Recombinant subunit vaccines are gaining traction in recent development efforts and will constitute the majority of vaccines in the future. One of the major problems in designing recombinant protein subunit vaccines is that the antigens are often membrane proteins found on the surface of the pathogens. Expression technologies commercially available are not effective in producing correctly folded and functional membrane proteins. When these protein expression methods are used to express membrane proteins, they will either fail to express or appear as insoluble materials, devoid of any biological activities. Correct and appropriate antigen presentation is crucial for efficient induction of immunity responses. In recombinant subunit vaccines or vaccine candidates made to date, the surface membrane protein antigens made using recombinant protein expression technologies have been obtained in the form of misfolded inclusion bodies. As a result of inappropriate presentation and incorrect epitopes displayed by incorrectly folded inclusion bodies as the antigen components, these vaccine formulations rarely provide full protection and ~80% effective rate is the norm.. Our proprietary powerful membrane protein expression technology can be used to prepare surface membrane protein antigens to develop vaccines against infectious diseases. Our correctly-folded and functional membrane protein antigens when used in vaccine formulations can be referred to as "live vaccine mimic". This term reflects the fact that these membrane protein antigens exhibit their true folding motifs as found on the surface of the pathogens. In confirmed to be highly effective, this aquaculture vaccine will serve as the prototype of many vaccines that can be made through our membrane protein production technology. The primary objective of this proposal is to develop subunit vaccine against Mycobacterium marinum infections for aquaculture. The vaccine we propose would prevent disease outbreaks, be logistically and economically appealing, and will lead to reduced environmental problems compared to antibiotics. Our vaccination technology will help to reduce mortality, improve the overall health and performance of fish exposed to the pathogen, and enhance the competitiveness of the U.S. aquaculture industry. The vaccine technology that we will develop to prevent bacterial infections in fish will have application in the culture of valuable finfish species, and economic advances in these industries through increased production and profitability and would stimulate further research, yield product improvements, and produce jobs in primarily rural areas suited for fish culture. In addition, this research will serve as the prototype for other vaccine development projects, particularly, those for mycobacteria diseases in human including tuberculosis, leprosy, and Buruli ulcer. These often neglected but devatasting diseases of the past are making a comeback with the emergence of drug resistant strains. Research into mycobacteria certainly carries additional significances in light of these renewed health threats. OBJECTIVES: The objective of this proposal is to develop highly effective formulations of vaccine - recombinant subunit formulation and/or its equivalent DNA vaccine to protect commercial hybrid strip bass from Mycobacterium marinum infections, an aquatic tuberculosis-like disease common in many fish species. Specifically, highly immunogenic surface membrane proteins from the pathogen will be identified and overexpressed using powerful proprietary membrane protein production technologies as well as the re-designed highly potent versions of these antigens. The native and re-designed antigens will be evaluated for their potency and efficacy as vaccine targets in experimental infection trials. APPROACH: For the Phase I project, a panel of high priority antigen targets including the M. marinum homologs of fbpA, pirG, Rv1477, Rv0125, mpt53, and ESAT6 has been assembled. This panel of six diverse M. marinum antigens that contain a representative member of different surface membrane protein groups is sufficient for the creation of prototype vaccines. TMB will attempt to overexpress the selected antigens as membrane-bound proteins first. Initially, the E. coli hosts will be used to quickly express the antigens for evaluation (from cloning gene to protein: ~ 2-4 weeks). Once potent antigens are identified, the methanotroph system then can be used to mass produce the membrane proteins. TMB will attempt to prepare the refolded solubilized inclusion bodies version of the selected antigens. Most of the recombinant antigens used in animal health vaccines have been the solubilized inclusion bodies since they are very cheap to make. These materials will be evaluated in mortality trials similar to the correctly folded membrane-bound versions. A study comparing the effectiveness of two different preparations should provide important insights for future vaccine design research. Besides preparing the antigens as recombinant proteins for mortality trials, the antigens will also be evaluated as DNA vaccine formulations. The antigen gene will be cloned into mammalian expression vectors, placed unto the control of the CMV promoter, and fused with the rainbow trout TGF-beta leader peptide. The rainbow trout TGF-beta leader peptide should allow the antigen(s) to be displayed on the fish cell surface or secreted into the intracellular space. The immune responses will be evaluated in similar manner as the purified recombinant protein version. Other approaches in antigen design and/or antigen presentation to enhance our chance of success will also be pursued. The immune responses to antigen candidates can be improved by using certain fusion constructs containing innocuous but extremely immunogenic proteins, the so-called T-cell epitopes, for instance, the small envelope protein of hepatitis B virus, or the small TCE peptides from tetanus toxin (TT) and measles virus fusion protein (MVF) (~15 amino acids each). The TT and MVF TCE peptides will be added in tandem into the C-terminus of M. marinum antigens to enhance the immune responses and these candidates will be evaluated in HSB mortality trials. The candidate vaccine antigens will be evaluated in mortality trials using an experimental disease model that has been previously developed by Kent SeaTech. Control fish will be vaccinated with either phosphate buffered saline or cells grown without the gene encoding the antigen of interest. Subsequently, duplicate groups of fish will each receive an injection of the antigen that has been formulated in an oil-based adjuvant. For bacterial challenge, fish will be anesthetized and experimentally injected with M. marinum by administration of a 0.1 ml challenge dose volume. Morbidity and mortality will collected once daily for the duration of each trial. Time to first mortality and cumulative mortality will be analyzed for each vaccine antigen
