Appendix 30 Preliminary results to evaluate cross-protection between O 1 Manisa and O 1 Campos in cattle V.A. Srinivasan 1, S.B.Nagendra Kumar 1, M.Madhan Mohan 1, V.Maroudam 1, P.Santha Kumar 1, S. Parida 2, J. Horsington 2, D.J. Paton 2* 1 Indian Immunologicals Limited, Rakshpuram, Gachibowli Post, Hyderabad 500 019, India 2 Pirbright Laboratory, Institute for Animal Health, Ash Road, Woking, Surrey GU24 0NF, UK Abstract: Introduction: Serology is used to predict vaccine induced protection against challenge with a heterologous strain of the same serotype of foot-and-mouth disease virus (FMDV). To evaluate the accuracy of such predictions, we compared the protection afforded to cattle vaccinated with the O 1 Manisa strain of FMDV against challenge with either a homologous (O 1 Manisa) or a heterologous strain (O 1 Campos). Serology by virus neutralization test (VNT) using O 1 Manisa antiserum predicted an acceptable protection (r 1 = 0.6) against such a challenge. Materials and Methods: Sixteen unvaccinated and FMDV-naive cattle were vaccinated with O 1 Manisa. They were challenged by intradermolingual inoculation with live FMDV, either O 1 Manisa (Group 1, n=8) or O 1 Campos (Group 2, n=8). Unvaccinated control cattle were challenged with either the O 1 Manisa (n=2) or O 1 Campos (n=2) viruses. The cattle were monitored for signs of FMD and samples were taken to measure viraemia and virus associated with nasal swabs and probangs. Blood and saliva were also collected for serology and to detect mucosal IgA antibody. Results: All control cattle developed generalized FMD. The 8 O 1 Manisa vaccinated and challenged cattle were protected from generalized FMD. In contrast, only 2 O 1 Manisa vaccinated and O 1 Campos challenged cattle were protected from generalized FMD and there was evidence of more virus replication in the O 1 Campos challenged cattle. Discussion: Despite relatively good cross-neutralization of O 1 Campos by O 1 Manisa antisera, O 1 Manisa vaccinated cattle were less well protected against challenge with O 1 Campos than with homologous O 1 Manisa. Another trial is planned to confirm/refute differences in virus replication between vaccinated and unvaccinated cattle and between O 1 Manisa and O 1 Campos challenged but unvaccinated cattle and to calculate PD 50 values for each vaccine/challenge combination. Introduction: FMD virus exists as seven different serotypes and as well as being serologically distinct, these serotypes elicit a serotype-specific immunity such that infection or vaccination with one serotype does not protect against the others (Brooksby, 1982; Cartwright et al., 1982). In addition, many antigenic subtypes are recognized within serotypes (Rweyemamu and Hingley, 1984; Alonso et al., 1993) and some of these differences may be important in relation to cross-protection. Therefore, serological vaccine matching tests are routinely used as part of the process for selecting the most appropriate vaccine strain for protection against a given field isolate (Kitching et al., 1988; Paton et al., 2005). However, the mechanisms of the immune protection elicited by vaccination are not fully understood (Dunn et al., 1998; McCullough et al., 1992) and relatively few studies have been published confirming the predictive value of serological vaccine matching tests (Aggarwal et al., 2002; Barteling and Swam, 1996; Mattion et al., 2004). Therefore, a study was undertaken to evaluate the accuracy of serologically predicted cross-protection within a serotype by vaccinating cattle with the O 1 Manisa strain of FMDV and then challenging them with either a homologous (O 1 Manisa) or a heterologous strain (O 1 Campos). Serology by virus neutralization test (VNT) using O 1 Manisa antiserum predicted an acceptable protection (r 1 = 0.64/0.62 at Hyderabad and Pirbright respectively, where >0.3 indicates that the field isolate is sufficiently similar to the vaccine strain that use of the vaccine is likely to confer protection against challenge with the field isolate) against such a challenge, whereas serology using ELISA predicted a borderline protection value (r 1 = 0.36/0.46 at Hyderabad and Pirbright respectively, where >0.39 signifies sufficient similarity and 0.2-0.39 signifies that the vaccine strain might be suitable for use if no closer match can be found provided that a potent vaccine is used and animals are preferably immunised more than once) (OIE Manual, 2006). 207
Materials and Methods: Experimental design: The O 1 Manisa strain of FMD virus is derived from a 1968 Turkish isolate that is of the Middle-East South-Asian topotype, whereas the O 1 Campos strain was originally isolated in Brazil in 1958 and is of the European South American topotype (Samuel and Knowles, 2001). Twenty cattle were obtained from a FMD-free herd and were screened by 3 rounds of testing for FMDV-NSP antibodies using Ceditest and UBI Kits. All the animals were negative in both the tests. Sixteen cattle were vaccinated with a monovalent oil adjuvant (ISA206) vaccine containing 15 µg of O 1 Manisa antigen. They were challenged by intradermolingual inoculation with live FMDV, either O 1 Manisa (Group 1, n=8) or O 1 Campos (Group 2, n=8). O 1 Manisa and O 1 Campos cattle challenge viruses were prepared by three cattle passages of cell culture adapted virus. The stock viruses had been titrated in seronegative healthy calves. The titres were log10 5.81 for O 1 Manisa and log10 8.67 for O 1 Campos. Unvaccinated control cattle were challenged with either the O 1 Manisa (Group 3, n=2) or O 1 Campos (Group 4, n=2) viruses. The cattle had their temperatures recorded daily and were monitored for signs of FMD. Probang and blood samples were taken until 35 days post infection to test for virus genome and antibodies respectively. Laboratory tests: A gel-based RT-PCR test (unpublished) was carried out at Hyderabad. Real-time RT-PCR (Reid et al., 2002) and serology by virus neutralization test and the liquid phase blocking ELISA were done at Pirbright. NSP serology using a commercial kit, Ceditest FMD-NS supplied by Cedi-Diagnostics, based on the assay of Sorensen et al. (2004) was done at both sites. The liquidphase blocking ELISA for FMDV antibodies was carried out as described in the OIE manual with antibody titres expressed as the final serum dilution giving 50% of the mean OD recorded in the virus control in the absence of serum (Hamblin et al., 1987). Virus neutralization tests used IBRS2 cells and pre-titrated O 1 Manisa or O 1 Campos viruses (OIE 2006). A partial VP1 gene sequence was obtained for each of the vaccine and challenge strains to confirm their authenticity. Results: At 21 days after vaccination, most of the cattle had seronverted to O 1 Manisa with homologous neutralising antibody titres averaging 1.6 and 1.5 log10 VN50 for Groups 1 and 2 respectively (Table 1). Sera from Group 2 cattle were also checked for their levels of neutralizing antibody to O 1 Campos and these were found to be comparable to the O 1 Manisa titres (Table 1). By liquid phase blocking ELISA, the relative log10 titres induced against O 1 Manisa and O 1 Campos by O 1 Manisa vaccination were 2.3 and 1.9 respectively (i.e. still not significantly different by t-test). The 8 cattle in Group 1 were protected from generalized FMD after challenge with O 1 Manisa virus while only 2 out of the 8 cattle in Group 2 (25%) were protected against challenge with O 1 Campos virus (Table 2). The control animals in Groups 3 and 4 all developed generalized FMD, although the onset of signs in one control animal challenged with O 1 Manisa was delayed (#1001, Table 2a). Fig 1 shows the rectal temperatures of all of the animals over the study and the marked difference between Groups 1 (O 1 Manisa challenge) and 2 (O 1 Campos challenge) are apparent. RT-PCR testing at Hyderabad detected O 1 Manisa virus genome in the probang samples from 2 Group 1 cattle on days 3 and 5 (#885) or day 35 (#971) respectively, whereas positive results were obtained on 3 (#983) or 5 (#1001) occasions from the two control unvaccinated cattle in Group 3. In contrast, all of the Campos challenged cattle were found positive by RT-PCR on days 3 and 5 post challenge and 6 further Group 2 cattle were positive on occasions thereafter. The two unvaccinated control cattle (Group 4) were positive from 3-21 days post challenge. Samples were not available at Pirbright from all of the Group 1 cattle for quantitative real-time RT-PCR testing. In Groups 1 and 3 (Manisa challenged cattle), high copy numbers of genome (>500) were detected in the unvaccinated controls and in two of the four tested vaccinated cattle (only at the third day after challenge). In contrast, high copy numbers of genome were detected at some point from all but one (#884) of the Campos challenged cattle. None of the animals used in the experiments had antibodies to NSPs on the day of vaccination and challenge indicating that the vaccine did not induce antibodies to NSPs. Subsequent to challenge with O 1 Manisa, four vaccinated cattle as well as the two unvaccinated animals developed antibodies to NSPs. All of the cattle challenged with O 1 Campos developed antibodies to NSPs from the 10th day post challenge onwards (Table 3). Discussion: Prior to the start of this cross-challenge experiment, serological cross reactivity between O 1 Manisa and O 1 Campos was examined independently at Hyderabad and Pirbright using separate stocks of viruses and antisera. The ability of bovine post vaccination sera to neutralize or bind to O 1 Campos virus was compared with the reactivity of the same sera in the same tests against parental O 1 208
Manisa virus. Very similar results were produced in the two laboratories resulting in calculated r 1 values of 0.64 and 0.62 for neutralization and 0.36 and 0.46 for ELISA. According to the standard interpretations used for these tests, this indicates that by neutralization, the two viruses are well matched, whereas by ELISA the match is more moderate. These differences, if real, may be due to the fact that the ELISA is detecting a broader range of antibodies including those without in vitro neutralizing activity. The question of whether or not antibodies that do not neutralize in vitro can nevertheless give protection in vivo remains controversial, although some authors consider that opsonisation of virus is the key mechanism by which antibodies clear virus in vivo and that this does not require antibodies to have in vitro neutralizing activity (McCullough et al., 1992). In the current study, vaccination of 16 calves resulted in moderate antibody responses by the time of challenge at 21 days post vaccination, although anomalous negative results were found for calves #761 and 913. Interestingly, there was no significant difference in the ability of these antibodies to neutralize O 1 Campos compared to O 1 Manisa, whereas ELISA titres were higher (but not significantly different) using the homologous antigen consistent with the earlier r value data. Comparison of the deduced capsid amino acid sequence of O 1 Manisa and O 1 Campos reveals a number of differences that are not located at known sites of antigenic significance, except for at position 159 in site 2 of VP2 where the Campos sequence has the soluble amino acid serine whilst that of Manisa has the hydrophobic amino acid proline. This amino acid change could contribute to the difference in reactivity of antisera by ELISA and might be of significance in cross-protection. An alternative explanation for the cross-protection findings are that the Campos challenge was more severe than that provided by the Manisa virus. A comparison between the Group 3 and 4 cattle (unvaccinated controls) in terms of the speed of onset of disease, the amount of virus shed and the severity of signs does not reveal any obvious support for this explanation, except that one of the Manisa cattle (#1001) had no fever spike and was slow to develop clinical lesions despite high levels of virus in probang fluids. However, the number of cattle in these groups is rather small to decide the matter. Another trial is planned to confirm or refute differences in virus replication between vaccinated and unvaccinated cattle and between O 1 Manisa and O 1 Campos challenged but unvaccinated cattle and to calculate PD 50 values for each vaccine/challenge combination. Conclusions: Despite a good level of in vitro cross-neutralization, clinical cross-protection afforded by O 1 Manisa vaccination against O 1 Campos challenge was incomplete. More control unvaccinated cattle are needed to confirm differences in the level of virus shedding between O 1 Manisa vaccinated and unvaccinated cattle when challenged with O 1 Campos. Explanations for the incomplete cross-protection in vivo could be either that the challenge with O 1 Campos was more severe than the O 1 Manisa challenge or that non-neutralizing antibodies or other immune responses to vaccination need to be measured to establish an in vitro correlation with protection. Recommendations: Further studies are required to investigate the correlation between in vitro and in vivo measures of within-serotype vaccine-induced cross-protection. Acknowledgements: Thanks are due to Nick Knowles for the partial VP1 sequencing work and to Pip Hamblin and Bob Statham for serology and r value work at Pirbright. Work at IAH-Pirbright was supported by FP6 grant SSPE-CT-2003-503603. References: Aggarwal N., Zhang Z., Cox S., Statham R., Alexandersen S., Kitching R.P., Barnett P.V. (2002). - Experimental studies with foot-and-mouth disease virus, strain O, responsible for the 2001 epidemic in the United Kingdom. Vaccine, 20:2508-15. Alonso A., Gomes M.P.D., Ramalho A.K., Allende R., Barahona H., Sondahl M. & Osório F. (1993). Characterization of foot-and-mouth disease virus by monoclonal antibodies. Viral Immunol., 6:219-228. 209
Barteling, S.J. and Swam, H. (1996). The potent aqueous and double oil emulsion foot-andmouth disease type O 1 vaccines from European Vaccine Banks probably protect against all other O 1 strains. Report of the European Commission for the Control of Foot-and-mouth disease, Session of the Research Group of the Standing Technical Committee, Kibbutz Ma ale Hachmisha, Israel, 2-6 September 2006, Appendix 13, pp 90-94. Brooksby J.B. (1982). Portraits of viruses: foot-and-mouth disease virus. Intervirology, 18:1-23. Cartwright B., Chapman W.G. & Sharpe R.T. (1982). Stimulation of heterotypic antigens of foot-and-mouth disease virus antibodies in vaccinated cattle. Res. vet. Sci., 32:338-342. Dunn C.S., Samuel A.R, Pullen L.A. & Anderson J. (1998). The biological relevance of virus neutralisation sites for virulence and vaccine protection in the guinea pig model of foot-and-mouth disease. Virology, 247:51-61. Hamblin, C., Kitching, R.P., Donaldson, A.I., Crowther, J.R. and Barnett, I.T.R. (1987). Enzyme-linked Immunosorbant assay for the detection of antibodies against foot-and-mouth disease virus III. Evaluation of antibodies after infection and vaccination. Epidemiol Infection, 99:733-744. Kitching R.P., Rendle R. & Ferris N.P. (1988). Rapid correlation between field isolates and vaccine strains of foot-and-mouth disease virus. Vaccine, 6:403-408. Mattion N., Konig G., Seki C., Smitsaart E., Maradei E., Robiolo B., Duffy S., Leon E., Piccone M., Sadir A., Bottini R., Cosentino B., Falczuk A., Maresca R., Periolo O., Bellinzoni R., Espinoza A., Torre J.L. & Palma E.L. (2004). Reintroduction of foot-and-mouth disease in Argentina: characterisation of the isolates and development of tools for the control and eradication of the disease. Vaccine, 22:4149-4162. McCullough K.C., De Simone F., Brocchi E., Capucci L., Crowther J.R. & Kihm U. (1992). Protective immune response against foot-and-mouth disease. J. Virol., 66:1835-1840. OIE (Office International des Epizooties/World Organisation for Animal Health). 2006. Foot and mouth disease. In: Manual of standards for diagnostic tests and vaccines. Ed. OIE Standards Commission. Paris, France, Office International des Epizooties. Chapter 2.1.1. http://www.oie.int/eng/normes/mmanual/a_summry.htm Paton, D.J., Valarcher, J.-F., Bergmann, I., Matlho, O.G., Zakharov, V.M., Palma, E.L., Thomson, G.R. (2005). Selection of foot-and-mouth disease vaccine strains a review. OIE Sci et Tech Rev, 24:981-993. Reid SM, Ferris NP, Hutchings GH, Zhang Z, Belsham GJ, Alexandersen S. (2002). Detection of all seven serotypes of foot-and-mouth disease virus by real-time, fluorogenic reverse transcription polymerase chain reaction assay. J Virol Methods, 105:67-80. Rweyemamu M.M and Hingley P.J. (1984): Foot-and-mouth disease virus strain differentiation: analysis of the serological data. J Biol Stand, 12:225-229. Samuel AR, Knowles NJ. (2001). Foot-and-mouth disease type O viruses exhibit genetically and geographically distinct evolutionary lineages (topotypes). J Gen Virol., 82:609-21. Sorensen KJ, de Stricker K, Dyrting KC, Grazioli S, Haas B. (2004). Differentiation of footand-mouth disease virus infected animals from vaccinated animals using a blocking ELISA based on baculovirus expressed FMDV 3ABC antigen and a 3ABC monoclonal antibody. Arch Virol., 150:805-14. 210
Table 1. Serology Results at 21 days post vaccination (day of challenge) Group Animal ID Manisa VNT Campos VNT Manisa LPBE Campos LPBE Results challenge 1 841 2.1 nd nd nd Protected 1 829 1.8 nd nd nd Protected 1 971 1.8 nd nd nd Protected 1 885 1.3 nd nd nd Protected 1 705 1.8 nd nd nd Protected 1 917 2.1 nd nd nd Protected 1 761 <0.9 nd nd nd Protected 1 706 1.8 nd nd nd Protected 1 Mean 1.6 - - - 100% protection 3 983 (C) <0.9 nd nd nd FMD 3 1001 (C) <0.9 nd nd nd FMD 2 884 2.1 2.3 2.9 2.6 FMD 2 702 2.3 2 2.4 1.7 FMD 2 913 <0.9 <0.9 <1.7 <1.7 Protected 2 770 1.3 1.3 1.8 <1.7 FMD 2 976 1.5 1.5 2.3 2.7 Protected 2 877 1.7 2.1 2.7 3 FMD 2 1006 1.2 1.5 2.3 1.7 FMD 2 876 1.8 1.5 3 1.7 FMD 2 Mean* 1.5 1.6 2.3 1.9 25% protection 4 895 (C) <0.9 nd nd nd FMD 4 1016 (C) <0.9 nd nd nd FMD * to calculate mean values, titres of <0.9 and <1.7 were considered as 0.45 and 0.85 respectively of 211
Table 2. Clinical Results of Cattle challenge experiments - Appearance of Foot (F) and Mouth (M) Lesions Post Challenge: Animal 0 1 2 3 4 5 6 7 8 9 Group ID F M F M F M F M F M F M F M F M F M F M 1 841 1 829 1 971 1 885 + ++ ++ ++ ++ ++ ++ ++ ++ 1 705 + + + + + + + 1 917 + + + + + + + + + 1 761 1 706 3 983 (C) + 3 ++ 3 +++ 4 +++ 4 +++ 4 +++ 4 +++ 4 +++ 4 +++ 1001 3 (C) 1 2 3 3 4 4 2 884 2 2 702 2 2 913 2 770 3 3 ++ 3 ++ 4 ++ 2 976 2 877 1 1 1 + 1 + 4 + 2 1006 + + + 1 + 1 + 1 1 2 2 876 + + 1 + 2 + 2 + 2 2 2 4 895 (C) 1 3 + 4 + 4 + 4 + 4 +++ 4 +++ 4 +++ 1016 4 (C) 2 4 + 4 + 4 + 4 + 4 4 4 M: Mouth Lesions Nil means only tracts were noticed; + means lesions noticed and scored as +; ++; +++; ++++; +++++ F: Foot lesions number of feet affected; : days post challenge; (C): unvaccinated control animal 212
Table 3: NSP serology results GROUP SAMPLE ID 0 14 21 0 5 10 15 21 35 0 14 21 0 5 10 15 21 35 PLACE HYDERABAD PIRBRIGHT 1 841 N N N N N N N N N N N ND P N N N 1 829 N N N N N N N N N N N ND N N N N 1 971 N N N N N N N N N N N ND N P N N 1 885 N N N N P P N P N N N ND P P P P 1 705 N N N N N N P P N N N ND N N N N 1 917 N N N N P P P P N N N ND P N P P 1 761 N N N N N N N N N N N ND N N N N 1 706 N N N N N N N N N N N ND N N N N 3 986 (C) N N N N P P P P N N N ND P P P P 3 1001 N N N N N P P P N N N ND N P P P (C) 2 884 N N N N N P P P N N N ND P P P P 2 702 N N N N N P P P N N N ND N P P P 2 913 N N N N P P P P N N N ND P P P P 2 770 N N N N P P P P N N N ND P P P P 2 976 N N N N P P P P N N N ND P P P P 2 877 N N N N P P P P N N N ND P P P P 2 1006 N N N N N N N P N N N ND P P P P 2 876 N N N N P P P P N N N ND P P P P 4 895 (C ) 4 1016 (C) N N N N P P P P N N N N P P P P N N N N N N Group 1: vaccinated with O 1 Manisa and challenged with O 1 Manisa Group 3: unvaccinated and challenged with O 1 Manisa Group2: vaccinated with O 1 Manisa and challenged with O 1 Campos Group 4: unvaccinated and challenged with O 1 Campos N: negative; P: positive; ND: not done : days post vaccination; : days post challenge; (C): unvaccinated control ND ND P P P P P P P P 213
Fig 1. Rectal temperatures in Fahrenheit following FMD challenge a) Group 1: O 1 Manisa challenge of vaccinated cattle 106 105 Rectal Temperature (Fahrenheit) 104 103 102 101 100 841 829 971 885 705 917 761 706 99 98 0 1 2 3 4 5 6 7 8 9 Days after challenge b) Group 2: O 1 Campos challenge of vaccinated cattle 106 105 104 Rectal Temperature (Fahrenheit) 103 102 101 884 702 913 770 976 877 1006 876 100 99 98 0 1 2 3 4 5 6 7 8 9 Dats after challenge c) Groups 3 and 4: O 1 Manisa and Campos challenge of unvaccinated cattle 106 105 Rectal Temperature (Fahrenheit) 104 103 102 101 100 99 983(Manisa) 1001(Manisa) 895(Campos) 1016(Campos) 98 0 1 2 3 4 5 6 7 8 9 Days after challenge 214