Vaccination against foot-and-mouth disease virus: strategies and effectiveness

347

Review

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Foot-and-mouth disease (FMD) is an economi-cally devastating and highly contagious disease of domestic and wild cloven-hoofed animals, including cattle, sheep, goats and pigs. It limits access to markets for developing countries and can cause costly outbreaks in formerly FMD-free countries, as evidenced in the UK and The Netherlands in 2001 and, more recently, in 2007 in the UK. The causative agent is the FMD virus (FMDV), which is a single-stranded positive-sense RNA virus belonging to the genus Aphthovirus in the family Picornaviridae. FMDV was the first virus discovered in ver-tebrates after the discovery of tobacco mosaic virus in plants [1]. The disease is characterized by fever, lameness and vesicular lesions on the mouth, tongue, feet, snout and teats of infected animals. FMD-infected cattle and pigs usually develop obvious signs of the disease; whereas, clinical diagnosis of FMD in sheep and goats is more difficult due to the mild manifestation of the disease. However, sheep proved to be effec-tive transmitters of the disease in the UK out-break in 2001 [2]. The average mortality from FMD is only approximately 1%, but morbidity is 100% in infected animals and the transmissi-bility is extremely high [3]. Although FMD does not result in high mortality in adult animals,

the disease has debilitating effects, including weight loss, decrease in milk production and loss of draught power, resulting in a loss in pro-ductivity for a considerable time. The virus has a high mutation rate and exists as seven distinct serotypes (O, A, C, Asia 1, South African ter-ritories [SAT]1, SAT2 and SAT3), as well as numerous and constantly evolving subtypes, showing a spectrum of antigenic diversity. In the past, outbreaks have occurred in every live-stock-containing region of the world, with the exception of New Zealand. Currently, FMD is enzootic in all continents except Australasia and North America [4]. At the moment, the EU is free from FMD, although, as mentioned earlier, recent outbreaks were observed in The Netherlands in 2001 and in the UK in 2001 and 2007.

FMD control by vaccinationThere are seven antigenically distinct serotypes of FMDV and each serotype has many intra-typic variants. This antigenic variation creates a major problem for the control of FMD, as infection or vaccination with one serotype of FMDV does not protect against other serotypes and may fail to protect fully against other sub-types within the same serotype [5–8]. However,

Satya ParidaInstitute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Surrey, GU24 0NF, UK Tel.: +44 148 323 2441 Fax: +44 148 323 2448 [email protected]

Although present conventional foot-and-mouth disease (FMD) vaccines can prevent clinical disease, protection is short lived (∼6 months), often requiring frequent revaccination for prophylactic control, and vaccination does not induce rapid protection against challenge or prevent the development of the carrier state. Furthermore, it is clear that the clinical protection depends upon the length of immunization and the duration of exposure/challenge methods. This review summarizes the present and future strategies for FMD control in endemic and FMD-free countries, the effectiveness of FMD vaccines in cattle, sheep and pigs, new methods for selecting vaccine strains, suggestions for alternative methods of vaccine testing, suggestions for the development of new-generation efficacious vaccines and their companion tests to differentiate infection in vaccinated animals.

Keywords: detection of FMD virus carrier animals • FMD control strategies in endemic and FMD-free countries • foot-and-mouth disease • foot-and-mouth disease vaccine • new-generation FMD vaccines • vaccine efficacy in cattle, sheep and pigs • vaccine matching and selection • vaccine testing

Vaccination against foot‑and‑mouth disease virus: strategies and effectivenessExpert Rev. Vaccines 8(3), 347–365 (2009)

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FMD control in some endemic areas is implemented by means of regular mass vaccination. In FMD-free countries (i.e., coun-tries of the EU and North America), the control policy has been primarily based upon slaughtering of infected and in-contact animals, together with restrictions on movement of animals and animal products. There has been provision to resort to vac-cination under emergency circumstances where the outbreak is extensive and the slaughtering of large numbers of animals becomes unmanageable [4]. During and following the 2001 FMD outbreak in the UK and The Netherlands, there has been a growing demand for ‘vaccination-to-live’ as an alternative to large-scale slaughter for the control of FMD. Although vaccina-tion was used in The Netherlands during the 2001 outbreak, all the vaccinated animals were killed (‘vaccinate-to-kill’) after the outbreak. According to the so-called ‘vaccinate-to-live’ pol-icy, the control of FMD outbreaks in the future in FMD-free countries could be controlled by a short period of emergency vaccination of surrounding infected herds, followed by detec-tion and elimination of infected herds. As a consequence, new regulations have been approved by the World Organization for Animal Health, the Office International des Epizooties (OIE) and the EU, according to which countries can now regain FMD-free status 6 months after the report of the last case following the use of the ‘vaccinate-to-live’ policy, rather than 1 year [9]. Countries using this approach will need to show the absence of virus circulation (in the case of ongoing vaccination) [10] and the absence of any infected animals in the case of short-lived emergency vaccination [9].

Current FMD vaccineCurrent FMD vaccines are produced by infecting baby hamster kidney-21 cells with virulent FMDV, under biosecure conditions, followed by chemical inactivation with binary ethyleneimine (BEI) and purification by ultrafiltration [11]. Some vaccine manufacturers also use polyethylene glycol (PEG) precipita-tion and ultrafiltration to purify the antigen. In some advanced vaccine companies, the relatively crude bulk of inactivated anti-gens are subjected to chromatography to obtain highly purified antigens, which are usually stored in a gaseous phase of liquid nitrogen until vaccine formulation [12]. At the time of vaccine formulation, the concentrated antigen is diluted with buffers and then blended with a suitable adjuvant, either oil or aluminum hydroxide/saponin. Oil–emulsion vaccines are now widely used for the immunization of pigs, sheep, goats and cattle; whereas, aluminum hydroxide/saponin-adjuvanted vaccines are only used for ruminants. Although modern BEI inactivation procedures make FMD vaccines safe to use in the field [12], the requirement for field strain tissue culture adaptation and large volumes of live virus production possess significant biocontainment and biosafety challenges.

Foot-and-mouth disease vaccines in endemic countries com-monly contain more than one serotype of virus, depending upon the epidemiological situation of the particular country. South Asian vaccines are trivalent, containing single isolates of serotype O, A and Asia I, selected from the region. As the C serotype

has not been reported in the Indian subcontinent since 1996, C strain has recently been excluded from the vaccine strain in India (Srinivasan AV, Pers. Comm.). However, South American vaccines still contain serotype C strain and they use a trivalent vaccine along with serotype O and A viruses. The most common vaccine used in South-East Asia is a monovalent type O virus and sometimes it contains several O strains appropriate for the South-East Asian countries [12]. O Manisa is the main vaccine strain in Middle East/South-East Asia; whereas, O Campos is mainly used in South America. A22 Iraq, A Iran 1996 and A Iran 1999 are the com-mon vaccine strains in Middle East; whereas, A24 Cruzeiro is the best known vaccine strain in South America. SAT vaccines are mainly bivalent (SAT1 and SAT2) or trivalent (SAT1, SAT2 and SAT3) and are commonly used in Africa. Vaccine formulations with SATs and serotypes O and A are also used. Monovalent SAT vaccines are rarely used in Africa, although they are available in some reserve vaccine banks.

The payload of each antigen generally varies from 1 to 10 µg, depending on the antigenicity of the strain. In case of sero-type O and SATs, more antigen is required compared with serotype A, Asia I and C, in order to achieve an equivalent potency [12]. Two recent potency tests (A and SAT2) conducted at Pirbright (Surrey, UK), where similar antigen payload per ml were administered, showed that many SAT2-vaccinated animals were clinically infected; whereas, all type A-vaccinated animals were protected upon homologous challenge [13]. This type of variation in potency may be attributed to the unequal stability of antigen in different serotypes of FMDV. The 146S particles have been considered as an immunogenic component of FMD vaccines and any degradation of 146S particles may reduce the potency of the vaccine [14].

Current FMD vaccine testingIn Europe, potency testing of FMD vaccine is carried out accord-ing to the European Pharmacopeia [15] and OIE Manual [16]. A minimum of 17 cattle are divided into four groups: three groups each containing five animals and the fourth group contain-ing two control animals. The vaccine antigen is administered intra muscularly or according to the vaccine manufacturer’s instruction at different doses per group (first three groups) with different volumes (using fractional volumes rather than dilu-tion of the product); for example, 2 ml for a full dose, 0.5 ml for a one quarter dose and 0.125 ml for a one sixteenth dose of vaccine. On the 21st day postvaccination, all the 15 vac-cinated and two control unvaccinated animals are challenged with intradermolingual inoculation of live, cattle-adapted, homologous virus (104 50% infectious dose [ID

50]) in two

sites (0.1 ml per site). The feet of the challenged animals are examined daily for 8 days. The total number of vaccinated ani-mals showing lesions on their feet are taken into account when calculating 50% bovine protective dose (PD

50) value, using the

Spearman–Karber method, and should be a minimum of three for prophylactic vaccine and a minimum of six for emergency vaccine. The Reed–Muench method is now rarely used, unless data sets are very symmetrical, with 0 and 100% end points [12].

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ReviewVaccination against foot-and-mouth disease virus

However, the PD50

test method has been questioned due to high variability of results, as observed in the ten identical potency tests conducted using the same batch of O1 Manisa vaccine [17]. This high variability and low repeatability and reproducibility between the PD

50 tests may be due to the small number of ani-

mals used in each group and, therefore, the authors suggested the need for alternative methods.

The protection against generalization (PG) test is used as a potency test in South and North America, where a group of 16 cattle are vaccinated with a single field dose (2 ml) and challenged after 1 month of vaccination with homologous virus [18]. Two unvaccinated controls are included in each potency trial. Both vaccinated and control cattle are challenged by inoculating 104 suckling mouse lethal dose (SMLD

50) of challenge virus intrader-

mally into four different sites (0.25 ml per site) on the upper surface of the tongue. At 7 days postchallenge, all animals are clinically checked for the appearance of feet lesions [301] and the protection against podal generalization (PPG) percentage is calculated using this formula:

%PPG = s/n × 100 (where s is the number of vaccinated-protected animals and n

is the total number of vaccinated animals).The vaccine is considered satisfactory if a minimum of 75%

protection is achieved. An option of repeating the test is required to obtain an overall 75% protection if the first test fails. 3 PD

50

in the European Pharmacopeia test corresponds to 78% PPG in the PG test for serotype A and O vaccines [18]. Recently it has been reported that the PPG test is more reliable than the European Pharmacopeia potency test [19]. However, the authors suggested that increasing the number of vaccinated animals may help to increase the test’s statistical power further. In vitro tests for vaccine potency and vaccine matching has been suggested to be considered as alternative methods in the future [19].

Potency tests in sheep, goats and buffalo have not yet been standardized. In general, a potency test passed for cattle is con-sidered suitable for vaccination in these species. Owing to the often inapparent nature of the disease in sheep and Asiatic and African buffaloes, potency results from a cattle test may be a more reliable indicator of vaccine quality than attempting a potency test reliant on the detection of clinical signs in these spe-cies [16]. A similar protocol to the test in cattle can be adopted for potency testing of FMD vaccines in pigs. However, 2-month-old pigs are used in the place of 6-month-old cattle and vaccination is carried out for a minimum period of 28 days. Challenge is usually performed by intradermal injection into the heel bulb of one foot with 10,000 50% tissue culture infective dose (TCID

50)

(0.2 ml) of homologous virus. Alternatively, the challenge virus may be administered into one site in the muscular part of the neck behind the ear. The animals are observed daily for 10 days after challenge for clinical signs of FMD. Likewise, a similar protocol to the PGP test in cattle can be adopted for pigs.

Indirect tests, including the measurement of virus-neutralizing antibodies in cell culture following vaccination, or ELISA anti-bodies, or serum-protecting antibodies in suckling mice, may be used to assess the potency of a vaccine, provided that a statistical

evaluation has established a satisfactory correlation between the results obtained by the test on the relevant vaccine serotype and the potency test in cattle [20].

Both PD and PPG potency tests employ the challenge of cattle with virulent virus that causes clinical manifestations of FMD with considerable pain to the animals. The other disadvantage is that the protected animals develop extensive primary lesions in the tongue, similar to the control unvaccinated animals. Furthermore, the costs of animals and containment facilities are very high and there is a high risk of the virus escaping, resulting in an outbreak. Multivalent vaccine tests are not easy to carry out, as only one virus strain can be handled at a time in a high-security unit. Moreover, the average 90% confidence interval for a reported PD

50 value lies between 45 and 220% [17,21,22].

Therefore, alternative methods may be considered for quan-tifying vaccine potency to eliminate the need for virus chal-lenge. Virus neutralizing antibody titer (VNT) is considered as a substitution of the PD/PPG test, provided a correlation with protection against challenge has been demonstrated [23]. Furthermore, a positive correlation between IFN-γ and VNT responses has been observed with vaccine-induced protection on the day of challenge [13,24], Therefore, there is the potential for the replacement of in vivo potency test with VNT [19] and IFN-γ in vitro assays [13]. However, further work is necessary to establish these facts before it can be adapted by regulatory bodies. In addition, any replacement of an in vivo potency test would have to be established for individual serotypes.

FMD vaccine selection & matchingEfficacy of vaccination is affected by the lack of crossprotection between serotypes, as well as incomplete protection between some subtypes [5,7]. In addition, new variant viruses are emerging peri-odically. Consequently, vaccine strain requirements differ accord-ing to the types and subtypes of virus prevailing globally and vaccines have to be selected with care, whether it is for prophylac-tic use in FMD-endemic countries or for incorporation into the antigenic reserves for emergency use in FMD-free countries.

In the case of FMD outbreaks, the immediate requirement is to detect the serotype of the circulating virus, which is gen-erally achieved by antigen-typing ELISA or by genetic typing (sequencing of the VP1 gene). Once the serotype of the virus is established, in vitro vaccine-matching assays are carried out to select a suitable vaccine strain. Currently, methods of vaccine strain selection mainly rely on serological approaches [8]. A commonly used method is to derive relationship values (‘r’ val-ues) between FMDVs using pools of antisera prepared against each vaccine strain to be matched. The antigenic similarities between vaccine strains and field isolates are estimated from their comparative reactivity with the appropriate serum pool using a neutralization test [25] or an ELISA-based method [26]. Alternatively, the complement fixation test (CFT) can be used for this purpose [27], although this test tends to be used only by South American countries. The advantages of ELISA over VNT are that the test is rapid and requires inactivated anti-gen [8]. If VNT is used to determine the antigenic similarity,


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an r1 value of 0.3 or greater indicates a close antigenic match

between the vaccine strain and the field isolate [28]. In the case of the two-step ELISA [26,29], an r

1 value of 0.4–1.0 indicates a

close match between the vaccine strain and the field isolate. If the r

1 value is less than 0.3 in the case of VNT and 0.4 in the

case of ELISA, the field isolates need to be examined against alternative vaccines and/or there may be a need to develop another vaccine strain [8].

There are various factors that may affect the results obtained from the in vitro matching tests. However, a major difficulty is the standardization of the polyclonal serum raised against the vaccine viruses, including both the sera collected from vaccinated target species (usually cattle) and the antisera raised in rabbits and guinea pigs for use as capture or detector antibodies in ELISA or the CFT. As the results of these tests are unreliable and poorly reproducible, and since there is uncertainty over their predic-tive value [8,28], attempts have been made to use consistent and replenishable reagents, such as monoclonal antibodies (mAbs), in an ELISA-based method to overcome these limitations [30,31]. Although mAb-based antigenic ELISA has been reported to be very promising, it does not work as well as the polyclonal anti-body (pAb)-based system at its current state and, therefore, needs further improvement [30]. Another approach could be finding out the antigenic relationship between the vaccine strain and field isolates by comparison of their capsid-gene sequence, which provides the genetic information regarding the epitopes playing a role in vaccine-induced protection. Presently, this approach is being investigated in various FMD laboratories [32].

The O serotype is the most widespread and certainly shows moderate levels of strain variation in the field with, occasionally, more extreme variants. Nevertheless, there are two main lineages of vaccine strains represented by the old (in excess of 30 years since their development) O strains of Europe and South America (O

1 BFS 1860, O

1 Lausanne and O

1 Campos) and the equally

old strains of the Middle East and Asia (represented by strains such as O

1 Manisa and O-3039). In the case of the latter group

of viruses, there are other valuable strains of O serotype in use. However, established virus strains, such as O

1 Manisa, provide

good cover for many first-occurrence situations [12,33]. The anti-genic diversity of serotype A viruses is well known and, therefore, requires a closely matched vaccine strain. The antigenic variation in serotypes Asia 1 and C is narrow compared with other sero-types, Asia 1 Shamir being very effective in cross-neutralizing a wide range of Asia 1 field isolates [12]. SAT2 virus has more sequence variation in the VP1 gene compared with viruses of serotypes O, A and C [34], for which it is considered as a broad antigenic variant. Therefore, a careful selection of the immu-nodominant vaccine strain is required in the case of SAT2 [35]. There are two reasons to develop a new vaccine strain. Firstly, there is the recognition by experts, including the vaccine produc-ers and the reference laboratories, that a significantly different virus has appeared in a region and may/will warrant the develop-ment of a new vaccine strain. This recognition is invariably based on a serological vaccine matching assay, such as the VNT, but may be supported by sequence analysis of the VP1 protein [36].

One such example was the emergence of a new A strain in Iran in 1996. A commercial vaccine manufacturer responded to it by developing a new vaccine strain referred to as A Iran 96, which is now used widely in the Middle East as well as being the choice of some antigen reserves. The second reason is concerned with those countries where regular vaccination programs are employed and the epidemiological situations are relatively stable. Such situ-ations support the obvious concept of preparing a vaccine strain from a local field isolate so that the field viruses and vaccine strains are matched as closely as possible.

FMD vaccine application & effectivenessControl of FMD is mainly carried out by controlling its spread from infected to susceptible animals, either by preventing the movement of the virus from the infected animals, animal prod-ucts, fomites and aerosol, or by reducing the number of suscep-tible animals by vaccination [37]. It has been observed from pre-vious outbreaks that both a restriction of the movement of the virus and a reduction of susceptible animals by vaccination syn-ergistically helps to control FMD [38]. Up until the 1960s there was a shortage of vaccine doses in Europe and, as a result, many FMD epidemics have occurred. However, after the availability of sufficient vaccine doses and with the implementation of annual vaccination programs along with the slaughter of infected and in-contact animals, the EU and Switzerland decided to intro-duce a nonvaccination policy in 1991–1992. During the 2001 outbreak in the UK, the slaughter of 2 million animals caused an economic loss of US$12 billion and a huge public uproar at the massacre of animals. This led to the re-evaluation of the non-vaccination policy, with an aim for a ‘vaccination-to-live’ policy using a highly potent emergency vaccine (minimum potency of 6 PD

50) in and around the infected farms.

In some endemic countries, prophylactic vaccination (mini-mum potency of 3 PD

50) is being carried out to control FMD. For

this purpose, an initial administration of vaccine followed by a booster around 4–6 weeks and followed subsequently by further boosts at either 4–6 months or annually, is required depending upon the species and epidemiological conditions. This vaccina-tion strategy allows sufficient time for the immune response to develop [39], in contrast to emergency vaccination [40–42]. A review on emergency vaccinations suggests that the risk of spread of infection decreases with an increase of intervals between vac-cination and virus exposure [43]. However, in endemic settings, this control program often fails, mainly due to the lack of a control measure on the transfer of virus by mechanical means (e.g., people and vehicles), and the movement of infected animals or animal products, despite an effective clinical protection often being observed in experimentally vaccinated and subsequently challenged animals. Therefore, the prohibition of the movement of infected livestock and their products along with restrictions on mechanical means of transmission should be imposed soon after the diagnosis of FMD [38].

During the 1960s–1980s, many experimental and commercial vaccines with a high concentration of the FMDV antigen as sin-gle-dose vaccine administration were tried successfully in cattle,



sheep and pigs [44–47]. During the 1990s, many experiments with high-potency doses (4.1–10 µg) of inactivated vaccines revealed full clinical protection in cattle by the end of first week of vac-cination upon indirect aerosol challenge from donor pigs [40,48]. Even 1.9 µg of serotype C antigen per vaccine dose was able to protect cattle 4 days postvaccination upon indirect aerosol chal-lenge from donor pigs [48]. A comparison of PD

50 with nanogram

quantities of antigen has been conducted using data from potency tests on 15 batches of monovalent FMD vaccine, comprising five batches each of type O, type A and type C [49]. Type O vaccines required a far higher level (220 ng) of 140S antigen to achieve a 50% protection level (PA

50) in cattle than type A (2.4 ng) and

type C (4.36 ng) vaccines.Along with the dose-comparison study, researchers were able

to demonstrate better protection in cattle vaccinated with oil-adjuvanted Asia 1 vaccine than cattle vaccinated with aqueous Asia 1 vaccine upon challenge [40]. However, protection in pigs requires a longer period following vaccination than in cattle, nd oil vaccines are mainly effective [41,43,48,50]. Although, histori-cally, sheep and goats are not included in vaccination programs in many countries, they represent a major population of FMDV-susceptible animals and the recent outbreaks of FMD within and around the EU have involved sheep [51–56]. The need to vaccinate sheep is uncertain [57], as ovine historically does not produce huge amounts of virus in aerosol and does not transmit the disease easily. Sheep have been reported to be protected from clinical disease as early as 3–4 days postvaccination [42] with a lesser dose of vaccine than cattle [58].

Although it is difficult to compare the results between the experiments conducted, owing to variation in the dose of vac-cine, virus serotype and challenge methods, it is still clear that a high-antigen payload vaccine could clinically protect all three major susceptible species. However, clinical protection depends upon the length of immunization and the duration of exposure/challenge methods [43]. Recent experiments with high-antigen payload emergency vaccine showed that cattle immunized for 21 days were protected clinically upon 5-day direct contact chal-lenge by infected cattle; whereas, some of the cattle immunized for 10 days were not protected [59–61]. Results of four cattle vac-cine–challenge experiments, in terms of protection and preva-lence of carrier status, varied from one to another based on the dose of the vaccine and the length of immunization (Table 1). Similarly, we showed that pigs vaccinated for 10 days with the same high-potency O

1 Manisa vaccine were not protected upon

9-h direct contact challenge by infected pigs, but on further increase of the immunization period up to 28 days postvaccina-tion, 75% of immunized pigs were protected [50]. Results of these two vaccine–challenge experiments in pigs are summarized in Figures 1 & 2. A recent study, showed that using half a dose of the same O

1 Manisa emergency vaccine, the sheep immunized for 4

and 10 days were clinically protected upon 9-h indirect aerosol challenge by infected pigs (Figure 3) [58]. However, the measurement of the virus quantities in saliva, blood, nasal swabs and probang samples of vaccinated and subsequently challenged animals (cattle, pigs and sheep) clearly revealed that vaccination reduces

virus excretions by several fold in comparison with unvaccinated animals, thereby, ultimately helping in the reduction of disease transmission [50,58–68]. A series of three homologous and eight heterologous challenge experiments showed that high-potency vaccines can induce protection against FMD serotype A viruses upon heterologous challenge [24]. Therefore, the present vaccine not only reduces the amount of virus excretions upon homolo-gous challenge, but also upon heterologous challenge, thereby helping in reduction of disease transmission.

An important aspect of effectiveness of vaccine application is reduction of persistent infection in ruminants. Up to 50% of FMD-recovered ruminants may become persistently infected, irrespective of their vaccination status. However, pigs usually clear FMDV within 3 weeks following infection and do not become carriers [50,69]. Although the mechanism of persistent infection is not clear yet, there is experimental evidence (Table 1) that increasing the vaccine dose may result in reduction of virus excretion and also the carrier status in vaccinated cattle [60,70]. Similar observations have also been recorded by the author, dur-ing many FMD vaccine potency tests conducted at Pirbright, UK following the European Pharmacopeia [15] and keeping ani-mals more than 28 days post-challenge, showing that the prob-ability of animals becoming carriers is higher in groups receiving a lower (one quarter and one sixteenth) vaccine dose [13,71].

A recent study involving immunization of sheep with FMD vaccine resulted in reduction in the number of carrier animals in comparison to unvaccinated sheep [58]. Furthermore, increasing the immunization period from 4 to 10 days in sheep resulted in less replication of virus, which was evident from the antibody level against nonstructural proteins (NSPs) following infec-tion [58]. The detailed virological and clinical results of the sheep experiment are summarized in Figure 3.

Another aspect of effective vaccination is the reduction of trans-mission of FMDV from vaccinated and subsequently infected animals to naive or other vaccinated animals by direct or indirect contact that may reduce the intra-herd and inter-herd spread of FMDV during field outbreaks. Researchers were able to demon-strate that 14 days vaccinated challenged animals were only able to transmit subclinical infection to naive cattle, without any signs of clinical lesions upon direct contact, whereas 4–7 days vaccinated animals could transmit the clinical disease to naive cattle upon direct contact [47]. Recently, we have demonstrated that 10 days vaccinated challenged cattle could transmit only subclinical infec-tion to vaccinated cattle, even when they were not in contact and separated a few meters apart [59]. In an experiment, four calves were vaccinated for 2 weeks and then two of them were inoculated intranasally with O/NET/2001 FMDV in a separate room. After 20 h of inoculation, when they mingled together, these inoculated cattle were able to transmit the virus to one of the two vaccinated non-inoculated animals [67].

Transmission studies in pigs indicated that vaccination would be beneficial to reduce the transmission of FMDV even within 1 week postvaccination [62–64,72]. Studies in vaccinated dairy cattle and sheep also revealed significant reduction in the transmis-sion of FMDV following vaccination [66,68]. Similar observations


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at Pirbright revealed that 7-day-vaccinated pigs upon challenge could not transmit the disease to naive in-contact pigs; whereas, 4-day-vaccinated challenged pigs could transmit disease to in-contact susceptible pigs [41]. Transmission of virus from 7-day- vaccinated challenged pigs to 7-day-vaccinated sentinel pigs has been recorded, but no transmission was demonstrated by doubling the vaccination period [62].

It has been observed that 3–10-day Asia 1-vaccinated, chal-lenged sheep could transmit the disease to in-contact sheep; whereas, C1 Oberbayern-vaccinated challenged sheep could not transmit the disease [42,73]. Recently, we have demonstrated that neither 4- nor 10-day O

1 Manisa-vaccinated challenged (by pig

aerosol challenge) sheep could transmit clinical disease to 4- and 10-day-vaccinated sentinel sheep, respectively, but could cause subclinical infection in a couple of vaccinated sentinel sheep in both groups (Figure 3) [58]. The variations of transmission in these experiments may be due to different serotype vaccines, vaccine doses, periods of vaccination and challenge methods. However, from the studies, it is clear that, although the vac-cine greatly reduces the virus transmission, all FMD susceptible animals should be vaccinated in the vaccination zone during the emergency vaccination strategies, and these vaccinated animals should be separated from the animals of the unvaccinated zone. Furthermore, studies have been carried out to determine whether vaccinated FMDV carrier cattle or sheep are able to transmit virus to either naive or vaccinated in-contact cattle when housed together for several months, but failed [58,65,70,74]. There is anec-dotal evidence available that shows that African carrier buffalo could transmit the disease to cattle, causing field outbreaks [75,76]. Recently, statistical tests have been performed on data from many published studies to find out the possibility of risk of virus transmission by carrier animals to susceptible animals [77]. An estimation of 0.0256 (likelihood-based CI: 0.008–0.059) infections per carrier per month has been estimated by these authors. Furthermore, a decrease in the proportion of carriers at the rate of 0.115 per month has been demonstrated [77].

Immunity induced by FMD virus & FMD vaccineImmune response against FMDV has been related to circulating humoral antibody titer, which is considered to be the most impor-tant factor in conferring protection against FMD [49,78,79]. IgM is the first serum-neutralizing antibody that appears at 3–4 days following infection or vaccination, and peaks in concentration approximately 10–14 days after infection and then declines [80–

82]. IgG is detected at 4–7 days postinfection or postvaccination and becomes the major neutralizing antibody by 2 weeks follow-ing immunization [83]. In both vaccinated and infected animals, the IgG

1 titer has been reported to be higher than IgG

2 [84]. The

major antibody subclasses found in secretions of upper respiratory and GI tracts are initially IgM, followed by IgA and IgG [84]. It is well known that parenterally administered inactivated FMD vaccine in cattle elicit very little or no IgA in mucosal secretions [85], but if the vaccinated or naturally infected animal becomes a carrier of FMDV, oropharyngeal replication of virus acts as a constant stimulus to produce a higher amount of IgA in saliva, Ta

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Review

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Vaccination against foot-and-mouth disease virus

nasal and oropharyngeal secretions [70,86]. The presence of IgA antibody in pharyngeal fluid, 20–60 days post infection, has been described to be produced at the mucosal surface rather than the serum transduction [87]. Although FMDV elicits a rapid humoral response in both naturally infected and vaccinated animals [88], it is slightly faster in natural infection. Protection has been correlated with high level of neutralizing antibody [13,23,24,79] and is reported to be serotype specific [79];+ however, occasional exceptions have been recorded. Animals with similar antibody titers can differ in their resistance to FMDV infection [13], that is, animals with low neutralizing antibody titers can resist challenge with live virus, while animals with acceptable titers of neutralizing antibody can be susceptible to FMDV infection [79]. Vaccination against FMD gives short-term serotype-specific protection [21,89] in comparison with several years in naturally infected animals [69]. Recently, it has been suggested that long-term antibody response detectable after FMDV infection is maintained due to the persistence of non-replicating FMDV antigen (structural proteins) in the fol-licular dendritic cells in the light zone of the germinal center of

the mandibular lymph node [90]. It is worth investigating the duration of presence of FMDV antigen in the follicular dendritic cells of these lymph nodes in vaccinated animals. Using SAT and A

22 Iraq oil–adjuvant vaccines minimum 6-month dura-

bility has been recorded [91,92] in contrast to 1.5 months in A24

oil–adjuvant vaccine [93]. However, oil–adjuvant A

22 Iraq and

O1 Manisa vaccine in sheep maintained neutralizing antibodies

up to 168 days postvaccination [94]. Recently it has been dem-onstrated that commercial emergency vaccines (O

1 Manisa, A

Turkey and Asia 1 Shamir) can provide immunity for at least 6 months in cattle, sheep and pigs [95]. Furthermore, in pigs, the duration of immunity has been demonstrated for at least 7 months [94]. The shorter duration of immunity in the case of vaccinated animals in comparison with naturally infected ani-mals may be due to vaccination with unstable, inactivated and nonreplicative virus particles that might not induce much cell-mediated immunity. Elimination of NSPs during the antigen-purification process of high-potency emergency vaccine may be another cause of lesser induction of cell-mediated immunity in

Figure 1. Comparative clinical and virological results of 10 days foot-and-mouth disease O1 Manisa-vaccinated pigs following direct contact challenge (foot-and-mouth disease virus [FMDV] O UKG 2001). Black color indicates severe clinical signs and gray color indicates mild clinical signs. Red, blue, purple and green colors indicate temperature >39.5°C, detection of viral RNA by real-time RT-PCR, detection of live virus by bovine thyroid cell culture and nonstructural seroconversion by Bommeli, Cedi and UBI NSP antibody tests, respectively. *: Samples not collected; -: Negative; Cont.: Controls; dpc: Days postchallenge; ns: No clinical signs; NSP: Nonstructural protein; RT: Reverse transcriptase. Reproduced from [49].


Review

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Parida

vaccinated animals. Many T-cell epitopes have been identified in NSPs of FMDV [96–99]. T-cell epitopes derived from the NSP 3A possess the capacity to induce Th activity and allow cooperative induction of anti-FMDV antibodies by B cells.

Identification and characterization of T-cell epitopes are important for understanding protection mediated by cell-me-diated immunity (i.e., responses of CD8+ and CD4+ T lympho-cytes). Th cells have been reported to recognize a number of viral epitopes, located both in capsids and NSPs [83,97]. Thus, it appears that T-cell responses mediated by CD4+ cells are required for protective immunity against FMDV by participating in the production of antiviral antibodies [100–104]. Subsets of CD4+ class II MHC-restricted T cells respond to activation by antigen-presenting cells and antigen by producing Th1 response (IFN-γ) and Th2 (IL-4, IL-5 and IL-13) response [82]. Previously, it has also been suggested that cell-mediated immunity is involved in the clearance of virus from persistently infected animals [105]. This is further supported by recent findings that there is a posi-tive correlation between IFN-γ response and FMDV persistence in cattle on the day of challenge [13]. Several publications dem-onstrate the presence of FMD-specific MHC class II-restricted responses in cattle and pigs [96,99,106–108]. Recently, an ex vivo IFN-γ assay has been developed to measure the quantity of IFN-γ in whole blood of FMDV-vaccinated and -infected cattle after restimulation with inactivated vaccine antigen [71]. Using this assay, a positive correlation between IFN-γ production and vaccine-induced clinical protection has been demonstrated in cattle on the day of challenge [13]. Furthermore, it has been dem-onstrated that CD4+ cells are the main source of IFN-γ produc-tion [13]. IFN-γ has antiviral activity against FMDV [109–111] and

also promotes natural killer and macrophage activation, which are likely to contribute to controlling FMDV replication and spread within the host [110]. γδ T cells are known to produce both Th1 and Th2 cytokines [112]. By showing the stimulation of purified naive pig γδ T cells and the induction of mRNA of various cytokines and chemokines by FMDV vaccine antigen, these authors suggested that γδ T cells probably contribute to the early phase of immune response to vaccination and infec-tion. Although CD8+ T-cell-mediated responses to FMDV have been reported [96,102,113], the role of CD8+ responses in terms of protective immunity is not clear [82]. Recently developed IFN-γ CD8+ ELISPOT assay detected antigen-specific MHC-restricted CD8+ T-cell response following vaccination and authors sug-gested that during FMDV infection, secretion of IFN-γ may be important to control the virus [114]. One interesting feature of the bovine T-cell response to FMDV vaccination and infec-tion is the crossreactivity between the viruses of different sero-type [103,115–117]. This is further supported by the fact that there is a reasonable amount of crossreactivity in IFN-γ production when blood from serotype A-vaccinated animals was restimu-lated with serotype O antigen [71]. This crossreactivity might have been attributed to the conserved epitopes within structural proteins of different serotype viruses [21].

Alternatives to conventional FMD vaccinesAlthough conventional FMD vaccines can prevent clinical infec-tion, they are incapable of inducing sterile immunity and may allow viral replication in the epithelial surface, giving rise to a carrier state in vaccinated animals following live virus chal-lenge [50,58–61,70,118–120]. Conventional FMD vaccine production

Figure 2. Comparative clinical and virological results of 28 days foot-and-mouth disease O1 Manisa-vaccinated pigs following direct contact challenge (FMDV, O UKG 2001). Black color indicates severe clinical signs and gray color indicates mild clinical signs. Red, blue, purple and green colors indicate temperature >39.5°C, detection of viral RNA by real-time RT-PCR, detection of live virus by bovine thyroid cell culture and nonstructural seroconversion by Bommeli, Cedi and UBI NSP antibody tests, respectively. *: Samples not collected; -: Negative; cont.: Controls; dpv: Days postvaccination; ns: No clinical signs; NSP: Nonstructural protein; RT: Reverse transcriptase. Reproduced from [49].


Review

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requires high containment facilities and the potential for escape of live virus from these facilities has led some FMD-free countries to prohibit vaccine manufacture. Moreover, it is very difficult to assess the potency of conventional vaccines, always requiring animal experiments to ensure required potency and also for vaccine reg-istration and batch control. Most vaccine preparations are concentrated cell-culture supernatants from FMDV-infected cells, containing variable amounts of viral NSPs, which make it difficult to identify infec-tion in vaccinated populations by use of NSP-antibody tests.

Alternative approaches to conventional vaccines, such as subunit vaccines based on highly immunogenic FMDV proteins, peptides or DNA vaccines, have been studied extensively in the past [121–128]. Recently, FMDV VP1 protein [129], the IgG-based chimeric protein [130], FMDV immunodominant epitopes [131], FMDV proteins/peptides expressed in plants [132–

134] and T-cell epitopes [96,135] were used for immunization of different susceptible animals with limited success. Synthetic consensus peptide, comprising the G–H loop of FMDV type O VP1 and non-VP1 Th epitope induced an antibody response in cattle, although it failed to confer pro-tection against FMDV challenge [136]. Experiments involving DNA vaccine for FMD resulted either in partial protection or failure [137–144]. DNA vaccine alone or modulated by coadministration of stimulatory molecules, such as cytokine constructs, induced enhanced immune responses [145–154]. To overcome the low level and slow development of humoral antibody response often observed with DNA vaccines recently, a prime–boost strategy has been followed, where FMD DNA vaccine P1-2A3C3D and pGM-CSF-primed pigs were boosted with inac-tivated FMDV antigen, along with recom-binant 3D protein [155]. As a result, a much higher anti-FMD antibody response was observed. All these above strategies presented limited subsets of viral immunogens [88] and do not necessarily express the full spectrum of neutralizing epitopes on the virion [156]. Therefore, attempts have been made to produce attenuated FMD vaccines by adaptation and further passage of FMDV in nonsusceptible hosts, but have been unsuccessful due to the reversion of the attenuated viruses to virulent forms [157]. Targeted deletion of the

L pro gene, which is not essential for virus replication, produced a vaccine that induced a good FMD-specific neutralizing antibody response, but could not provide complete protection [158].

Another approach has been to produce an empty capsid vaccine that contains all of the immunogenic sites present on the intact virus but lacks the infectious nucleic acids [159–165]. Using this strategy, scientists at Plum Island (NY, USA) expressed a FMDV

Figure 3. Comparative clinical and virological results of 10-day O1 Manisa-vaccinated, 4-day O1 Manisa-vaccinated and unvaccinated control sheep following indirect aerosol challenge by pig (foot-and-mouth disease virus [FMDV], O UKG 2001). Red, black, blue and purple colors indicate vesicular lesions, temperature >39.5°C, detection of viral RNA by real-time RT-PCR/live virus by bovine thyroid cell culture and NSP antibody seroconversion by Bommeli, Cedi and UBI NS tests, respectively. ‘v’ indicates vaccinated animals and ‘s’ indicates sentinels. -: Negative; dpc: Days postchallenge; NSP: Nonstructural protein; RT: Reverse transcriptase. Reproduced from [57].


Review Parida

subunit vaccine in a live replication-defective human adenovirus AdV5 vector [166–170]. A single parenteral inoculation of this vac-cine in pigs induced serum antibodies within 7 days and protected pigs against FMD [158]. AdV vaccines have not yet been evaluated in vivo on a large scale, require large doses (5 × 109) of virus and, since AdV infection is widespread in cattle and pigs, their efficacy may be limited by pre-existing, crossreactive immunity and needs to be validated in the field. Recently, FMDV capsid pentamers expressed in a Baculovirus expression system were demonstrated to be immunogenic in mice [171]. More recently, FMDV capsid pro-teins were expressed in a silkworm–Baculovirus expression system that achieved the potency of 6.34 PD

50 per dose in cattle [172].

Protective immunity in response to live vaccines has been found to be more efficient than that of inactivated vaccines [173]. This can be explained as the requirement for a broad spectrum of immune responses for the achievement of efficient protec-tion [174]. This strategy is used in viral vector-based vaccines by expressing antigen as a replicative form of the genome encoded in them. Adenovirus [176], vaccinia virus [175–177] and pseudorabies virus [178–180] vectors expressing FMDV proteins were extensively tested as vaccine candidates for FMD. Mice boosted once with recombinant vaccinia FMD capsid precursor protein (P1) virus were protected against homologous challenge [176]. Similarly, recombinant fowl pox FMD P1+3C virus demonstrated protec-tion against viral challenge in three out of four guinea pigs [177]. Recently, it has been demonstrated that pseudorabies virus, expressing FMD P1 and a multiepitope peptide, protects pigs from FMD infection [178].

Work in pigs has demonstrated that full protection against FMD can be achieved following vaccination with chimeric FMDV vac-cines, in which the VP1 G–H loop had been substituted with that from another serotype [181]. As an alternative to adapt field isolates for vaccine purposes, construction of recombinant FMDVs for pro-duction of inactivated virus vaccines could be advantageous. The antigenicity of these viruses can be manipulated by substituting the antigenic coding regions (the structural proteins) in a cDNA clone of a suitable strain [128,174,181–183]. Quick substitution of the capsid sequence of a cDNA clone with that of a foreign strain/serotype (suitable match with field isolates) using modern technologies may help to prepare the vaccine strain and the subsequent production of a potent and efficacious inactivated vaccine.

Antiviral approaches using RNA interference have been attempted by many researchers, in vitro [184–189] and in vivo [190,191], and have shown successful inhibition of viral replica-tion. However, it has not yet been evaluated in vivo in target animals. The pyrazinecarboxamide derivative T-1105 is a viral RNA-dependent RNA polymerase inhibitor and this compound has been used as an antiviral agent in FMDV-inoculated pigs, which prevented generalization of the disease [192]. Similarly, oligodeoxynucleotides (ODNs) containing unmethylated CpG reduced viremia and disease in FMDV-inoculated Balb/c mice [193]. Recently, anti viral activity of the ribonucleoside analog 2´-C-MetCyt has been demonstrated in vitro against FMDV and, therefore, has the potential to be used in vivo to reduce the disease [194].

Type I α/β interferons play a key role in the innate control of virus replication. Virus-infected cells are induced to express and secrete IFN-α/β, which bind to the receptors on neighboring cells, inducing resistance to virus infection. FMDV is highly sen-sitive to IFN-α/β, suggesting that they may be useful in vivo to protect against FMDV. Parenteral inoculation of pigs with AdV-expressing porcine IFN-α was able to protect pigs from FMDV viremia [158]. In addition to providing a first line of defense against virus infection, IFN-α/β have been demonstrated to potently enhance immune responses, at least in mice [195].

Future FMD control strategies in FMD-free & FMD-endemic countriesFor efficient control of FMD, vaccination and restriction of the movement of infected animals and animal products are crucial. In endemic situations, prophylactic vaccination every 6 months or more depending on the local epidemiological situation, and in FMD-free countries, emergency vaccination (vaccinate-to-live) along with culling of infected animals, may be a suitable option to control FMD. Emergency ring vaccination may increase the likeli-hood of circulating excreted virus in comparison with emergency blanket vaccination, where the animals across an entire region or epidemic focus should be vaccinated [196–198]. However, culling of all infected and susceptible animals may reduce the time needed to control FMD compared with blanket emergency vaccination, but it is not economical or ethical. This emergency blanket vaccina-tion may be a control tool in semi-endemic regions, while culling and emergency ring vaccination may be suitable to control the disease in FMD-free countries. ‘Vaccination-to-cull’ is another option to control disease in FMD-free areas where all the vac-cinated animals need to be culled after the outbreak has ceased, but this option is neither economical nor ethical in comparison with the ‘vaccinate-to-live’ policy. Historically, different susceptible species are treated in different ways to control FMD. Cattle are more susceptible to aerosols and should be vaccinated with single or multiple administration, as per requirement in free or endemic areas. As pigs excrete large amounts of virus in aerosol, they should be vaccinated to control the spread of virus and then culled quickly. Sheep do not produce large quantities of viruses in aerosol and do not transmit the disease easily. Therefore, in FMD-free countries, culling of sheep is usually carried out on suspected farms without vaccination; whereas, in some endemic countries, vaccination of sheep is a usual strategy. According to the author, vaccination of all the susceptible animals would be beneficial for the FMD-control policy. However, consideration of availability of vaccine doses for all susceptible animals should be taken into account. Therefore, priority of vaccination may be considered for highly susceptible animals. Water buffaloes and susceptible wildlife in national zoos may need to be incorporated in the vaccination program.

In the case of vaccination in an outbreak situation, it is necessary to conduct serosurveillance to prove there is no further virus circula-tion. The 2001 outbreak in the UK showed that strict movement control combined with the stamping out of infected and in-contact animals are not always sufficient to eradicate FMD quickly, have high economic costs and cause great public alarm. In future FMD



outbreaks, countries may adopt a ‘vaccinate-to-live’ control policy, after which only vaccinated herds that are carrying FMDV need to be identified for removal using postoutbreak serosurveillance. Detection of serum antibodies against FMDV NSPs in FMD-vaccinated and, subsequently, infected animals usually indicates infection (Figures 1, 2 & 3), since vaccination alone mainly elicits anti-bodies against the FMDV structural proteins. However, the pres-ently available NSP antibody tests are not able to detect absolute infection (Figures 1, 2 & 3) in the vaccinated population [70,120,199]. A suggestion was made to use more than one NSP assay to differenti-ate infection in vaccinated population to increase the sensitivity and specificity of the NSP tests [199,200]. Alternatively, the confidence of detection of infection may be increased by using multiple antigens in ELISA [201,202]. However, in endemic countries, owing to application of vaccines that are not purified from NSP and also because of mul-tiple vaccinations, NSP tests may detect many seropositive animals against NSP antibodies that are not all FMDV carriers. Testing of saliva for FMDV-specific IgA (Table 1) [70,86] is also a promising tool for the detection of carrier status in ruminants, when conventional vaccines are administered by the parenteral route [4]. The application of a salivary IgA test in serosurveillance may avoid the difficulty of using impure vaccines in FMD-endemic countries and, thus, avoid detection of false NSP seropositive animals. Furthermore, IgA anti-body in saliva indicates the presence of FMDV in the oro-pharynx of ruminants (carrier); whereas, NSP antibody in serum indicates either past infection or presence of FMDV in the oro-pharynx. For example, in total, 32 FMDV-carrier cattle were detected by RT-PCR and virus isolation in four vaccine–challenge experiments, but a NSP antibody test detected 46 seropositive animals (Table 1). However, only 30 RT-PCR/virus isolation carrier animals were detected by NSP test and the remaining 16 animals are detected due to past infection, but not due to the status of FMDV carrier. By contrast, 29 out of 32 RT-PCR/virus isolation carrier cattle were detected by salivary IgA test (Table 1).

Other major problems of conventional vaccines are: instability of antigen outside the range of 2–8°C [11], short-lived immunity that persists for approximately 6 months [91,92,94,95] and inabil-ity to stimulate high cell-mediated immunity. The stability of the vaccines could be increased by using a potential stabilizer along with the vaccine or by increasing the physical and chemi-cal stability of the antigen by mutating the unstable amino acids in the capsid proteins. Using reverse genetics techniques, the capsid sequence of an FMDV infectious clone can be rapidly substituted with a stabilized/thermostable version from other strains/serotypes, depending on the circulating field isolates, which may help to formulate a robust and efficacious vaccine. Recently, a FMDV vaccine has been engineered with increased thermostability by site-directed mutagenesis [203]. As the con-ventional FMD vaccines contain inactivated and nonreplicative virus particles, they do not stimulate much cell-mediated immu-nity. This non-replicative nature of vaccine antigen may impair expression of NSPs, limit MHC class I presentation and may also limit the spread and distribution of FMDV antigen in the animal body. Purified FMD vaccines lose NSPs during the purification process of vaccine production. This may be another reason why

T-cell-mediated epitopes present in the NSPs may not be available for the stimulation of cell-mediated antibodies in vaccinated ani-mals, in contrast to naturally infected animals. Therefore, alter-native methods need to be developed to provide FMDV T-cell epitopes of nonstructural and structural proteins extraneously to the present vaccines. Furthermore, the oil adjuvant in the current vaccines favors induction of humoral immunity with little or no stimulation of cell-mediated immunity, while aluminum hydrox-ide favors Th2-type immune responses, enhancing only humoral immune responses [204]. For this reason oil or alum are not opti-mal choices as adjuvants for vaccines against pathogens, as both Th1 and Th2 responses are required for protection. Therefore, there is an urgent need for the selection/trial of novel alternative or additional adjuvants. Many new adjuvants have recently been identified as potent immune enhancers, including ligands for Toll-like receptors (TLRs) [205–207], and their incorporation may increase both cellular and humoral immune responses, which may help to further increase the duration of immunity. Recently a consortium consisting of members from the Institute for Animal Health (UK), the joint US department of Agriculture and US Department of Homeland Security at Plum Island (USA), The Australian Animal Health Laboratory (Geelong, Australia), The National Centre for Foreign Animal Diseases and the International Live Stock Research Institute (Kenya), known as the Global FMD Research Alliance (GFRA), has been formed [11]. The GFRA recognizes the different problems associated with controlling FMD globally and is moving forward, with the main goals to develop new-generation efficacious vaccines, diagnostics and antiviral agents for the future control of FMD.

Expert commentaryVaccination for FMD has been proven to be an efficient method of disease control. Although current FMD vaccines can protect animals from clinical infection, there remains the possibility of viral replication in the oro-pharynx of subclinically infected ani-mals, leading to the carrier status in ruminants. To date, there is no experimental evidence of virus transmission from carrier animals to naive or vaccinated sentinel animals, but there is anecdotal evidence available that shows that African carrier buffalo could transmit disease to cattle, causing field outbreaks. Therefore, the epidemiological threat of virus transmission from carrier animals should not be ignored. FMD vaccines work as marker vaccines when purified from NSPs. However, the vaccines used in endemic countries are not completely purified from NSPs. Similarly, cur-rently available differentiating infection in vaccinated animals (DIVA) tests are not 100% efficient in detecting infection in vac-cinated populations. Therefore, as in other diseases, development of an improved marker vaccine along with robust companion test may help to control FMD. More fundamental immunological research is needed on host–pathogen interactions in order to understand the mechanism of protection. There is also a need for research on novel adjuvants for FMD vaccines that could provide high enough Th1 and Th2 responses, thereby making the vac-cine more efficacious. Stabilization of FMDV capsid proteins in vaccine antigen may help to induce stronger and longer immune


Review Parida

response, which may further help to control FMD. As antigenic diversity of FMDV is a major concern for FMD control, vac-cine matching and selection are important areas that need to be assessed for easy and rapid selection of vaccine strains in an out-break situation. Finally, insufficient vaccine doses to cover all the susceptible animals, particularly in endemic countries, is a major problem for FMD control by vaccination. To overcome this, fur-ther planning and support from the national and international agencies are essential.

Five-year viewStabilization of capsid antigen by genetic engineering may avoid the requirement of cold chains in the field, and also prevent the dissociation of capsid proteins in vivo, producing a faster and stronger immune response. By using reverse genetics techniques and substituting stabilized/thermostable versions of casid pro-teins, a new generation of efficacious vaccines may be developed. The addition of new-generation Toll-like receptors as adjuvant may increase both Th1 and Th2 responses, which are impor-tant for protection. Supplement of additional T-cell epitopes of FMDV structural proteins and NSPs may help to increase the cell-mediated response against FMDV. New methods of vac-cine selection using capsid sequence data may allow rapid selec-tion of the appropriate vaccine strain. Quick substitution of the

capsid sequence of a cDNA clone with that of a foreign strain/serotype (suitable match with field isolates) using modern tech-nologies may help to prepare the appropriate vaccine strain and the subsequent production of a potent and efficacious vaccine. As a positive correlation of vaccine-induced protection has been observed with VNT and IFN-γ responses on the day of challenge, a mathematical model using data from in vitro assays (VNT and IFN-γ) may replace the in vivo potency test, but further work is needed to establish the model. Empty capsid-based marker vac-cines and a robust DIVA test that could detect absolute infection in vaccinated populations may increase the efficiency of the FMD control policy.

Financial & competing interests disclosureSatya Parida is funded by Defra, UK (Project SE1122 and SE1125). Parida is a WP (4.3) member of EU Network of Excellence, EPIZONE (contact number Foot-CT-2006-016236) and adjunct professor to Murdoch University, Australia. Thanks are due to Mana Mahapatra for her careful reading and Aravindh Babu for his help in end note preparation. The author has no other relevant affiliations or financial involvement with any organiza-tion or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Key issues

Although conventional vaccines can prevent clinical infection, they do not induce sterile immunity. The protection lasts approximately • 6 months, often requiring frequent revaccination in the prophylactic control program and does not induce rapid protection against challenge. Furthermore, it may allow viral replication in the epithelial surface, giving rise to the carrier state in vaccinated animals following live virus challenge.

Although vaccination could not provide complete clinical or virological protection in animals immunized for a shorter period, it reduces • the severity of the disease, virus excretion and virus replication. Inoculation route and dose of challenge virus may play a major role in vaccine-induced protection.

Enhanced stability of the capsid antigen may give a stronger immune response and durable immunity.•

High antigenic variation in foot and mouth disease viurs (FMDV) causes a major problem in vaccine strain selection to control • the disease.

Comparison of sequence data generated from FMDV capsids may help in the rapid selection of a vaccine strain for better protection.•

In vitro• assays may replace in vivo potency tests, which may reduce the cost of animal experiments, maintenance of expensive biocontainment facilities and possibility of virus escape.

New-generation viral vector-based marker vaccines expressing stabilized capsid and robust tests to differentiate infection in vaccinated • animals may increase the efficiency of vaccination in foot and mouth disease (FMD) control policy.

In endemic countries, sufficient doses of foor and FMD vaccine should be made available to vaccinate susceptible animals. Water • buffaloes in South-East Asia, susceptible wildlife in Africa, particularly in national zoos, and susceptible zoo animals in FMD-free countries may need regular vaccination to control the disease.

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Website

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(SENASA). In: Boletin Official No. 30.940-945/7/06 (2006). Act number 351/2006 http://infoleg.mecon.gov.ar/infoleg Internet/anexos/11500-119999/117636/norma.htm

AffiliationSatya Parida •Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Surrey, GU24 0NF, UK Tel.: +44 148 323 2441 Fax: +44 148 323 2448 [email protected]

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Vaccination against foot-and-mouth disease virus: strategies and effectiveness

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