key: cord-259299-z3o4t7mz authors: Chinsangaram, Jarasvech; Mason, Peter W.; Grubman, Marvin J. title: Protection of swine by live and inactivated vaccines prepared from a leader proteinase-deficient serotype A12 foot-and-mouth disease virus date: 1998-10-31 journal: Vaccine DOI: 10.1016/s0264-410x(98)00029-2 sha: doc_id: 259299 cord_uid: z3o4t7mz Abstract Previously, we demonstrated that a genetically engineered variant of foot-and-mouth disease virus (FMDV) serotype A12 lacking the leader proteinase-coding region (A12-LLV2) was attenuated and induced an immune response that partially protected cattle from FMD. In this study, A12-LLV2 was tested in swine as a live or chemically inactivated vaccine. Animals vaccinated with chemically inactivated A12-LLV2 or wild-type (WT) virus in oil adjuvant developed high levels of neutralizing antibodies and were protected from FMD upon challenge. Animals vaccinated with live A12-LLV2 did not exhibit signs of FMD, did not spread virus to other animals, developed a neutralizing antibody response and antibodies to nonstructural protein 3D, and were partially protected from FMD. Animals given a similar dose of chemically inactivated A12-LLV2 in the absence of adjuvant developed a poor immune response and were not protected from FMD, indicating that limited replication was responsible for the improved immune response found in animals vaccinated with live A12-LLV2. The results demonstrate the potential of A12-LLV2 as a live-attenuated vaccine as well as a safe source of antigen for chemically inactivated vaccines. Foot-and-mouth disease (FMD) is a debilitating disease of cloven-hoofed animals including swine and cattle. The disease is highly contagious and characterized by fever, vesicular lesions (mainly on the feet, but also on the mouth, nose, and sometimes teats), as well as abortions, and neonatal deaths. Infected animals rapidly spread the virus via aerosol to others, resulting in a dramatic loss of production by the herd. FMD outbreaks are so devastating that countries which do not have the disease restrict the importation of animals and animal products from countries where the disease is present. When FMD is found, infected and exposed animals are slaughtered and ring vaccination surrounding the affected area may be implemented'.'. Vaccines prepared by chemical inactivation of FMDV have been used to successfully control FMD3. However, there is an association of disease outbreaks with incomplete chemical inactivation and escape of USDA virus from vaccine manufacturing facilities4.'. Furthermore, this conventional vaccine induces relatively short-lived immunity and some vaccinated animals can develop a carrier state following contact with FMDV6.'. Over the last 20 yr, a number of alternative approaches in FMD vaccine development have been examined. One of the approaches we have examined is the liveattenuated vaccine. A vaccine based on stably attenuated FMDV could provide improved safety over the existing product?. Moreover, work on other viral diseases has shown that live-attenuated vaccines can induce longer lasting immunity, present a greater antigenic mass, as a result of limited replication, than inactivated vaccine, and if delivered by the natural route can induce mucosal immunity'. FMDV is an Aphthovirus in the Family Picomaviridae'". The single-stranded positive-sense RNA genome is surrounded by an icosahedral capsid composed of four structural proteins, VPl, VP2, VP3 and VP4. The RNA genome contains a long translational open reading frame that codes for a polyprotein which is processed by virally encoded enzymes into mature structural and nonstructural proteins. The FMDV leader (L) protein, a papain-like proteinase"-'4, is associated with viral virulence8,'5.'6. In infected cells, the L proteinase autocatalytically cleaves itself from the Proteinase-deficient FMD vaccines: J. Chinsangaram et al. were immunoprecipitated with 14 and 56 dpv serum from swine vaccinated with; lanes 3-8, ; lanes 9-14, BEI-inactivated A12-IC (no. [23] [24] [25] ; lanes 15-18, control swine (no. 26 and 27) . Lane 1, immunoprecipitation with bovine convalescent serum. Lane 2, immunoprecipitation with 0 dpv serum from swine no. 20. The products were examined by SDS-PAGE on a 15% gel. viral polyprotein'7 and shuts off cap-dependent host protein synthesis by cleaving eIF-4G, a component of the cap-binding complex, eIF-4F'8-20. As a result, FMDV, which initiates translation by a cap-independent mechanism, does not need to compete with host mRNAs for use of the cellular translation machinery. As a part of our program to identify attenuated vaccine candidates, we have genetically altered the genome of FMDV seroXpe Al2 by deleting the L proteinase coding region . In contrast to attenuated viruses selected by serial passage which are genetically unstable', deletion of the L protein coding region makes it highly unlikely that the leader proteinasedeficient virus (A12-LLV2) will revert to virulence. Our previous studies have shown that A12-LLV2 replicates more slowly than parental virus (A12-IC) in baby hamster kidney (BHK) cellsn and is attenuated in cattle8'16. After aerosol inoculation, A12-LLV2 was only found localized in the respiratory bronchioles and did not produce clinical signs in cattle, in contrast to A12-IC which caused classical FMD including fever and vesicular lesions"'. Furthermore, we have demonstrated that a single subcutaneous vaccination with live A12-LLV2 induced a partially protective immune response in cattle against a severe challenge by intradermal inoculation in the tongue with virulent cattlepassaged FMDV'. It has been shown that cattle and swine do not necessarily res ond to FMD vaccines or infection in a similar fashior$e'3. Differences in disease susceptibility of cattle and swine has also been suggested by a recent FMD outbreak in Taiwan where the swine population was devastated, but no significant disease in cattle was reportedz3. In the present study, the safety and efficacy of live or inactivated vaccine preparations of A12-LLV2 were tested in swine. Proteinase-deficient FMD vaccines: J. Chinsangaram et al. Baby hamster kidney (BHK) cells (strain 21, clone 13) were used to propagate virus stocks and for plaque reduction neutralization (PRN) assays. Secondary lamb kidney (LK) cells (provided by Dr C. House, Foreign Animal Disease Diagnostic Laboratory, USDA, Plum Island, USA) were used to produce antigen for radioimmunoprecipitation (RIP). Parental wild-type (WT) FMDV Al2 119 (A12-IC), A12-LLV2, and a virulent cattle-passa ed g strain of FMDV Al2 were described previously". 4-26. The A12-IC and A12-LLV2 viruses were purified by sucrose-gradient centrifugation and chemically inactivated by treatment with binary ethylenimine (BEI) as previously described"'. Sixteen 20-30 kg Yorkshire cross or Yorkshire/ bluepoint gilts were divided into four vaccine groups of three animals each and two groups of two control animals. These animals were housed in two separate rooms. The first room contained one group of animals vaccinated subcutaneously with 2 pg of live A12-LLV2 (lo7 p.f.u.) and two control animals. The second room contained three groups of animals and the remaining two control animals. Each group of animals in the second room was vaccinated either subcutaneously with I I I I I I I I I 0 10 20 30 40 50 60 70 80 days postvaccination 2 pug of BEI-inactivated A12-LLV2 or intramuscularly with 2 pg of BEI-inactivated A12-LLV2 in mineral oil (9:l Marco1 5UMontanide 888) or 2 pg BEI-inactivated A12-IC in the above adjuvant. At 56 days postvaccination (dpv), all animals were combined into a single room and challenged with live FMDV by inoculating a single control animal intradermally in the snout and by a combination of intradermal (coronary band and heel bulb) and skin scarification (coronary band) on one foot, with a total of 5 x lo4 infectious units of virulent cattle-passaged FMDV type A12. Rectal temperature data and clinical signs, including lameness and vesicular lesions were recorded daily. Temperature of over 40°C for two or more consecutive days was considered to constitute a fever. At the time of necropsy, the number of digits affected by the disease were recorded as a score for each animal (see Table I ). Serum samples were collected every week and used for PRN and RIP assays. RIP was performed using cytoplasmic extracts from [35S] methionine-labeled FMDV-infected LK cells as antigen15. Serum samples were screened for antibodies to FMDV structural and nonstructural proteins at 1:40 dilution by RIP and the precipitated products were analyzed by SDS-PAGE on 15% gels. Serum samples were serially diluted and assayed for neutralizing antibodies by PRN in BHK cells". Neutralization titers were reported as the log of the serum dilution yielding a 70% reduction in p.f.u. (PRN,"). Intramuscular vaccination of BEI-inactivated virus in adjuvant (both induced high neutralizing antibody titers (Table I) and these animals had antibodies to the viral structural proteins as detected by RIP (Figure 1) . Neutralizing antibody was first detected in these animals at 7 dpv and plateaued at 14-21 dpv (Figure 2 ). Subcutaneous vaccination of BEI-inactivated virus in the absence of adjuvant (no. 17-19) induced a lower neutralizing antibody response (Table 1) the titer appeared to decline slightly over time ( Figure 2 ). This latter group of animals did not develop antibodies to the viral structural proteins that could be detected by RIP (lanes 9-14 in Figure 3 ). As expected all animals Figure 3 Immunoprecipitation of viral proteins with serum from swine vaccinated with live or BEI-inactivated A12-LLV2 in the absence of adjuvant. Cytoplasmic extracts from FMDV-infected LK cells radiolabeled with [35S:Jmethionine were immunoprecipitated with 14 and 56 dpv serum from swine vaccinated with; lanes 3-8, live Al2-LLV2 (no. 14-16); lanes 9-14, BEI-inactivated Al 2-LLV2 in the absence of adjuvant (no. 17-19) ; lanes 15-18, control swine (no. 34 and 35). Lane 1, immunoprecipitation with bovine convalescent serum. Lane 2, immunoprecipitation with 0 dpv serum from swine no. 14. The products were examined by SDS-PAGE on a 15% gel. given inactivated viruses did not produce any detectable antibodies to nonstructural proteins, including 3D (Figures I and 3, Table 1 ). In contrast to animals given inactivated virus preparations, animals vaccinated with live A12-LLV2 ( no. 14-16) developed antibodies to nonstructural protein 3D and the structural proteins at 14 dpv (lanes 3-8 in Figure 3) . The presence of antibodies to a viral nonstructural protein is indicative of virus replication in these animals. However, these animals did not show any clinical signs of FMD including lameness, fever or any apparent lesions on the feet or mouth. In addition, the two control animals (no. 34 and 35) which were housed in the same room with swine no. 14-16 for 56 days did not seroconvert or show any signs of FMD, indicating that there was no spread of virus from A12-LLV2-vaccinated animals (Table 1 , lanes 15-18 in Figure 3 ). Following commingling of all animals in a single room, one control animal (no. 35) was infected with virulent cattle-passaged Al2 virus on the snout and one foot. Vesicular lesions were observed on the foot and snout at 1 and 2 days postchallenge (dpc), respectively, and vesicles developed in the following days on all four feet of this animal, and temperatures over 40°C persisted from two to four and from seven to 10 dpc [ Figure 4(C) ]. All the remaining control animals (no. 26, 27, and 34) and vesicular lesions at 3 dpc; one of these animals (no. 34) also developed a vesicle on the snout. Animals vaccinated subcutaneously with BEI-inactivated A12-LLV2 in the absence of adjuvant ( no. 17-19) were not protected from challenge (Table l) , although all three animals had a delayed appearance of vesicles (4-5 dpc) as compared to control swine (no. 26, 27, and 34) . Animals vaccinated subcutaneously with live A12-LLV2 (no. 14-16) were partially protected from challenge (Table I) . These animals did not develop a fever [ Figure 4 (A)], had a delayed appearance of vesicles (5-6 dpc), and only one animal in this group (no. 14) developed vesicles on all four feet (although with a low score; see Table 1 ). No fevers [ Figure 4 (Table I) were found in any of the animals vaccinated with BEI-inactivated A12-IC (no. [20] [21] [22] or A12-LLV2 (no. [23] [24] [25] in adjuvant for the period of observation (12 days). Sera collected 12 dpc was evaluated to determine the extent of challenge virus replication in the exposed animals. The results revealed a strong boost in neutralizing antibody titers in animals vaccinated subcutaneously with either live or BEI-inactivated A12-LLV2 and no increase in neutralizing titers in animals vaccinated intramuscularly with BEI-inactivated A12-LLV2 or A12-IC (Figure 2, Table 2 ). Moreover, RIP analyses revealed that only the latter two groups of animals failed to develop antibodies to the nonstructural protein 3D following exposure, indicating that replication of the challenge virus was absent or limited in these animals (Figure 5 ). In this study, we have shown that a leader proteinasedeficient derivative of FMDV serotype Al2 can be safely administered to swine, and that vaccinated swine produce neutralizing antibodies and are partially protected from virulent virus challenge. Replication of live A12-LLV2 in swine was confirmed by the detection of antibody to nonstructural protein 3D, and presumably improved the immune response relative to that observed in animals vaccinated with inactivated A12-LLV2 by the same route (subcutaneous vaccination) in the absence of adjuvant. In spite of the evidence of virus replication, vaccination with live A12-LLV2 did not produce signs of disease and A12-LLV2 did not spread to control animals. These results together with our previous results in cattle (see Section 1) demonstrate the safety of A12-LLV2 as a live-attenuated vaccine candidate for livestock. However, an extensive study involving animals of various ages and immune status is required to unequivocally address the safety of this type of vaccine candidate. The severity of disease judged by lesion score and rectal temperature correlated well with the neutralizing antibody titer measured at the day of challenge (Table I) . Animals vaccinated with BEI-inactivated A12-IC or A12-LLV2 in an oil adjuvant developed high titers of neutralizing antibody and were all protected against challenge. Thus, as expected, the deletion of the L proteinase coding region did not alter the antigenic structure of the virion. Animals vacci-1520 Vaccine 1998 Volume 16 Number 16 nated with live A12-LLV2 had lower levels of neutralizing antibody as compared to animals vaccinated with BEI-treated virus in the presence of adjuvant and were partially protected from virulent virus challenge, as determined by the absence of fever, delayed lesion appearance, and low lesion score. Animals vaccinated with BEI-inactivated A12-LLV2 in the absence of adjuvant displayed low or non-detectable neutralizing antibody titers at the day of challenge and although lesions appeared later in these animals than in control animals, by the end of the challenge period, no difference in disease severity was noticed between these two groups. Subcutaneous vaccination of live A12-LLV2 was chosen for this study to allow a comparison with our previous study in cattle*. In addition, this route of inoculation was selected based on a study in cattle indicating that subcutaneous was better than intramuscular vaccination in terms of ability to induce neutralizing antibodies (unpublished observation). Subcutaneous inoculation in cattle induced neutralizing antibody titers (PRN,,,) of 2.0, 2.6 and 2.9 and the animal with the highest titer (2.9) was protected against challenge, the animal with the PRNTO 2.6 developed a fever but no pedal lesions, and the third animal (PRN," 2.0) developed fever and mild lesion?. In the present study, we showed that vaccination of swine by the same route induced similar neutralizing antibody titers, and these swine were partially protected from challenge (animals did not develop fever and displayed a low lesion score). Since the immune response to live A12-LLV2 given by the with 12 dpc serum from swine no. 14-27, 34, and 35, respectively. Lane 1, immunoprecipitation with bovine convalescent serum. Lane 2, immunoprecipitation with Cl dpv serum from swine no. 14. The products were examined by SDS-PAGE on a 15% gel. subcutaneous route was superior to that seen in animals given the same antigenic mass of inactivated A12-LLV2 by the same route, these studies demonstrated that replication of live A12-LLV2 virus in these animals was responsible, in part, for the increased immune response. Problems associated with the conventional FMD vaccine have led us to search for safer vaccine candidates. The development of attenuated FMD vaccines by passage in unnatural hosts to obtain viruses which are both innocuous and yet able to induce a protective immune response has proven difficult6,z8. In addition, viruses which are selected by this procedure can revert to virulence'. We have taken the approach of deleting the entire L coding region of FMDV to produce an attenuated virus suitable for use as a live vaccine. This virus has a deletion of a complete coding region which significantly reduces the risk of reversion to virulence as compared to attenuated viruses produced by conventional means. M.oreover, we have demonstrated that this virus can be used as a source of antigen in traditional BEI-inactivated vaccines; since A12-LLV2 is non-pathogenic and does not spread between animals, its use in vaccine production could reduce the risk associated with current vaccine manufacture. The results of this study, in conjunction with our previous result?, suggest that vaccination of cattle and swine with live A12-LLV2 is feasible. However, the limited replication of this attenuated virus resulted in an increase in antigenic mass which was insufficient to induce a completely protective immune response. Additional studies are currently ongoing to further demonstrate sifety, improve efficacy, and more completely understand the host response to attenuated virus infection. 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