Vaccines against East Coast fever: re-assessment of p67C and identification of new antigens

Anna Lacasta8 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

Why we want to re-asses p67C protein?

Re-assessment of p67C protein

Re-assessment of 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

Why are we re-assessing the p67C protein?

Re-assessment of p67 proteinPrime 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…

Looking for new antigens

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)

PlasmodiumBabesiaTheileria

Toxoplasma

CryptosporidiumGregarine

Eimeria

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

Combine different antigens in one vaccine in order to achieve better protection levels

VHVL sequence VHVL sequence

VHVL sequence

VHVL sequence VHVL sequence

VHVL sequenceHost 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:

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