Incorporation of Complement C3d into Immune Stimulating Complexes as a Dual Adjuvant System for Early Calfhood Vaccination
Shipping fever pneumonia is the most costly production disease of cattle in North America, with annual losses due to treatment, death and lost production estimated at one billion dollars per year. Although vaccines against both viral and bacterial pathogens are marketed and used extensively, there is continuing and heavy dependence on antibiotics in prevention and treatment of pneumonia in the feedlot. More efficacious vaccines are needed, because of the need to reduce the costs of treatment, because of increasing bacterial resistance to antibiotics, and because of increasing pressure from society to reduce the use of antibiotics in food producing animals.
One way of improving existing vaccines is to develop next generation adjuvant (vaccine additive) systems, that not only induce more potent immune responses following vaccination, but also permit flexibility of the timing of administration. Vaccination of calves on feedlot arrival in many cases induces immune responses too late to provide protection in the critical first two weeks after arrival. Early calfhood vaccination not only would induce immunity before the stresses of weaning and shipping, but would also reduce the incidence of enzootic pneumonia in suckling calves. Unfortunately, the value of conventional vaccines is reduced when administered to very young calves or to calves with maternal antibodies (antibodies absorbed by the calf from the dam’s first milk [colostrum]). An improved vaccine adjuvant system should permit effective vaccination from birth regardless of whether maternal antibodies are present. It is expected that a vaccine which is potent enough to be effective in this age group is potent enough to be used at any age. Our long-term objective therefore is to develop a flexible and potent adjuvant system that can be adapted for use with many different vaccines and that can be used effectively in the first weeks of life. Our short-term objective is to develop an improved vaccine against Mannheimia (Pasteurella) haemolytica, the major bacterial pathogen causing shipping fever, as a model for the improved adjuvant system.
In this ongoing project we are investigating a dual adjuvant system that uses submicroscopic lipid particles (immune stimulating complexes or ISCOMs) as a platform to exhibit multiple copies of an immune enhancing serum protein called complement component C3, fragment d (C3d). Experimental evidence suggests that multiple copies of C3d need to be attached to vaccine antigen (protein) to induce maximal immune responses. Although ISCOM particles themselves have adjuvant activity, it is anticipated that addition of bovine C3d to ISCOM particles will be needed to achieve the required vaccine performance. We have chosen the leukotoxin of M. haemolytica as the primary vaccine antigen of interest because of its documented importance in protective vaccines against shipping fever.
Preliminary work in this project involved cloning and sequencing of the gene fragment coding for bovine complement component C3d, since this sequence had not been published previously. The cDNA sequence determined by our laboratory was accepted into the GenBank database in July 2004 (Accession number AY630404). Various plasmid constructs were assembled using this bovine C3d sequence, and recombinant C3d-containing fusion proteins were produced in the bacterium E.coli. Several different C3d fusion proteins were produced and purified (using a combination of chromatographic techniques) before a construct was found that would bind to ISCOM vaccine particles.
ISCOM particles were produced containing antigens secreted or released by M.haemolytica during culture. The ISCOM particles were examined using an electron microscope to confirm their characteristic size and structure, and analysed in the laboratory to characterize their components. Vaccination of experimental rabbits confirmed that the ISCOM particles (without C3d) could drive immune responses to leukotoxin and other antigens of M.haemolytica. Recently we were able to demonstrate that C3d and vaccine antigens could be incorporated simultaneously into ISCOMs.
Our preliminary studies in calves using experimental ISCOM vaccines (without C3d), indicate that a M.haemolytica ISCOM vaccine can drive a strong antibody response in young calves, following injection by the subcutaneous route. Local reactions at the injection site however have been excessive with the formulations tested to date. Further studies are needed to optimise ISCOM formulation and to determine the adjuvant effects of C3d when presented in ISCOM particles.
Benefits to the cattle industry:
Shipping fever pneumonia has been a costly disease for the cattle industry in North America for many decades. The use of antibiotics for prevention and treatment of pneumonia has become ever more expensive as bacteria have developed resistance to older, cheaper drugs. Concerns over transfer of antibiotic resistance to pathogens of humans may soon restrict the use of antibiotics in food producing animals. Vaccines to date have shown some benefit in preventing respiratory disease, but considerable improvement is needed before they can be considered the primary means of defence. Improved adjuvants (vaccine additives) can improve vaccine performance by enhancing the magnitude of immune responses and also by improving the flexibility of timing of vaccination to provide more management options. We have investigated a potentially potent combination of adjuvants for use in cattle. Although the present project has focused on a vaccine for M.haemolytica, it is expected that most vaccines could be adapted to an alternative adjuvant system. If cattlemen would have effective vaccines that could be used in more effective ways, it is hard to imagine how much more healthy cattle would be!
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