Recombinant human adenovirus-5 expressing capsid proteins of Indian vaccine strains of foot-and-mouth disease virus elicits effective antibody response in cattle.

Recombinant adenovirus-5 vectored foot-and-mouth disease constructs (Ad5- FMD) were made for three Indian vaccine virus serotypes O, A and Asia 1. Constructs co-expressing foot-and- mouth disease virus (FMDV) capsid and viral 3C protease sequences, were evaluated for their ability to induce a neutralizing antibody response in indigenous cattle (Bos indicus). Purified Ad5-FMD viruses were inoculated in cattle as monovalent (5×109 pfu/animal) or trivalent (5×109 pfu/animal per serotype) vaccines. Animals vaccinated with monovalent Ad5-FMD vaccines were boosted 63days later with the same dose. After primary immunization, virus neutralization tests (VNT) showed seroconversion in 83, 67 and 33% of animals vaccinated with Ad5-FMD O, A and Asia 1, respectively. Booster immunization elicited seroconversion in all of the animals (100%) in the monovalent groups. When used in a trivalent form, the Ad5-FMD vaccine induced neutralizing antibodies in only 33, 50 and 16% of animals against serotypes O, A and Asia 1, respectively on primo-vaccination, and titers were significantly lower than when the same vectors were used in monovalent form. Neutralizing antibody titers differed by serotype for both Ad5-FMD monovalent and trivalent vaccines, with Asia 1 serotype inducing the lowest titers. Antibody response to Ad5 vector in immunized cattle was also assessed by VNT. It appeared that the vector immunity did not impact the recall responses to expressed FMDV antigens on booster immunization. In summary, the study suggested that the recombinant Ad5-FMD vaccine has a potential use in monovalent form, while its application in multivalent form is not currently encouraging.

The pathogenesis of foot-and-mouth disease II: viral pathways in swine, small ruminants, and wildlife; myotropism, chronic syndromes, and molecular virus-host interactions.

Investigation into the pathogenesis of foot-and-mouth disease (FMD) has focused on the study of the disease in cattle with less emphasis on pigs, small ruminants and wildlife. 'Atypical' FMD-associated syndromes such as myocarditis, reproductive losses and chronic heat intolerance have also received little attention. Yet, all of these manifestations of FMD are reflections of distinct pathogenesis events. For example, naturally occurring porcinophilic strains and unique virus-host combinations that result in high-mortality outbreaks surely have their basis in molecular-, cellular- and tissue-level interactions between host and virus (i.e. pathogenesis). The goal of this review is to emphasize how the less commonly studied FMD syndromes and host species contribute to the overall understanding of pathogenesis and how extensive in vitro studies have contributed to our understanding of disease processes in live animals.

Evidence of activation and suppression during the early immune response to foot-and-mouth disease virus.

Foot-and-mouth disease virus causes a serious disease of livestock species, threatening free global trade and food security. The disease spreads rapidly between animals, and to ensure a window of opportunity for such spread, the virus has evolved multiple mechanisms to subvert the early immune response. The cycle of infection in the individual animal is very short, infection is initiated, disseminated throughout the body and infectious virus produced in <7 days. Foot-and-mouth disease virus has been shown to disrupt the innate response in vitro and also interacts directly with antigen-presenting cells and their precursors. This interaction results in suboptimal immune function, favouring viral replication and the delayed onset of specific adaptive T-cell responses. Detailed understanding of this cycle is crucial to effectively control disease in livestock populations. Knowledge-based vaccine design would specifically target and induce the immunological mechanisms of early protection and of robust memory induction. Specifically, information on the contribution of cytokines and interferon, innate immune cells as well as humoral and cellular immunity can be employed to design vaccines promoting such responses. Furthermore, understanding of viral escape mechanisms of immunity can be used to create attenuated viruses that could be used to develop novel vaccines and to study viral pathogenesis.

Marked differences between MARC-145 cells and swine alveolar macrophages in IFNß-induced activation of antiviral state against PRRSV.

The activation of antiviral activity induced by recombinant swine interferon beta (rswIFNß) against PRRSV was comparatively examined in MARC-145 cells and porcine alveolar macrophages (PAMs). A dose-response analysis showed, in MARC-145 cells, that isolate Mo25544 was highly sensitive to rswIFNß while a vaccine strain and isolate PDV130-9301 were resistant to different extents. In contrast, all three viruses were equally sensitive to rswIFNß in PAMs even at the lowest dose of IFN utilized in the bioassays. To analyze potential differences in mechanisms of antiviral activation between these cells, treatment with 2-aminopurine (2-AP), an inhibitor of double-stranded RNA-dependent protein kinase (PKR), was performed in rswIFNß-treated cells. Addition of 2-AP to rswIFNß-primed MARC-145 cells restored replication of the Mo25544 isolate, and to some extent that of vaccine virus and PDV130-9301. In contrast, virus replication could not be rescued for any of the three viruses with 2-AP in rswIFNß-treated PAMs. The differences in sensitivity of PRRSV to rswIFNß as well as the effects of 2-AP strongly suggest that MARC-145 cells and PAMs utilize different rswIFNß-associated antiviral pathways. Therefore, studies to understand virus-host cell interactions performed in MARC-145 cells require additional scrutiny when utilized as a host cell model for immunologic responses to PRRSV.

Adenovirus-mediated expression of interferon-alpha delays viral replication and reduces disease signs in swine challenged with porcine reproductive and respiratory syndrome virus.

In this study, pigs were injected with a nonreplicating human adenovirus type 5 vector expressing porcine interferon-alpha (Ad5-pIFN-alpha) and then challenged with porcine reproductive and respiratory syndrome virus (PRRSV) to determine whether the presence of increased levels of IFN-alpha would decrease viral replication and/or disease. Groups of 10 pigs each were inoculated with Ad5-pIFN-alpha and not challenged, Ad5-pIFN-alpha and challenged with PRRSV 1 d later, or inoculated with a control adenovirus that does not express IFN-alpha (Ad5-null) and challenged 1 d later with PRRSV. IFN-alpha levels in all pigs inoculated with the Ad5-pIFN-alpha were elevated the day of challenge (1 d after inoculation), but were undetectable by 3 d after inoculation in the pigs that were not challenged with PRRSV. Pigs inoculated with Ad5-pIFN-alpha and challenged with PRRSV had lower febrile responses, a decreased percentage of lung involvement at 10 d post-infection, delayed viremia and antibody response, and higher serum IFN-alpha levels as a result of PRRSV infection, compared to pigs inoculated with Ad5-null and challenged with PRRSV. These results indicate that IFN-alpha can have protective effects if present during the time of infection with PRRSV.

Recombinant swine beta interferon protects swine alveolar macrophages and MARC-145 cells from infection with Porcine reproductive and respiratory syndrome virus.

Swine beta interferon (swIFN-beta) produced in HEK 293 cells infected with a recombinant, replication-defective human adenovirus 5 (Ad5) encoding the swIFN-beta gene was tested for antiviral activity against Porcine reproductive and respiratory syndrome virus (PRRSV). MARC-145 cells were incubated overnight with dilutions of supernatant fluids from HEK 293 cells infected with Ad5-swIFN-beta or with an Ad5 control virus (Ad5-Blue). Treated cells were infected with PRRSV; MARC-145 cells incubated with Ad5-Blue supernatants developed cytopathic effects (CPE), whereas those incubated with swIFN-beta showed no CPE. To confirm the antiviral activity of swIFN-beta, culture fluids from Ad5-swIFN-beta-infected cells were affinity-purified on a Sepharose-anti-swIFN-beta matrix, and the resulting fractions exhibited antiviral activity upon infection with PRRSV. The antiviral effects were specific, as they were blocked by mAbs against swIFN-beta. Additional cultures of MARC-145 cells treated with swIFN-beta-containing supernatants or affinity-purified swIFN-beta were infected with PRRSV and tested by real-time RT-PCR for viral RNA in culture supernatants at various times post-inoculation. These experiments confirmed the protective effects of swIFN-beta. swIFN-beta was also tested for antiviral activity on porcine alveolar macrophages (PAMs) obtained by bronchoalveolar lavage from PRRSV-negative swine. PAMs were treated with dilutions of swIFN-beta or Ad5-Blue culture fluids, infected with PRRSV and tested for viral RNA by real-time RT-PCR. The viral load data showed a dose-dependent protection in swIFN-beta-treated PAMs, whereas no protection was evident from Ad5-Blue culture fluids. The data demonstrate that swIFN-beta protects both MARC-145 cells and PAMs from PRRSV infection.

Engineering better vaccines for foot-and-mouth disease.

Although efficacious and safe, current vaccines for FMD suffer from drawbacks. Among these are that the immune response to the vaccine interferes with the ability to detect vaccinated animals that have subsequently become infected and could carry and shed the virus, creating an obstacle to re-instating disease-free status to countries/regions that vaccinate to control outbreaks. Multiple diagnostic tests are available to identify animals that have been infected with FMDV by detection of antibodies to viral non-structural proteins (NSP) that are present in low concentration in traditional vaccines and are poorly immunogenic in vaccine preparations. However, these tests are not 100% reliable. To circumvent this problem, we have developed a new generation of vaccines that express the "empty capsid" subunit of the virus, in the absence of one of the most immunogenic NSPs, 3Dpol. Here we describe delivery of the empty capsid subunits by recombinant replication-defective human adenovirus type 5 (Ad5). These Ad5-vectored empty capsid vaccines can protect pigs from FMDV challenge as early as 7 days post-vaccination. A second problem with current FMD vaccines is that they do not induce protective immunity quickly, a drawback that is likely to be shared by our Ad5-vectored empty capsid vaccine. To overcome this problem, we have developed a prophylactic antiviral treatment consisting of an Ad5 encoding porcine interferon alpha (pIFNalpha). Administration of Ad5-pIFNalpha protects swine from FMD as early as one day post-administration. The combination of this antiviral treatment and the empty capsid subunit vaccine should induce rapid and complete protection from FMD, and could overcome current diagnostic problems.

Early protection against homologous challenge after a single dose of replication-defective human adenovirus type 5 expressing capsid proteins of foot-and-mouth disease virus (FMDV) strain A24.

Previously we demonstrated that two doses of a replication-defective human adenovirus serotype 5 (Ad5) carrying the capsid (P1) and 3C protease coding regions of a laboratory strain of FMDV (A12) completely protected five of six swine challenged with homologous virus. The objective of the current study was to evaluate the efficacy of one dose of an Ad5-vectored vaccine expressing the P1 coding region of an FMDV field strain. A replication-defective Ad5 containing the P1 coding region of FMDV A24 and the 3C coding region of A12 (Ad5A24) was constructed and evaluated for its ability to induce neutralizing antibodies and protect swine against homologous challenge after a single vaccination. Animals were challenged 7, 14 or 42 days after vaccination. Control groups included animals inoculated with commercial vaccine or phosphate-buffered saline. All vaccinated swine were completely protected against homologous challenge at 7, 14 or 42 days after vaccination. Based on these results, we conclude that a single inoculation of Ad5-vectored vaccines could be used as a tool to control FMD in outbreak situations.

Prospects, including time-frames, for improved foot and mouth disease vaccines.

Inactivated foot and mouth disease (FMD) vaccines have been used successfully as part of eradication programmes. However, there are a number of concerns with the use of such vaccines and the recent outbreaks of FMD in disease-free countries have increased the need for improved FMD control strategies. To address this requirement, new generation FMD vaccines are being developed. Currently, one of the most promising of these vaccine candidates utilises an empty viral capsid subunit delivered to animals by a live virus vector. This candidate, a replication-defective recombinant human adenovirus containing the capsid and 3C proteinase coding regions of FMD virus (FMDV), induces an FMDV-specific neutralising antibody response in inoculated animals. Upon challenge with a virulent animal-passaged homologous virus, swine and cattle vaccinated with this recombinant adenovirus are protected from clinical signs of FMD as well as from FMDV replication. One inoculation of a high dose of this vaccine candidate protected swine from challenge as early as seven days after vaccination.

pAd5-Blue: direct ligation system for engineering recombinant adenovirus constructs.

We have engineered a new vector that makes use of direct ligation for the generation of replication-defective recombinant adenovirus constructs. In the pAd5-Blue vector, unique yet common restriction endonuclease sites exist, that allow cloning in a directional manner of a gene of interest under control of a cytomegalovirus promoter, upstream of a simian virus 40 polyadenylation signal. The insertion of the new gene replaces the beta-galactosidase alpha gene fragment in the pAd5-Blue vector, allowing the identification of recombinants in bacterial culture by the selection of white colonies. Plasmid DNA from white colonies is digested with PacI and transfected into 293 cells, resulting in the generation of a homogenous population of adenovirus containing the gene of interest. The pAd5-Blue vector system does not rely on recombination either in mammalian or bacterial cells. Furthermore, because of compatible overhangs, the variety of restriction endonucleases that can be used to generate the inserted gene gives flexibility to the process for greater ease of use. The system is quick and straightforward, allowing the generation of recombinant adenoviruses within three weeks of obtaining an appropriate insert. This new vector should greatly enhance the ease and speed with which new recombinant adenovirus constructs can be made.

Inhibition of L-deleted foot-and-mouth disease virus replication by alpha/beta interferon involves double-stranded RNA-dependent protein kinase.

We previously demonstrated that the ability of foot-and-mouth disease virus (FMDV) to form plaques in cell culture is associated with the suppression of alpha/beta interferon (IFN-alpha/beta). In the present study, we used Escherichia coli-expressed porcine and bovine IFN-alpha or -beta individually to demonstrate that each was equally effective in inhibiting FMDV replication. The block in FMDV replication appeared to be at the level of protein translation, suggesting a role for double-stranded RNA-dependent protein kinase (PKR). In support of these findings, treatment of porcine and bovine cells with 2-aminopurine, an inhibitor of PKR, increased the yield of virus 8.8- and 11.2-fold, respectively, compared to that in untreated infected cells. In addition, results of FMDV infection in mouse embryonic fibroblast cells derived from gene knockout mice lacking the gene for RNase L(-/-) or PKR(-/-) or both indicated an important role for PKR in the inhibition of FMDV replication.

Immune responses and protection against foot-and-mouth disease virus (FMDV) challenge in swine vaccinated with adenovirus-FMDV constructs.

A replication-defective adenovirus 5 encoding foot-and-mouth disease virus (FMDV) capsid and 3C proteinase coding regions (Ad5-FMDV3CWT) was used to vaccinate swine. A single inoculation utilizing 1 x 10(8) plaque forming units (pfu) or an inoculation of 1 x 10(8) followed by a boost of 5 x 10(8) pfu Ad5-FMDV3CWT were tested, along with an inoculation and boost using an adenovirus encoding the FMDV capsid coding region and an inactive form of the 3C proteinase (Ad5-FMDV3CMUT). Sera collected from these animals were examined for the presence of FMDV-specific antibodies using immunoprecipitation, neutralization, and ELISA assays specific for IgM, IgG1 and IgG2. Efficacy studies were performed by placing the vaccinated swine in contact with an FMDV-infected swine and monitoring for signs of disease and changes in serum antibody levels. Ad5-FMDV3CMUT, which is unable to produce FMDV capsid structures, did not elicit FMDV-neutralizing antibodies or protect against FMD. Single inoculation with Ad5-FMDV3CWT generated FMDV-specific neutralizing antibodies, and reduced clinical signs in challenged swine, but failed to completely protect the majority of swine from FMD. Swine which received a primary vaccination with Ad5-FMDV3CWT followed by the boost at 4 weeks generated high levels of FMDV-neutralizing antibodies resulting in complete protection of five of the six swine and limited disease in the remaining animal. Increased efficacy of the two-dose regimen was associated with heightened levels of FMDV-specific IgG1 and IgG2 antibodies.

Type I interferon production in cattle infected with 2 strains of foot-and-mouth disease virus, as determined by in situ hybridization.

Four calves were exposed via aerosol to 1 of 2 strains of foot-and-mouth disease virus. Two animals received virus derived from an infectious clone virus (A12-IC) and 2 received virus derived from the same clone but which lacked the leader coding region (A12-LLV2) that codes for a protein responsible for turning off host protein synthesis. Animals were euthanized at 24 and 72 h post exposure. Cattle receiving A12-IC had a rapid course of disease with more virus in tissues while A12-LLV2-infected cattle did not develop clinical signs of disease. Formalin-fixed, paraffin-embedded tissue sections were probed with digoxigenin-labeled riboprobes corresponding to the coding sequence for bovine interferon (IFN) alpha and IFNbeta. Staining for IFNalpha mRNA was noted in mononuclear cells of the lungs of all animals and in respiratory lymph nodes of cattle receiving A12-IC. Staining for IFNbeta mRNA was confined to bronchiolar epithelium and present only in the animals infected with A12-IC. Inability of the A12-LLV2 virus to achieve levels of spread seen with A12-IC may be related to translation of IFNalpha in A12-LLV2-infected cells, which renders adjacent cells less susceptible to productive infection.

Development of replication-defective adenovirus serotype 5 containing the capsid and 3C protease coding regions of foot-and-mouth disease virus as a vaccine candidate.

A recombinant replication-defective human adenovirus serotype 5 vector containing FMDV capsid, P1-2A, and viral 3C protease coding regions was constructed. Two viral clones were isolated, Ad5-P12X3CWT, containing the wild-type (WT) 3C protease that processes capsid polyprotein precursor into mature capsid proteins, and Ad5-P12X3CMUT, containing a point mutation in the protease coding region that inhibits processing. In 293 cells infected with either virus, synthesis of the FMDV capsid polyprotein precursor occurred, but processing of the polyprotein into structural proteins VP0, VP3, and VP1 occurred only in 3CWT virus-infected cells. Immunoprecipitation with monospecific and monoclonal antibodies indicates possible higher order structure formation in Ad5-P12X3CWT virus-infected cells. The viruses were used to elicit immune responses in mice inoculated intramuscularly (im). Only virus containing the 3CWT elicited a neutralizing antibody response. After boosting, this neutralizing antibody response increased. Swine inoculated im with Ad5-P12X3CWT virus developed a neutralizing antibody response and were either completely or partially protected from contact challenge with an animal directly inoculated with virulent FMDV. This adenovirus vector may be an efficient system for the delivery of FMDV cDNA into animals, leading to a high level of neutralizing antibody production and protection from FMDV challenge.

Ability of foot-and-mouth disease virus to form plaques in cell culture is associated with suppression of alpha/beta interferon.

A genetic variant of foot-and-mouth disease virus lacking the leader proteinase coding region (A12-LLV2) is attenuated in both cattle and swine and, in contrast to wild-type virus (A12-IC), does not spread from the initial site of infection after aerosol exposure of bovines. We have identified secondary cells from susceptible animals, i.e., bovine, ovine, and porcine animals, in which infection with A12-LLV2, in contrast to A12-IC infection, does not produce plaques; this result indicates that this virus cannot spread from the site of initial infection to neighboring cells. Nevertheless, A12-LLV2 can infect these cells, but cytopathic effects and virus yields are significantly reduced compared to those seen with A12-IC infection. Reverse transcription-PCR analysis demonstrates that both A12-LLV2 and A12-IC induce the production of alpha/beta interferon (IFN-alpha/beta) mRNA in host cells. However, only supernatants from A12-LLV2-infected cells have significant antiviral activity. The antiviral activity in supernatants from A12-LLV2-infected embryonic bovine kidney cells is IFN-alpha/beta specific, as assayed with mouse embryonic fibroblast cells with or without IFN-alpha/beta receptors. The results obtained with cell cultures demonstrate that the ability of A12-IC to form plaques is associated with the suppression of IFN-alpha/beta expression and suggest a role for this host factor in the inability of A12-LLV2 to spread and cause disease in susceptible animals.

Development of DNA vaccines for foot-and-mouth disease, evaluation of vaccines encoding replicating and non-replicating nucleic acids in swine.

We have developed naked DNA vaccine candidates for foot-and-mouth disease (FMD), an important disease of domestic animals. The virus that causes this disease, FMDV, is a member of the picornavirus family, which includes many important human pathogens, such as poliovirus, hepatitis A virus, and rhinovirus. Picornaviruses are characterized by a small (7-9000 nucleotide) RNA genome that encodes capsid proteins, processing proteinases, and enzymes required for RNA replication. We have developed two different types of DNA vaccines for FMD. The first DNA vaccine, pP12X3C, encodes the viral capsid gene (P1) and the processing proteinase (3C). Cells transfected with this DNA produce processed viral antigen, and animals inoculated with this DNA using a gene gun produced detectable antiviral immune responses. Mouse inoculations with this plasmid, and with a derivative containing a mutation in the 3C proteinase, indicated that capsid assembly was essential for induction of neutralizing antibody responses. The second DNA vaccine candidate, pWRMHX, encodes the entire FMDV genome, including the RNA-dependent RNA polymerase, permitting the plasmid-encoded viral genomes to undergo amplification in susceptible cells. pWRMHX encodes a mutation at the cell binding site, preventing the replicated genomes from causing disease. Swine inoculated with this vaccine candidate produce viral particles lacking the cell binding site, and neutralizing antibodies that recognize the virus. Comparison of the immune responses elicited by pP12X3C and pWRMHX in swine indicate that the plasmid encoding the replicating genome stimulated a stronger immune response, and swine inoculated with pWRMHX by the intramuscular, intradermal, or gene gun routes were partially protected from a highly virulent FMD challenge.

Development of chicken antibodies to bovine interferon alpha.

We have developed chicken polyclonal antibody to bovine interferon alpha (IFNalpha). Five hundred microg of recombinant bovine IFNalpha suspended with complete Freund's adjuvant was used in the first immunization round. A suspension of the same amount of IFNalpha and incomplete Freund's adjuvant was used for all subsequent boosters. The antibody was purified from egg yolks using polyethylene glycol precipitation. The first reactive antibody appeared several weeks after the first immunization. The antibody is specific for IFNalpha in immunoblotting, it is also useful in ELISA and immunohistochemistry. This method provides a fast, cheap and efficient alternative to development of monoclonal antibodies to conserved mammalian antigens.

Protection of swine by live and inactivated vaccines prepared from a leader proteinase-deficient serotype A12 foot-and-mouth disease virus.

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.

Construction and evaluation of an attenuated vaccine for foot-and-mouth disease: difficulty adapting the leader proteinase-deleted strategy to the serotype O1 virus.

Over the last few years we have utilized a system to genetically engineer foot-and-mouth disease virus (FMDV) to produce live-attenuated vaccine candidates. These candidates have been generated in the genetic background of a tissue culture-adapted strain of serotype A12 virus. Based on this A12 system, we created a virus lacking the sequence encoding the leader (L) proteinase (Piccone et al., 1995), and demonstrated that this leaderless virus, A12-LLV2 was avirulent in bovine and swine, and could be used as an attenuated vaccine (Mason et al., 1997; Chinsangaram et al., 1998). The current study shows that a similar leader-deleted chimeric virus containing the genome of the type A12 virus with a substituted type O1 capsid coding region from a bovine-virulent virus can be constructed, and that the virus has low, but detectable virulence in swine. A second chimera specifying a tissue culture-adapted type O1 capsid lacking the RGD cell binding site, was avirulent in swine, but was not sufficiently immunogenic to provide protection from challenge. These results are described with respect to mechanisms of attenuation and antigen formation in live-attenuated virus-inoculated animals.