Vaccine 27 (2009) 21992201

Contents lists available at ScienceDirect

Vaccine
journal homepage: www.elsevier.com/locate/vaccine

Editorial

Thermostable foot-and-mouth disease virus as a vaccine candidate for endemic countries: A perspective
a r t i c l e i n f o

Keywords: Foot-and-mouth disease virus Foot-and-mouth disease Vaccine Thermostability

Foot-and-mouth disease (FMD) is the most contagious and economically burdensome disease of animals. The disease affects all cloven-footed animals, and is endemic in Africa, Asia and South America, with frequent incursions into Europe [1]. Whereas culling is advocated in FMD-free nations, vaccination strategy is practiced in endemic countries to combat, contain and eventually eradicate the disease. The only vaccine marketed for FMD is inactivated crude or puried virus preparation, adjuvanted with either oil or aluminum salts. However, such inactivated vaccines are associated with several drawbacks, including short shelf-life, engendering suboptimal immune responses, and short duration of protective immunity. An extremely critical component of vaccination programmes is the maintenance of cold chain, a not so easy task owing to the fact that most of the endemic countries simply do not have the infrastructure to achieve it. Considering that endemic nations are contiguous with other land-locked countries, FMD becomes a continental or a global problem for international trade, animal movement and maintenance of disease-free zones, requiring a concerted international effort to control and eradicate this disease [2]. The development of potent new generation vaccines for FMD has been the subject of intense research during the last few decades. Inline with other medical and veterinary viral vaccines, research and development on FMD vaccines have mostly focused on subunit and vectored vaccines. Peptides or puried proteins, recombinant DNA, viral vectors and plants expressing FMD virus (FMDV) structural proteins, with or without immunopotentiators, have been demonstrated to elicit humoral and cell-mediated immune responses in experimental animals and shown to protect natural hosts to varying degrees in enclosed settings [311]. However, while these approaches show promise for use in FMD-free zones, none of them have so far been subjected to large-scale eld trials in endemic countries. Whereas peptides and puried proteins have failed to command an important place in vaccinology due to various reasons, the use of recombinant DNA in large domestic and wild animals may be difcult due to the necessity of injecting large and multiple doses, the period needed to attain optimal responses (compared to the rapidity with which FMD spreads through herds), delivery issues, and the potential for vaccinated animals to be carriers
0264-410X/$ see front matter 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2009.01.032

(e.g., suboptimal responses may suppress superinfection with a eld virus but may not be able to prevent infection or virus shedding). Recombinant adeno, pox and pseudorabies virus vectors have been developed for vaccination purpose, and alphavirus vectors are under development, but the potential for these in restricting FMD outbreaks or to eradicate them in endemic countries is not clear. In this regard, a recent report by Mateo et al. [12] on engineering FMDV to achieve increased thermostability is of great interest for vaccination programmes. The authors have rationally engineered FMDV to increase its stability against thermal dissociation into subunits without disrupting the many biological functions needed for its infectivity. Amino acid side chains located near the capsid inter-subunit interfaces and either predicted or found to be dispensable for infectivity were replaced by others that could establish new disulde bonds or electrostatic interactions between subunits [12]. Two engineered mutants A2065H and D3069E/T2188A were normally infectious, genetically stable, and antigenically indistinguishable from nonmutated FMDV C-S8c1 but showed substantially increased stability against irreversible dissociation. The results of the present study show, in fact, that virion thermostability against dissociation into subunits may not be selectively constrained by functional requirements for infectivity [12]. Typically, FMD vaccine is produced in bulk, inactivated, potency assessed and frozen at 80 C or in liquid nitrogen vapours. When required, the virus concentrate is thawed and formulated. Standard vaccines are formulated to contain a minimum of 3.0 PD50 (potency dose-50) of antigen per vial [13,14]. The shelf-life after formulation is generally 12 years, but once formulated, the vaccine needs to be administered as quickly as possible due to the rapid loss of antigenic load. The crucial immunogenic component of FMD vaccines is the 146S particle of the virus. Any degradation of the particle will signicantly reduce the potency of the vaccine. This is where a thermostable vaccine would be critical. The data from Mateo et al. [12] show that a unit log dissociation of the wild type virus occurs in about 20 h at 42 C, and 3040 days at 4 C, suggesting and reafrming that viral structure is lost in less than a day upon exposure to temperatures close to eld conditions. If loss of antigen correlates with dissociation, then antigen derived from the

2200

Editorial / Vaccine 27 (2009) 21992201

engineered FMDV would be much more stable than the traditional vaccines derived from wild type strains. In this regard, a change in amino acid from Ala to His at position 65 in the VP2 protein (A2065H) is of particular interest since the rate of unit log dissociation of this variant is about 60 h at 42 C, and several months at 4 C (extrapolated from 20% loss in 2 months). However, the A2065H mutant needs to be evaluated for long-term stability both with and without formulation. Further, retention of antigenicity (as assessed by immunoblot) or gaining resistance to dissociation may not always translate to immunogenicity i.e., ability to induce protective immune responses. Therefore, potency tests with A2065H mutant in parallel with wild type are required to conrm that resistance to dissociation corroborates with improvement in duration and quality of protective immune responses. FMDV exists as seven serotypes: O, A, C, Asia-1 and South African Territory (SAT)-1, -2 and -3 of which, serotype O predominates in most countries [15]. The level of protection attained with present vaccines is about 7080% for serotype O, while it is nearly 100% for serotype C [16]. Several factors play a role in dictating protection conferred by vaccine [17]. Mateo et al. [12] used structural proteins from a serotype C virus in their studies. Although this virus is a chimera between types C and O [18], all its structural proteins are from serotype C, making it likely that the results predict outcome with a virus containing the complete C type genome. Similar mutation with serotype O may benet vaccination programs in endemic countries. Irrespective of the vaccination status, animals recovered from acute FMD will carry the virus persistently and can act as reservoirs for other animals. Recent ndings suggest that increasing the vaccine antigen payload can reduce sub-clinical infection following virus challenge, leading to fewer persistently infected carrier animals [19]. Thus, we can infer that a vaccine virus with greater stability should signicantly reduce persistent virus infection during disease outbreaks. Therefore, it will be of interest to study the role of engineered FMD vaccines on virus persistence and secretion. Another interesting observation is that thermostable variants are not represented among the quasispecies of FMDV serotypes [20]. It is therefore tempting to speculate that FMDV has evolved to undergo persistency by avoiding establishment of thermostable species. A potential link between acquisition and loss of heparin binding characteristics and persistence of FMDV has been propounded [21,22]. In other words, thermostability might be detrimental to survival, persistence and spread of FMDV in nature (in fact, tropism and the spread of the virus during cold seasons could be a direct consequence of this). If so, this evolutionary adaptation would be blocked by a thermostable vaccine. In this context, two engineered mutants A2065H and D3069E/T2188A studied by Mateo et al. showed a substantially increased stability against irreversible dissociation as compared to nonmutated FMDV C-S8c1 [12]. A bioinformatics analysis revealed that no natural isolate of FMDV contains His65 in VP2, and only one isolate (C1Arg85) carries Lys whereas Thr appears to be permissible at this position. Apart from the potential ramications to evolution or persistence of FMDV, combined with the fact that inter-serotype recombination is infrequent in FMDV structural region [23], the rarity of natural occurrence of all these variants provides an avenue to detect outbreaks resulting from improper inactivation or accidental release (i.e., the mutation would be present in the outbreak virus). Formalin and binary ethyleneimine, used for the inactivation of FMDV, are known to affect the integrity of the virus epitopes and may promote dissociation of viral structural proteins [23]. Since the integrity of proteins (VP1VP3) on the virus is one of the major contributing factors for vaccine potency, studies have to be performed to conrm that A2065H mutant maintains epitope integrity during viral inactivation process. In this context, we suggest that FMDV inactivation by non-chemical methods such as induction of virion-

associated endonuclease activity [24,25], which degrades the RNA genome but maintains epitope integrity of the virus [26], can complement thermostable mutant-based vaccines. In addition, use of new generation molecular adjuvants might enhance the quality of the immune responses [27,28]. The method described by Mateo et al. [12] has broad and important vaccine application potential for both FMD-free and FMD-endemic countries. Increased stability could at least mean longer storage, reduced production and quality testing costs, and less dependence on cold chain, not to mention other potential advantages like further increase in stability following viral inactivation process, reduction in persistent state, and signicant enhancement in immune responses. Of particular interest is the potential solution to cold chain-related problems. In most endemic countries, the vaccination drive is during the hotter season of the year and a lag time of 2448 h is typical between the time the vaccine is removed from the major distribution centres and the time of actual vaccination. Increasing the thermostability may completely eliminate maintenance of cold chain beyond the distribution centres. The A2065H mutant dissociates at the rate of 1 log every 60 h at 42 C, and this property alone makes A2065H variant a formidable vaccine candidate. In this regard, it is worthwhile noting that thermostability of the vaccine strain played a critical role in the global eradication of rinderpest [29]. Finally, the duration of immunity following natural infection is much longer compared to that following vaccination ([30] and eld observations), and engendering long-term immunity through immunization is still a black box for FMD. Although live vaccines might be an answer, they are not acceptable due to the possibility of reversion to virulence and the inability to differentiate infected and vaccinated animals. The work by Mateo et al. [12] has laid the foundation for a thermostable vaccine for FMD, but further research, especially with respect to eld studies and immune responses, is warranted. Acknowledgements We apologize to those whose work could not be cited due to space constraints. NRH and PPR are supported by a Grant (BT/PR10382/GBD/27/105/2007) from the Department of Biotechnology, Ministry of Science and Technology, Government of India. MSM, SVK and JB are supported by grants from Institut National de la Sant et de la Recherche Mdicale (INSERM), Centre National de la Recherche Scientique (CNRS) and UPMC-Paris VI. JB is also supported by Coopration INSERM-ICMR-AO 2009/2010. References
[1] Grubman MJ, Baxt B. Foot-and-mouth disease. Clin Microbiol Rev 2004;17(2): 46593. [2] Kitching P, Hammond J, Jeggo M, Charleston B, Paton D, Rodriguez L, et al. Global FMD controlis it an option? Vaccine 2007;25(30):56604. [3] Balamurugan V, Renji R, Venkatesh G, Reddy GR, Nair SP, Ganesh K, et al. Protective immune response against foot-and-mouth disease virus challenge in guinea pigs vaccinated with recombinant P1 polyprotein expressed in Pichia pastoris. Arch Virol 2005;150(5):96779. [4] Wang X, Zhang X, Kang Y, Jin H, Du X, Zhao G, et al. Interleukin-15 enhance DNA vaccine elicited mucosal and systemic immunity against foot and mouth disease virus. Vaccine 2008;26(40):513544. [5] Su B, Wang J, Wang X, Jin H, Zhao G, Ding Z, et al. The effects of IL-6 and TNFalpha as molecular adjuvants on immune responses to FMDV and maturation of dendritic cells by DNA vaccination. Vaccine 2008;26(40):511122. [6] Cubillos C, de la Torre BG, Jakab A, Clementi G, Borras E, Barcena J, et al. Enhanced mucosal immunoglobulin A response and solid protection against foot-andmouth disease virus challenge induced by a novel dendrimeric peptide. J Virol 2008;82(14):722330. [7] Li Y, Stirling CM, Denyer MS, Hamblin P, Hutchings G, Takamatsu HH, et al. Dramatic improvement in FMD DNA vaccine efcacy and cross-serotype antibody induction in pigs following a protein boost. Vaccine 2008;26(21):264756. [8] Choudary S, Ravikumar P, Ashok Kumar C, Suryanarayana VV, Reddy GR. Enhanced immune response of DNA vaccine (VP1-pCDNA) adsorbed on cationic PLG for foot and mouth disease in guinea pigs. Virus Genes 2008;37(1):817.

Editorial / Vaccine 27 (2009) 21992201 [9] Yang B, Lan X, Li X, Yin X, Li B, Han X, et al. A novel bi-functional DNA vaccine expressing VP1 protein and producing antisense RNA targeted to 5 UTR of footand-mouth disease virus can induce both rapid inhibitory effect and specic immune response in mice. Vaccine 2008;26(43):547783. [10] Greenwood DL, Dynon K, Kalkanidis M, Xiang S, Plebanski M, Scheerlinck JP. Vaccination against foot-and-mouth disease virus using peptides conjugated to nano-beads. Vaccine 2008;26(22):270613. [11] Du Y, Jiang P, Li Y, He H, Jiang W, Wang X, et al. Immune responses of two recombinant adenoviruses expressing VP1 antigens of FMDV fused with porcine granulocyte macrophage colony-stimulating factor. Vaccine 2007;25(49):820919. [12] Mateo R, Luna E, Rincon V, Mateu MG. Engineering viable foot-and-mouth disease viruses with increased thermostability as a step in the development of improved vaccines. J Virol 2008;82(24):1223240. [13] Doel TR. FMD vaccines. Virus Res 2003;91(1):8199. [14] Goris N, Merkelbach-Peters P, Diev VI, Verloo D, Zakharov VM, Kraft HP, et al. European Pharmacopoeia foot-and-mouth disease vaccine potency testing in cattle: between test variability and its consequences. Vaccine 2007;25(17): 33739. [15] Knowles NJ, Samuel AR, Davies PR, Midgley RJ, Valarcher JF. Pandemic strain of foot-and-mouth disease virus serotype O. Emerg Infect Dis 2005;11(12): 188793. [16] Patil PK, Bayry J, Nair SP, Gopalakrishna S, Sajjanar CM, Misra LD, et al. Early antibody responses of cattle for foot-and-mouth disease quadrivalent double oil emulsion vaccine. Vet Microbiol 2002;87(2):1039. [17] Jamal SM, Bouma A, van den Broek J, Stegeman A, Chenard G, Dekker A. Foot-and-mouth disease vaccine potency testing: the inuence of serotype, type of adjuvant, valency, fractionation method, and virus culture on the doseresponse curve in cattle. Vaccine 2008;26(50):631721. [18] Mateo R, Diaz A, Baranowski E, Mateu MG. Complete alanine scanning of intersubunit interfaces in a foot-and-mouth disease virus capsid reveals critical contributions of many side chains to particle stability and viral function. J Biol Chem 2003;278(42):4101927. [19] Cox SJ, Voyce C, Parida S, Reid SM, Hamblin PA, Hutchings G, et al. Effect of emergency FMD vaccine antigen payload on protection, sub-clinical infection and persistence following direct contact challenge of cattle. Vaccine 2006;24(16):318490. [20] Mateo R, Luna E, Mateu MG. Thermostable variants are not generally represented in foot-and-mouth disease virus quasispecies. J Gen Virol 2007;88(Pt 3):85964. [21] Sa-Carvalho D, Rieder E, Baxt B, Rodarte R, Tanuri A, Mason PW. Tissue culture adaptation of foot-and-mouth disease virus selects viruses that bind to heparin and are attenuated in cattle. J Virol 1997;71(7):511523. [22] Fry EE, Lea SM, Jackson T, Newman JW, Ellard FM, Blakemore WE, et al. The structure and function of a foot-and-mouth disease virus-oligosaccharide receptor complex. EMBO J 1999;18(3):54354. [23] Jackson AL, ONeill H, Maree F, Blignaut B, Carrillo C, Rodriguez L, et al. Mosaic structure of foot-and-mouth disease virus genomes. J Gen Virol 2007;88(Pt 2):48792.

2201

[24] Scodeller EA, Lebendiker MA, Dubra MS, Crespo OA, Basarab O, La Torre JL, et al. Inactivation of foot-and-mouth disease virus vaccine strains by activation of virus-associated endonuclease. J Gen Virol 1984;65(Pt 9):1567 73. [25] Amadori M, Barei S, Melegari M, Panina GF. Safety and efcacy of foot-andmouth disease vaccines containing endonuclease-inactivated virions. Vaccine 1987;5(3):21922. [26] Patil PK, Suryanarayana V, Bist P, Bayry J, Natarajan C. Integrity of GH-loop of foot-and-mouth disease virus during virus inactivation: detection by epitope specic antibodies. Vaccine 2002;20(78):11638. [27] Guy B. The perfect mix: recent progress in adjuvant research. Nat Rev Microbiol 2007;5(7):50517. [28] Bayry J, Tchilian EZ, Davies MN, Forbes EK, Draper SJ, Kaveri SV, et al. In silico identied CCR4 antagonists target regulatory T cells and exert adjuvant activity in vaccination. Proc Natl Acad Sci USA 2008;105(29):102216. [29] Roeder PL. The animal story. BMJ 2005;331(7527):12624. [30] Doel TR. Natural and vaccine induced immunity to FMD. Curr Top Microbiol Immunol 2005;288:10331.

Nagendra R. Hegde a Mohan S. Maddur b,c Pavuluri Panduranga Rao a Srini V. Kaveri b,c,d Jagadeesh Bayry b,c,d, a Bharat Biotech Foundation, Genome Valley, Shameerpet Mandal, Hyderabad 500078, India b Unit 872, Institut National de la Sant et de la Recherche Mdicale, F-75006 Paris, France c Centre de Recherche des Cordeliers, Universite Pierre et Marie Curie, Unit Mixte de Recherche-Sant 872, F-75006 Paris, France d Universit Paris Descartes, Unit Mixte de Recherche-Sant 872, F-75006 Paris, France author at: INSERM U 872, Equipe 16, Centre de Recherche des Cordeliers, 15 rue de lEcole de Mdicine, Paris F-75006, France. Tel.: +33 1 55 42 82 66; fax: +33 1 55 42 82 62. E-mail address: jagadeesh.bayry@crc.jussieu.fr (J. Bayry) 9 January 2009 Available online 2 February 2009
Corresponding