a tumor antigen-cytokine fusion, with or without a co-delivered tumor CTL epitope, can improve anti-tumor immunity to a self antigen. Our initial studies demonstrated that a Semliki Forest Virus (SFV) bGal RNA expression vector could be modified to allow for in vitro coat protein RNA encapsidation by a distant alphavirus family member, Tobacco Mosaic Virus (TMV). Encapsidated RNA stimulated robust targeted immune cell uptake and activation, including dendritic cells. In response to vaccination, encapsidation significantly improved humoral and cellular immune responses compared to naked RNA, including IFNg activation , and protection from challenge with a lethal dose of bGal expressing tumor. More recently, our goal is to test efficacy of encapsidated RNA in a Non-hodgkin’s murine lymphoma tumor self-antigen system using the A20 expressing tumor. We have constructed a variety of SFV A20 scFv antigen-cytokine fusions, and are co-developing an A20 CTL surface antigen fusion. In vitro expression will be confirmed, and mice will be immunized and tested by ELISA and ELISpot for functional B and T cell immune activation. This nanoparticle RNA system generates vaccine compositions that are easy to customize, easy to produce, and facilitates intracellular delivery of unique nucleic acid/protein antigen combinations that should improve vaccine efficacy against a weakly immunogenic tumor.
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