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Vaccines against East Coast fever: Re-assessment of p67C and identification of new antigens
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Presentation by Anna Lacasta at the 12th Biennial Conference of the Society for Tropical Veterinary Medicine (STVM) and the VIII International Conference on Ticks and Tick-borne Pathogens (TTP-8) Cape Town, South Africa 24 to 29 August 2014.
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Vaccines against East Coast fever: Re-assessment of p67C and identification of new antigens
- Vaccines against East Coast fever: re-assessment of p67C and identification of new antigens Anna Lacasta 8 International Congress on Ticks and Tick-Borne Pathogens (TTP8) and 12 Biennial Conference, South Africa, 24-29 August 2014
- East Coast fever • Economically important in endemic areas: Eastern, Central and Southern Africa • Caused by the parasite Theileria parva • Rhipicephalus appendiculatus act as a vector • Lymphoproliferative disease • The severity of the infection is dose dependent
- Immunity against ECF Hyperimmune sera from cattle infected several times could neutralize sporozoites Cattle that survive to natural infection developed specific CTL response Ab CTL
- Neutralizing antibodies • The hyperimmune sera from cattle infected several times with T. parva has neutralizing antibodies against sporozoites (Musoke et al., 1982) • Monoclonal antibodies to sporozoites were made and it was found that they were neutralizing. These were recognizing a 67 kDa protein (Dobbelaere et al., 1985)
- p67 protein • p67 was discovered as a potential immunoprotective antigen in 1992 (Musoke et al., 1992) • Very conserved among cattle derived T. parva strains. Polymorphic in buffalo derived parasites (Nene et al., 1996) • p67 is present in sporozoites and also in schizonts in lower amounts (Honda et al., 1998) • Antibodies and recombinant p67 protein inhibit in vitro infection • p67 protects the 50% of the cattle against an LD70 challenge
- p67 protein fragments Murine and cattle neutralizing antibodies only recognize epitopes in p67Nt and p67Ct p67 full length protein is unstable p67C has the same level of protection as p67 full length and it is stable expressed in E. coli
- Re-assessment of p67C protein Why we want to re-asses p67C protein?
- Re-assessment of p67C protein Why are we re-assessing the p67C protein? • The last experiment was done more than 10 years ago starting point • Laboratory experiments and field experiments didn’t get the same results • different p67 protein construct • different adjuvant • different route of infection • Compare two versus three immunizations
- Re-assessment of p67 protein Prime boost experiment using p67C protein with ISA206 adjuvant, followed by an LD70 challenge with T. parva Muguga, using Boran cattle Antibody titration by ELISA Seroneutralization assay B-cell ELISPOT Proliferation assays FACs analysis Cytokine expression by ELISA and RT-qPCR RNA sequence
- Re-assessment of p67 protein Starting point for further improving the protective capacity of p67 protein: • TMV (tobacco mosaic virus) method for protein expression • Hepatitis B core antigen • Different adjuvants Correlate the protection achieved with the titer of antibodies and their activity: • New neutralization assay: ELISA and qPCR facilitate the read-out of the assay • Antibody isotypes • Other antibody activities: opsonization, complement-dependent cytotoxicity…
- New antigens Looking for new antigens
- New antigens • Bioinformatics search in T. parva genome: • Cell to cell interaction proteins • Proteins involved in the binding and entry of the sporozoites, comparing with other apicomplexan parasites (Plasmodium) Plasmodium Babesia Theileria Toxoplasma Gregarine Eimeria Cryptosporidium Sarcocystis Some T. parva proteins are homologous to cell binding proteins Cells bind to other cells with proteins called CD
- New antigens • Antibody Phage Display VHVL sequence VHVL sequence VHVL sequence VHVL sequence VHVL sequence VHVL sequence Combine different antigens in one vaccine in order to achieve better protection levels Host animal
- Acknowledgements Stephen Munyao Elias Awino Rosemary Saya Charity Muthoni Simeon Otieno Thomas Njoroge Gideon Ndambuki Roger Pelle Lucilla Steinaa Vish Nene This project is funded by:
- better lives through livestock ilri.org The presentation has a Creative Commons licence. You are free to re-use or distribute this work, provided credit is given to ILRI.
Editor's Notes
- Good morning everyone. I am Anna Lacasta and I am going to talk about the vaccines against East Coast fever: re-assessment of p67C and identification of new antigens.
- First of all I want to introduce to you the disease. East Coast fever (ECF) is a lethal disease of cattle, that is economically important in eastern, central and southern Africa. It is caused by Theileria parva a tick-transmitted protozoan. The disease is a lympho-proliferative disorder that typically results in death 2–4 weeks after infection, particularly in exotic cattle breeds. A very important concept is that the severity of the infection is totally dose dependant, this differ from other apicomplexan infections, like in Plasmodium.
- Ivan Morrison explained widely in the Monday presentation the immunity of ECF but I want to briefly remind it to the audience. There is two main stages of the parasite involved in natural immunity: sporozoite and schizont stage. It was demonstrated that after several re-infections the cattle developed neutralizing antibodies against the sporozoites. Then avoiding that the sporozoite infect the cells. On the other hand, after natural infection cattle generate an specific CTL response against the schizont stage. Those are the main parasite stages where the subunit vaccine research is focused and in this presentation I am going to focus in the Antibody immunity.
- Neutralizing antibodies against the sporozoites were discovered by Musoke in 1982, when it was observed that the hyperimmune sera from cattle infected several times with T.parva could neutralize the T. parva infection. Demonstrating that is possible to develop a humoral immune response able to block the infection. After that, it was demonstrated that monoclonal antibodies that neutralize the parasite infection recognize a 67 kDa protein. Afterwards this protein was called p67.
- The potential of p67 to confer protection in cattle was demonstrated in 1992 in a prime and two boost experiment with a posterior LD70 challenge. Some characteristics of the protein are that P67 protein is a very conserved protein among cattle derived strains and it is the major surface coat protein in the sporozoite and it is also expressed in schizont in lower amounts. But more important is that the presence of antibodies or the recombinant p67 protein inhibits in vitro infection, and that p67 protects the 50% of the animals under laboratory conditions conditions, alone, with no presence of other antigens.
- Then what was the problem? The protein is unstable in E.coli system expression, as we can see in this figure. What is the solution? By PepScan analysis was demonstrated that murine antibodies with sporozoite neutralizing activity and hyperimmune sera from cattle also with neutralizing activity only recognize epitopes in the Nt and Ct of the protein, not in the middle of the protein. Anyway, this most polymorphic part of the protein. Afterwards Bishop demonstrate the protective capacity of p67C and p67N proteins, being the p67C the one that gave the same level of protection as p67 full length. p67N also confers protection but not at the same level. The major advantage of using p67C instead of p67 full length was that it is stable in expression in E.coli.
- It is known that p67 and p67C protect the animals against the LD70 challenge. Then… why we want to re-asses the protein?
- It is important to stress that the last experiment was done more than 10 years ago and this is our starting point for the new funded project. In future we want to improve the p67 protection level with different formulas, but we are going to discuss afterwards. Another reason to repeat the experiment is that the results achieved in the lab and in the field didn’t correlate, maybe because of: different p67 protein constructions, different adjuvant or different route of infection that had been used. The last reason why we want to repeat the experiment is because we want to compare the level of protection between two and three immunizations, to make the vaccination procedure more feasible and realistic for a future application of the vaccine.
- Currently we are running the experiment. It is a prime boost experiment using two or three immunizations depending on the group followed by an LD70 challenge using T.parva Muguga strain. The adjuvant used is Montanide ISA206 and the cattle used are Boran. Today we are in day 72 in the experiment and the only preliminary data that we can share is that the animals are responding to the immunizations as we expected. There are many assays that we want to implement in this experiment, some of them are listened below, some related to the antibody generation and activity and some other focused in the cell response, especially the CD4 T-cell response. As you can see it is a very complete panel of assays.
- As I said before the p67 re-assessment is our starting point for future improvement of the level of protection achieved with this protein. In future experiments we are going to test the protection capacity of p67 expressed in TMV method that it is know that enhance the antibody response. We also will try different adjuvants to enhance the generation of antibodies. In a final step we will try to correlate the level of protection with the antibody titers and activity. It was never done before. To do this we are developing a new neutralization assay more feasible. For the moment we improved a lot the read-out of the assay using a cellular ELISA quantification method instead of the Giemsa staining that previously was used and also want to set-up a qPCR to quantify the parasite. We also want to take into account the importance of the isotypes of the antibodies in protection and other possible antibody activities like opsonization, complement-dependent cytotoxicity…
- In our current research we are also looking for new antigens that can generate protective antibodies.
- Bioinformatics is a very powerful tool for the antigen searching taking the whole T.parva genome into consideration. There are two approaches that we want to use. On one side, it is known that the cells bind to each others with proteins called CD and Theileria parva has some homologous genes to the CDs. Blocking those surface proteins maybe we can neutralize the infection. On the other side we are looking for genes that are orthologous to other apicomplexan parasites, like Plasmodium, genes that code proteins most probably involved in the binding and entry of the parasite. Another time, blocking those proteins we can avoid the infection of the cells.
- Another approach, a part from the bioinformatics, is the construction of an antibody phage display. How it works? The idea is to immunize a host animal with purified sporozoites, when the specific B cells are generated the variant heavy and light chain will be sequenced and cloned to finally be expressed in a phage system. The final phage collection will help us to identify new sporozoite antigens in a widely way. Under our perspective the future protective vaccine will combine different antigens in order to achieve better protection and most probably combining protection against the sporozoite stage and also against the schizont stage of the parasite.
- I would like to thanks the ILRI people help and the Bill and Melinda Gates foundation for their economical support.
- Thank you very much for you attention.
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