Efficacy of foot-and-mouth disease vaccines A22 Iraq 64 and A Malaysia 97 against challenge with a recent South East Asian serotype A field strain in cattle and sheep Jacquelyn Horsington1, Nagendrakumar Singanallur1, Aldo Dekker2, Soren Alexandersen3, Wilna Vosloo1 1Australian Animal Health Laboratory, CSIRO, 5 Portarlington Rd, Geelong, Victoria, Australia 2Central Veterinary Institute (CVI), Wageningen UR, Lelystad, The Netherlands. 3National Centres for Animal Disease, Canadian Food Inspection Agency, 1015 Arlington St, Winnipeg, Manitoba, Canada HEALTH AND BIOSECURITY

In memory.. Claudia Perez, SENASA, Buenos Aires, Argentina

FMDV Serotype A Widespread Antigenically and genetically heterogenous 26 regional genotypes within three continental topotypes

Serotype A in South East Asia: are there reasons for concern?

Vaccine Matching Studies with Serotype A Major project objectives increase our real-time understanding of the epidemiology of FMD in South East Asia (SEA) perform vaccine matching studies and build capacity in both Australia and SEA OIE Regional Reference Laboratory, based in Pakchong, Thailand major collaborator access to current viruses circulating in the region Vaccines must protect against viruses circulating in the field Australia has a limited number of vaccine strains in our FMD vaccine bank Limited number of strains can be tested in in vivo challenge studies Laboratory (in vitro) assays are used to predict which viruses should be prioritised for live animal studies

Vaccine matching or ‘r’-value of FMDV type A in SEA region A22 IRAQ 64 BVS A MAY 97 BVS TAI/161/12 VIT/3/13 TAI/20/13 TAI/26-3/13 TAI /21-2/14 TAI/94-3/11 LAO/5/14 TAI/5/14 TAI/89-2/11 TAI/99-2/11 TAI/17-1/12 VIT/1/13 TAI/58/13 TAI/44/14 VIT/2/13 TAI/39/13 The in vitro assays determine the match between the vaccine strains and field viruses by establishing the relative homology or r1 values Two serotype A vaccine strains in the vaccine bank were used as references (A MAY 97 and A22 IRQ) for the vaccine matching assays LAO/2/14 TAI/1-2/14 TAI/2/14 TAI/65-1/14 TAI/47-2/13 TAI/53/13 TAI/60/13 TAI/10/14 TAI/13-2/14

Vaccine efficacy trials with A MAY 97 and A22 Iraq monovalent vaccine against challenge with A/VIT/15/2012 in cattle and sheep A virus from the 2010–2013 cluster selected to test against the vaccine strains A22 IRQ and A MAY 97 Vietnamese serotype A isolate - A/VIT/15/2012 In vitro vaccine matching A value of >0.3 is considered a match Field isolate r1-value A22 IRQ A MAY 97 A/VIT/15/2012 0.17 0.16

Unvaccinated controls Testing efficacy A MAY 97 and A22 IRQ monovalent vaccines against challenge with A/VIT/15/2012 in cattle 5 cattle/test group Vaccine - A22 Iraq or A MAY 97 Vaccination 7 or 21 days prior to challenge High potency >6 PD50 Virus - A/VIT/15/2012 Donors inoculated by intradermal lingual (IDL) injection Vaccine Group Day of challenge A22 IRQ 1 (5 cattle) 21 dpv   3 (5 cattle) 7 dpv A MAY 97 2 (5 cattle) 4 (5 cattle) Unvaccinated controls 5 (3 cattle) Vaccination Day: -7 10 14 35 Sampling Weekly Sampling Daily Termination Challenge -21

Clinical Signs Complete Protection! Complete Protection! A22 IRQ 64; challenged 21 dpv A MAY 97; challenged 21 dpv Complete Protection! Complete Protection! UV Controls A22 IRQ 64; challenged 7 dpv A MAY 97; challenged 7 dpv Partial Protection! Partial Protection! Clinical disease

Immune response to non-structural protein Group Animal ID 0 dpc 1 dpc 2 dpc 3 dpc 4 dpc 5 dpc 6 dpc 7 dpc 10 dpc 14 dpc 21 dpc 28 dpc 35 dpc 1 (A22 IRQ, 21 dpv) 8092 32* 24 33 35 45 66 78 80 81 8093 39 38 34 37 36 59 79 73 84 8094 22 21 20 14 60 85 83 8095 30 25 28 62 76 86 8096 41 53 91 90 82 88 2 (A MAY97, 21 dpv) 8097 40 43 44 50 65 87 8098 27 31 55 69 8099 47 74 8100 26 32 70 71 8101 3 (A22 IRQ, 7 dpv) 8102 42 63 8103 68  NS 8104 54 8105 49 67 93 94 8106 46 48 89 4 (A MAY97, 7 dpv) 8107 58 8108 9 12 13 16 17 23 64 75 8109 77 8110 19 8111 18 61 5 8112 72 8113 57 56 52 8114 NS NS – No sample as the animals were euthanized or died; green = positive; blue = possible false positive; * values shown as percent inhibition

Virus detection in oro-pharyngeal fluid Virus isolation indicates carrier Group -7 7 10 14 21 28 30 35   V A22 Iraq 21 dpv A MAY 7 dpv UV controls ND ND  RT-qPCR positive V = virus isolated

Testing efficacy A MAY 97 and A22 IRQ monovalent vaccines against challenge with A/VIT/15/2012 in cattle Summary Both vaccines protected 100% of cattle, 21 dpv High potency A22 Iraq vaccine protected 80% (4 of 5) of cattle, 7 dpv High potency A MAY 97 vaccine protected 60% (3 of 5) of cattle, 7 dpv Virus excretion in oral and nasal fluids in all groups NSP antibody in all cattle in all groups Virus persistence in all groups

Testing efficacy A22 IRQ monovalent vaccine against challenge with A/VIT/15/2012 in sheep 6 replicate rooms 3 donors, 1 vaccinated contact and 1 unvaccinated contact per room Vaccine - A22 Iraq Vaccination 4 days prior to challenge High potency >6 PD50 Virus - A/VIT/15/2012 Donors inoculated by coronary band (CB) injection Vaccinated/unvaccinated control sheep challenged by direct contact exposure = natural route of infection Vaccination Day: -4 Contact 10 14 35 Sampling Weekly Sampling Daily Termination

Clinical Signs Donors Vaccinated Contacts 1/6 Diseased 18/18 Diseased *diseased sheep euthanized day 9 Unvaccinated Contacts 5/6 Diseased *donors euthanized on day 9 *UVs euthanized between day 9 and 14

Immune response to non-structural protein Group Sheep ID NSP antibody ELISA -4–5 dpc 6 dpc 7 dpc 8 dpc 9 dpc 10 dpc 12 dpc 14 dpc 21 dpc 28 dpc VC 7 - P 8 9 10 11 12 UC 13 14 15 16 17 18 Donor 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 (-) – Negative; P– Positive; VC – vaccinated contact; UI – unvaccinated infected; Grey = dead

Virus detection in oro-pharyngeal fluid Vaccinated Unvaccinated Carrier Carrier DPC -4 1 2 3 4 5 6 7 9 12 14 21 28 35 Sheep 7   V Sheep 8 Sheep 9 Sheep 10 Sheep 11 Sheep 12 DPC -4 1 2 3 4 5 6 7 9 12 14 21 28 35 Sheep 13   V Sheep 14 Sheep 15 Sheep 16 Sheep 17 Sheep 18 2/6 vaccinated sheep became carriers RT-qPCR positive V = virus isolated

Testing efficacy A22 IRQ monovalent vaccine against challenge with A/VIT/15/2012 in sheep Summary The A/VIT/15/2012 virus was pathogenic in sheep High potency A22 Iraq vaccine protected 83% (5 of 6) of sheep, 4 dpv Vaccinated animals had Virus excretion in oral and nasal fluids NSP antibody Virus persistence

Summary A May 97 A22 Iraq Species Cattle Sheep Challenged at 21 dpv 7 dpv 4 dpv Route IDL Direct Contact Protection 100% 60% 80% 83% NSP Ab Yes Carriers Virus in nasal and oral swabs High potency serotype A vaccines provide protection from clinical disease against heterologous challenge Even at early time points - But is that good enough? Vaccination does not prevent virus replication However, the challenge route may be important Vaccination does not prevent the development of carriers What is the importance of these carriers?

Conclusions Antigenic matching does not always accurately predict protection, especially with high potency vaccines Other measures of control will be important during an outbreak, vaccination alone is not the golden bullet Transmission studies would answer a lot of questions

Thank you Acknowledgements Soren Alexandersen Kurtis Swekla Zhidong Zhang Jaime Bernstein Charles Nfon Margaret Forbes Kate Hole Marlee Phair Hilary Bittner Cory Nakamura Melissa Goolia Tim Salo FMD Risk Management Project Jacquelyn Horsington (PhD, MSc, BSc) Research Scientist t +61 3 5227 5127 e jacquelyn.horsington@csiro.au w www.csiro.au Aldo Dekker Klaas Weerdmeester Phaedra Eble Meindert Bleijenberg Froukje van Hemert-Kluitenberg HEALTH AND BIOSECURITY