- 4.1Vaccine Development
- Programme Title:Immunology of bovine tuberculosis
- Programme Title:Possum immunology
- Programme Title:Testing of naked DNA and peptide vaccine strategies for the induction of CD8 T cell immune responses and TB development of protective immunity
4. DEVELOPMENT OF VACCINES AGAINST TB
4.1Vaccine Development
4.1.1
Programme Title: Immunology of bovine tuberculosisResearch Leader:Dr B Buddle Institution:AgResearch, Wallaceville |
Summary
This programme aims to evaluate tuberculosis vaccines in cattle and possums and identify immune responses associated with immunopathology or resistance to bovine tuberculosis.
Studies of the effect of bovine cytokines (activators of macrophages) on the growth of Mycobacterium bovis in macrophages (type of defence cell) have shown that a combination of cytokines is necessary to inhibit the growth of M. bovis. A marked difference between the growth of BCG (M. bovis derived from a vaccine) and virulent M. bovis in bovine macrophages was found.
An induction of a strong immune response in possums to M. bovis antigens and enhanced protection against bovine tuberculosis following intraduodenal vaccination with BCG is encouraging. This result indicates that encapsulating a tuberculosis vaccine to protect against degradation in the stomach may be possible and lead to an effective oral bait tuberculosis vaccine.
Description
To continue to evaluate tuberculosis vaccines in cattle and possums, and identify immune responses associated with immunopathology or resistance to bovine tuberculosis by:
- purifying recombinant bovine interferon-y and interleukin-2, determine the effect of recombinant bovine cytokines on survival of M. bovis isolates in cultured bovine macrophages, and produce monoclonal antibodies against these cytokines;
- determining whether the virulence of M. bovis isolates can be predicted from in vitro infection in bovine macrophages;·
- comparing the peripheral blood and pulmonary lymphocyte and monocyte/macrophage responses of calves vaccinated with BCG by different routes;
- establishing whether strong humoral or cellular immune responses can be induced in cattle vaccinated with mycobacterial culture filtrate vaccines; and
- vaccinating possums with BCG using a range of different oral delivery systems and determine the level of protection against bovine tuberculosis.
Approach & Outcomes
This year research continued to evaluate tuberculosis vaccines in cattle and possums and identify immune responses associated with immunopathology or resistance to bovine tuberculosis.
Studies of the effect of bovine cytokines on the growth of M. bovis in macrophages have shown that a combination of cytokines including IFN-_ and LPS-induced cytokines are necessary to inhibit the growth of M. bovis. Usually live bacterial vaccines are required to stimulate these types of cytokines.
There was a marked difference between the growth of BCG and virulent M. bovis in bovine alveolar macrophages.
Intranasal BCG vaccination of cattle stimulated regional lymph node immune responses, but not systemic immune responses. This finding suggests that intranasal BCG vaccination may not induce a tuberculin skin test, although may still protect cattle against bovine tuberculosis.
The induction of interleukin-2 (IL~2) and antibody responses but no tuberculin skin test response following vaccination with a mycobacterial short term culture filtrate + DEAE dextran vaccine provides researchers with a second opportunity to develop a tuberculosis vaccine which does not interfere with skin testing. This vaccine will also be tested for protection of cattle against bovine tuberculosis.
The induction of strong immune responses in possums to M. bovis antigens and enhanced protection against bovine tuberculosis following intraduodenal vaccination with BCG is very encouraging. This result indicates that micro-encapsulation of a tuberculosis vaccine to protect against degradation in the stomach may lead to an effective oral bait tuberculosis vaccine.
Publications
Buddle, B.M.; de Lisle, G.W.; Pfeiffer, A.T. and Aldwell, F.E. (1995): Immunological responses and protection against Mycobacterium bovis in calves vaccinated with a low dose of BCG. Vaccine, 13, pp.1123-1130.
Ng, K.H.; Watson, J.D.; Prestidge, R. and Buddle, B.M. (1995): Cytokine mRNA expressed in tuberculin skin test biopsies from BCG-vaccinated and Mycobacterium bovis inoculated cattle. Immunology and Cell Biology, 73, pp.362-368.
Buddle, B.M.; Nolan, A.; McCarthy, A.R.; Heslop, J.; Aldwell, F.E.; Jackson, R. and Pfeiffer, D.U. (1995): Evaluation of three serological assays for the diagnosis of Mycobacterium bovis infection in brushtail possums. New Zealand Veterinary Journal, 43, pp.91-95.
Buddle, B.M.; Keen, D.; Thomson, A.; Jowett, G.; McCarthy, A.R.; Heslop, J.; de Lisle, G.W.; Stanford, J.L. and Aldwell, F.E. (1995): Protection of cattle from bovine tuberculosis by vaccination with BCG by the respiratory or subcutaneous route, but not by vaccination with killed Mycobacterium vaccae. Research in Veterinary Science, 59, pp.10-16.
Aldwell, F.E.; Keen, D.L.; Stent, V.C.; Thomson, A.; Yates, G.F.; de Lisle, G.W. and Buddle, B.M. (1995): Route of BCG administration in possums affects protection against bovine tuberculosis. New Zealand Veterinary Journal. 43, pp.356-359.
Wedlock, D.N.; Aldwell, F.E. and Buddle, B.M. (1996): Molecular cloning and characterisation of tumour necrosis factor alpha (TNF-_) from the Australian common brushtail possum, Trichosurus vulpecula. Immunology and Cell Biology, 74, pp.151-158.
Aldwell, F.E.; Wedlock, D.N. and Buddle, B.M. (1996): Bacterial metabolism, cytokine mRNA transcription and viability of bovine alveolar macrophages infected with Mycobacterium bovis BCG or virulent M. bovis. Immunology and Cell Biology, 74, pp.45-51.
Ng, K.H.; Aldwell, F.E.; Wedlock, D.N.; Watson, J.D. and Buddle, B.M. (1996): Antigen-induced interferon-_ and interleukin-2 from periphial blood cultures of cattle inoculated with Mycobacterium bovis. Veterinary Immunology and Immunopathology (submitted).
A paper describing the molecular cloning and characterisation of possum TNF-_ was published in Immunology and Cell Biology (vol 74:151-158).
Conferences
Buddle, B.M.; Aldwell, F.E.; Wards, B.J. and de Lisle, G.W. (1995): Vaccination with novel Mycobaterium bovis strains protect cattle against bovine tuberculosis. Proceedings of the 9th International Congress of Immunology, San Francisco, U.S.A., July 23-29, p. 817.
Aldwell, F.E. and Buddle, B.M. (1995): Growth of virulent Mycobacterium bovis but not M. bovis BCG in cultured bovine macrophages. Proceedings of the 9th International Congress of Immunology, San Francisco, U.S.A., July 23-29, p.818.
Buddle, B.M.; Aldwell, F.E. and de Lisle, G.W. (1995): Comparison of immunological responses of cattle vaccinated with BCG or novel tuberculosis vaccines. Proceedings of the 4th International Veterinary Immunology Symposium, Davis, U.S.A., July 16-21, p.119.
Papers published in proceedings of the Second International Conference on Mycobacterium bovis, University of Otago, Dunedin, 28 August - 1 September, 1995;
Wedlock, D.N.; Aldwell, F.E.; Stent, V.C. and Buddle, B.M. (1995): Expression of bovine cytokines in the yeast Saccharomyces cerevisiae. pp.35-36.
Wedlock, D.N.; Aldwell, F.E. and Buddle, B.M. (1995): Cloning and characterisation of tumour necrosis factor alpha (TNF-_) from the Australian brushtail possum, Trichosurus vulpecula. pp.37-39.
de Lisle, G.W.; Buddle, B.M. and Collins, D. (1995): The measurement of virulence of mycobacteria. pp.67-69.
Aldwell, F.E.; Wedlock, D.N., Ng, K.H. and Atkinson, P.A. (1995): Modulation of bovine cytokines by lipoarabinomannan from Mycobacterium bovis. pp.153-154.
Aldwell, F.E.; Wedlock, D.N. and Buddle, B.M. (1995): Mycobacterium bovis /Macrophage interactions: bacterial survival and cytokine induction. pp.92-93.
Buddle, B.M., Aldwell, F.E. and Wedlock, D.N. (1995): Vaccination of cattle and possums against bovine tuberculosis. pp.111-115.
Lugton, I.W.; Jackson, R.; Pfeiffer, D.U.; Buddle, B.M.; Morris, R.S.; McCarthy, A.R. and Heslop, J. (1995): Evaluation of ELISA for diagnosis of tuberculosis in possums. pp.243-246.
Aldwell, F.E.; Wedlock, D.N. and Buddle, B.M. (1995): Growth of Mycobacterium bovis in bovine macrophages - the role of interferon-_ and nitric oxide. Proceedings of Australasian Society for Immunology, Gold Coast, Australia, 3-7 December, p.9.
Wedlock, D.N.; Aldwell, F.E. and Buddle B.M. (1995): Interferon-_-induced nitric oxide production by LPS-stimulated bovine alveolar macrophages. Proceedings of Australasian Society for Immunology, Gold Coast, Australia, 3-7 December, p.119.
Buddle, B.M. (1996): Biotechnology and disease control. Proceedings of OECD Conference on Biotechnology in Dairying, Wellington, 7-9 February.
Buddle, B.M. (1996): BCG vaccination against tuberculosis in animals. Report of the meeting of an OIE ad hoc group on bovine tuberculosis control in developing countries, Paris, 20-21 February, pp.5-6.
Adlwell, F.E.; Wedlock, D.N. and Buddle B.M. (1996): Sequential activation of alveolar macrophages by interferon-_ and LPS is required for growth inhibition of Mycobactenum bovis. Proceedings of the 3rd Conference on the Pathogenesis of Mycobacterial Infections, Stockholm, Sweden, June 27-30.
General Articles
Buddle, B.M. (1996): New doors opening to disease control. Dairy Exporter, March, pp 132-133.
4.1.2
Programme Title: Possum immunologyResearch Leader:Dr B Buddle Institution:AgResearch, Wallaceville |
Summary
This programme aims to develop vaccine delivery systems in possums which will induce high antibodies or cellular immune responses indicating immunity, including isolating the genes of possum cytokines which are important in regulation of immune responses.
A trial was undertaken to compare a range of different methods to administer oral vaccines to possums for induction of antibody responses. Ovalbumin was used as a model toxic substance. Possums administered ovalbumin orally did not demonstrate antibody responses.
Description
To continue to develop vaccine delivery systems in possums which will induce high antibodies or cellular immune responses indicating immunity, including isolating the genes of possum cytokines which are important in regulation of immune responses by:
- comparing methods for oral delivery of antigens to possums to induce high antibody responses;
- producing recombinant tumour necrosis factor-a in both Escherichia coli and yeast expression systems and determine biological activity;
- screening cDNA from possum macrophages for cytokines using primers designed from mammalian cytokine sequences; and
- developing biological assays for interferon-y and IL~2 for use in screening cDNA libraries for these cytokines.
Approach & Outcomes
A trial was undertaken to compare a range of different methods to administer oral vaccines to possums for induction of antibody responses (responses by the body to a toxic substance [antigen]). Ovalbumin was used as a model toxic substance. Possums administered orally with ovalbumin did not demonstrate antibody responses.
Biologically active recombinant possum TNF-_ (tumour necrosis factor) has now been produced. When suitable quantities of this purified cytokine are available, the adjuvant activity of this cytokine will be investigated.
The cloning of possum IL-1ß and IL-10 is important since it has been shown in other animals that both these cytokines play an important role in production of antibodies. The expression of recombinant possum IL-1ß and IL-10 and development of techniques to monitor their mRNA expression should enable researchers to prove whether they play a similar role in possums.
Publications, Conferences and General Articles
Refer to those listed under the previous project "Immunology of bovine tuberculosis".
4.1.3
Summary
This research clearly showed that using known CD8+ T immune cells as vaccines elicits a protective immune response against Mycobacteria in mice, and this could be applicable to protection of cattle.
Objective 1 Identify Class-1 MHC restricted M. bovis epitopes
Research Leader:Dr K Erb
Objective 2 Test naked DNA M. bovis vaccine strategy
Research Leader:Dr K Erb
Objective 3 Test peptide on beads vaccine strategy
Research Leader:Dr K Erb
Description - Objective 1: Identify Class-1 MHC restricted M .bovis epitopes
Identify Class-1 MHC restricted M. bovis epitopes (the part of an antigen molecule that determines the specific antibody molecule that will attach to it) by using computer programmes to identify sequences using available immunological techniques.
Description - Objective 2: Test naked DNA M. bovis vaccine strategy
Test naked DNA M. bovis vaccine strategy by:
- constructing naked DNA vector containing M. bovis 19kD gene fragment; and
- testing this vector in mice for the development of CD8+ and CD4+ T cell immune responses using identified peptide fragments in in vitro immune response assays.
Approach & Outcomes
There is strong evidence that CD8+ T cells are an important class of immune cells in the development of protective immunity against Mycobacteria in humans, cattle, and mice. CD8+ T cells only recognise 9 amino acid peptide fragments from the whole antigen.
The data showed that DNA vaccination can efficiently be used to endogenously express the 19 kD and AhpC mycobacterial proteins in mice. Furthermore potential CD8+ T cell epitopes were easily identified. A recently published report showed that DNA vaccination with the 65 kD and 36 kD genes from M. tuberculosis elicited both T cell responses and protection against M. tuberculosis in mice. Using the 19 kD or ahpC genes no T cell response was detected and no protective effect to M. bovis-BCG after DNA vaccination was observed. Therefore, the 19 kD and AhpC proteins do not seem to be good candidates for subunit vaccines.
Description - Objective 3: Test peptide on beads vaccine strategy
Test peptide on beads vaccine strategy to study whether the new peptide - coupled to latex beads vaccine strategy, is capable of inducing M. bovis specific CD8 immune responses.
Approach & Outcomes
There is strong evidence that CD8+T cells can elicit a protective immune response against Mycobacteria in humans, cattle, and mice.
The results clearly show that using known CD8+ T cell epitopes as peptide vaccines in alum injected s.c. can prime CD8+ T cell responses. In influenza and lymphocytic choriomeningitis infections, this approach has led to the protection of these animals, clearly showing that this approach can be successfully used. If the CD8+ T cell epitopes are not known (as in Tb) peptides or proteins bound to beads can be used to induce both a CD4 and CD8+ T cell response.
Publications
Watson, J.; Sato, T.; Chuk, D.; Prestidge, R.L.; Erb, K.J.; LeGros, G.; and Tan, P. (1995): Role of cytokines in immune reponses to mycobacterial antigens. Tuberculosis in Wildlife and Domestic Animals, University of Otago Press, Dunedin.
Erb, K.J.; Watson, J. and LeGros, G. (1995): Class I restricted epitopes of M. bovis and M. tuberculosis as components of DNA and peptide vaccines. Tuberculosis in Wildlife and Domestic Animals, University of Otago Press, Dunedin.
Erb, K.J.; Woodfield, L.; Wilson, T.; Collins, D.M.; Watson, J.D. and LeGros, G. (1996): The identification of Class I MHC restricted epitopes of the l9kD and AhpC proteins from M. tuberculosis. Their use as components in DNA vaccines. Vaccines 1997, Cold Spring Harbor Press, USA.
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