Evaluation of DNA Vaccine Candidates against Foot-and-Mouth Disease Virus in Cattle.

We evaluated four DNA vaccine candidates for their ability to produce virus-like particles (VLPs) and elicit a protective immune response against Foot-and-mouth disease virus (FMDV) in cattle. Two traditional DNA plasmids and two DNA minicircle constructs were evaluated. Both the pTarget O1P1-3C plasmid and O1P1-3C minicircle encoded a wild-type FMDV 3C protease to process the P1-2A polypeptide, whereas the O1P1-HIV-3CT minicircle used an HIV-1 ribosomal frameshift to down-regulate expression of a mutant 3C protease. A modified pTarget plasmid with a reduced backbone size, mpTarget O1P1-3CLT, used a 3C protease containing two mutations reported to enhance expression. All constructs produced mature FMDV P1 cleavage products in transfected cells, as seen by western blot analysis. Three constructs, O1P1-3C minicircles, pTarget O1P1-3C, and mpTarget O1P1-3CLT plasmids, produced intracellular VLP crystalline arrays detected by electron microscopy. Despite VLP formation in vitro, none of the DNA vaccine candidates elicited protection from clinical disease when administered independently. Administration of pTarget O1P1-3C plasmid enhanced neutralizing antibody titers when used as a priming dose prior to administration of a conditionally licensed adenovirus-vectored FMD vaccine. Further work is needed to develop these DNA plasmid-based constructs into standalone FMD vaccines in cattle.

Evaluation of modified Vaccinia Ankara-based vaccines against foot-and-mouth disease serotype A24 in cattle.

To prepare foot-and-mouth disease (FMD) recombinant vaccines in response to newly emerging FMD virus (FMDV) field strains, we evaluated Modified Vaccinia virus Ankara-Bavarian Nordic (MVA-BN®) as an FMD vaccine vector platform. The MVA-BN vector has the capacity to carry and express numerous foreign genes and thereby has the potential to encode antigens from multiple FMDV strains. Moreover, this vector has an extensive safety record in humans. All MVA-BN-FMD constructs expressed the FMDV A24 Cruzeiro P1 capsid polyprotein as antigen and the FMDV 3C protease required for processing of the polyprotein. Because the FMDV wild-type 3C protease is detrimental to mammalian cells, one of four FMDV 3C protease variants were utilized: wild-type, or one of three previously reported mutants intended to dampen protease activity (C142T, C142L) or to increase specificity and thereby reduce adverse effects (L127P). These 3C coding sequences were expressed under the control of different promoters selected to reduce 3C protease expression. Four MVA-BN-FMD constructs were evaluated in vitro for acceptable vector stability, FMDV P1 polyprotein expression, processing, and the potential for vaccine scale-up production. Two MVA-BN FMD constructs met the in vitro selection criteria to qualify for clinical studies: MVA-mBN360B (carrying a C142T mutant 3C protease and an HIV frameshift for reduced expression) and MVA-mBN386B (carrying a L127P mutant 3C protease). Both vaccines were safe in cattle and elicited low to moderate serum neutralization titers to FMDV following multiple dose administrations. Following FMDV homologous challenge, both vaccines conferred 100% protection against clinical FMD and viremia using single dose or prime-boost immunization regimens. The MVA-BN FMD vaccine platform was capable of differentiating infected from vaccinated animals (DIVA). The demonstration of the successful application of MVA-BN as an FMD vaccine vector provides a platform for further FMD vaccine development against more epidemiologically relevant FMDV strains.

Plant-made E2 glycoprotein single-dose vaccine protects pigs against classical swine fever.

Classical Swine Fever Virus (CSFV) causes classical swine fever, a highly contagious hemorrhagic fever affecting both feral and domesticated pigs. Outbreaks of CSF in Europe, Asia, Africa and South America had significant adverse impacts on animal health, food security and the pig industry. The disease is generally contained by prevention of exposure through import restrictions (e.g. banning import of live pigs and pork products), localized vaccination programmes and culling of infected or at-risk animals, often at very high cost. Current CSFV-modified live virus vaccines are protective, but do not allow differentiation of infected from vaccinated animals (DIVA), a critical aspect of disease surveillance programmes. Alternatively, first-generation subunit vaccines using the viral protein E2 allow for use of DIVA diagnostic tests, but are slow to induce a protective response, provide limited prevention of vertical transmission and may fail to block viral shedding. CSFV E2 subunit vaccines from a baculovirus/insect cell system have been developed for several vaccination campaigns in Europe and Asia. However, this expression system is considered expensive for a veterinary vaccine and is not ideal for wide-spread deployment. To address the issues of scalability, cost of production and immunogenicity, we have employed an Agrobacterium-mediated transient expression platform in Nicotiana benthamiana and formulated the purified antigen in novel oil-in-water emulsion adjuvants. We report the manufacturing of adjuvanted, plant-made CSFV E2 subunit vaccine. The vaccine provided complete protection in challenged pigs, even after single-dose vaccination, which was accompanied by strong virus neutralization antibody responses.

Versatility of the adenovirus-vectored foot-and-mouth disease vaccine platform across multiple foot-and-mouth disease virus serotypes and topotypes using a vaccine dose representative of the AdtA24 conditionally licensed vaccine.

We investigated the serotype- and topotype versatility of a replication-deficient human adenovirus serotype 5 vectored foot-and-mouth disease (FMD) vaccine platform (AdtFMD). Sixteen AdtFMD recombinant subunit monovalent vaccines targeting twelve distinct FMD virus (FMDV) serotype/topotypes in FMD Regional Pools I-VII were constructed. The AdtA24 serotype conditionally licensed vaccine served as the basis for vaccine design and target dose for cattle clinical trials. Several vaccines contained an additional RGD motif genetic insertion in the adenovector fiber knob, and/or a full-length 2B gene insertion in the FMDV P1 gene cassette. In 13 of the 22 efficacy studies conducted, naïve control and AdtFMD vaccinated cattle were challenged intradermolingually at 2 weeks post-vaccination using a FMDV strain homologous to the AdtFMD vaccine strain. Each of the 16 AdtFMD vaccines were immunogenic based on the presence of homologous neutralizing antibodies in the serum of approximately 90% of total vaccinates (n = 375) on the day of challenge. Importantly, for 75% of vaccines tested, the effective dose that conferred 100% protection against clinical FMD was identical to or in some cases lower than, the minimum protective dose for the conditionally licensed AdtA24 vaccine formulated with ENABL® adjuvant. Results also confirmed the capability of the AdtFMD vaccine platform to differentiate infected from vaccinated animals (DIVA) across the five FMDV serotypes evaluated. Collectively, this comprehensive set of FMD cattle vaccine dose ranging studies highlights the serotype- and topotype versatility of the AdtFMD vaccine platform for further development, licensure, and application in FMD outbreak control and disease eradication efforts.

Smart Card Decontamination in a High-Containment Laboratory.

Validated procedures for decontamination of laboratory surfaces and equipment are essential to biosafety and biorisk programs at high-containment laboratories. Each high-containment laboratory contains a unique combination of surfaces, procedures, and biological agents that require decontamination methods tailored to specific facility practices. The Plum Island Animal Disease Center (PIADC) is a high-containment laboratory operating multiple biosafety level (BSL)-3, ABSL-3, and BSL-3 Ag spaces. The PIADC facility requires the use of federally issued smart cards, called personal identity verification (PIV) cards, to access information technology (IT) networks both outside and within the high-containment laboratory. Because PIV cards may require transit from the BSL-3 to office spaces, a validated procedure for disinfecting PIV card surfaces prior to removal from the laboratory is critical to ensure biosafety and biosecurity. Two high-risk select agents used in the PIADC high-containment laboratory are foot-and-mouth disease virus (FMDV) and swine vesicular disease virus (SVDV). We evaluated disinfection of PIV cards intentionally spotted with FMDV and SVDV using a modified quantitative carrier test and the liquid chemical disinfectant Virkon® S. Our experimental design modeled a worst-case scenario of PIV card contamination and disinfection by combining high concentrations of virus dried with an organic soil load and use of aged Virkon® S prepared in hard water. Results showed that FMDV and SVDV dried on PIV card surfaces were completely inactivated after immersion for 30 and 60 seconds, respectively, in a 5-day-old solution of 1% Virkon® S. Therefore, this study provided internal validation of PIADC biosafety protocols by demonstrating the efficacy of Virkon® S to inactivate viruses on contaminated smart cards at short contact times.

Efficacy of an adenovirus-vectored foot-and-mouth disease virus serotype A subunit vaccine in cattle using a direct contact transmission model.

BACKGROUND: A direct contact transmission challenge model was used to simulate natural foot-and-mouth disease virus (FMDV) spread from FMDV A24/Cruzeiro/BRA/55 infected 'seeder' steers to naïve or vaccinated steers previously immunized with a replication-deficient human adenovirus-vectored FMDV A24/Cruzeiro/BRA/55 capsid-based subunit vaccine (AdtA24). In two independent vaccine efficacy trials, AdtA24 was administered once intramuscularly in the neck 7 days prior to contact with FMDV A24/Cruzeiro/BRA/55-infected seeder steers. RESULTS: In Efficacy Study 1, we evaluated three doses of AdtA24 to estimate the 50%/90% bovine protective dose (BPD50/90) for prevention of clinical FMD. In vaccinated, contact-challenged steers, the BPD50/90 was 3.1 × 1010 / 5.5 × 1010 AdtA24 particles formulated without adjuvant. In Efficacy Study 2, steers vaccinated with 5 × 1010 AdtA24 particles, exposed to FMDV A24/Cruzeiro/BRA/55-infected seeder steers, did not develop clinical FMD or transmit FMDV to other vaccinated or naïve, non-vaccinated steers. In contrast, naïve, non-vaccinated steers that were subsequently exposed to FMDV A24/Cruzeiro/BRA/55-infected seeder steers developed clinical FMD and transmitted FMDV by contact to additional naïve, non-vaccinated steers. The AdtA24 vaccine differentiated infected from vaccinated animals (DIVA) because no antibodies to FMDV nonstructural proteins were detected prior to FMDV exposure. CONCLUSIONS: A single dose of the AdtA24 non-adjuvanted vaccine conferred protection against clinical FMD at 7 days post-vaccination following direct contact transmission from FMDV-infected, naïve, non-vaccinated steers. The AdtA24 vaccine was effective in preventing FMDV transmission from homologous challenged, contact-exposed, AdtA24-vaccinated, protected steers to co-mingled, susceptible steers, suggesting that the vaccine may be beneficial in reducing both the magnitude and duration of a FMDV outbreak in a commercial cattle production setting.

Production of foot-and-mouth disease virus capsid proteins by the TEV protease.

Protective immunity to viral pathogens often includes production of neutralizing antibodies to virus capsid proteins. Many viruses produce capsid proteins by expressing a precursor polyprotein and related protease from a single open reading frame. The foot-and-mouth disease virus (FMDV) expresses a 3C protease (3Cpro) that cleaves a P1 polyprotein intermediate into individual capsid proteins, but the FMDV 3Cpro also degrades many host cell proteins and reduces the viability of host cells, including subunit vaccine production cells. To overcome the limitations of using the a wild-type 3Cpro in FMDV subunit vaccine expression systems, we altered the protease restriction sequences within a FMDV P1 polyprotein to enable production of FMDV capsid proteins by the Tobacco Etch Virus NIa protease (TEVpro). Separate TEVpro and modified FMDV P1 proteins were produced from a single open reading frame by an intervening FMDV 2A sequence. The modified FMDV P1 polyprotein was successfully processed by the TEVpro in both mammalian and bacterial cells. More broadly, this method of polyprotein production and processing may be adapted to other recombinant expression systems, especially plant-based expression.

Foot-and-Mouth Disease (FMD) Virus 3C Protease Mutant L127P: Implications for FMD Vaccine Development.

The foot-and-mouth disease virus (FMDV) afflicts livestock in more than 80 countries, limiting food production and global trade. Production of foot-and-mouth disease (FMD) vaccines requires cytosolic expression of the FMDV 3C protease to cleave the P1 polyprotein into mature capsid proteins, but the FMDV 3C protease is toxic to host cells. To identify less-toxic isoforms of the FMDV 3C protease, we screened 3C mutants for increased transgene output in comparison to wild-type 3C using a Gaussia luciferase reporter system. The novel point mutation 3C(L127P) increased yields of recombinant FMDV subunit proteins in mammalian and bacterial cells expressing P1-3C transgenes and retained the ability to process P1 polyproteins from multiple FMDV serotypes. The 3C(L127P) mutant produced crystalline arrays of FMDV-like particles in mammalian and bacterial cells, potentially providing a practical method of rapid, inexpensive FMD vaccine production in bacteria.IMPORTANCE The mutant FMDV 3C protease L127P significantly increased yields of recombinant FMDV subunit antigens and produced virus-like particles in mammalian and bacterial cells. The L127P mutation represents a novel advancement for economical FMD vaccine production.

Evaluation of Gaussia luciferase and foot-and-mouth disease virus 2A translational interrupter chimeras as polycistronic reporters for transgene expression.

BACKGROUND: The Gaussia princeps luciferase is used as a stand-alone reporter of transgene expression for in vitro and in vivo expression systems due to the rapid and easy monitoring of luciferase activity. We sought to simultaneously quantitate production of other recombinant proteins by transcriptionally linking the Gaussia princeps luciferase gene to other genes of interest through the foot-and-mouth disease virus 2A translational interrupter sequence. RESULTS: We produced six plasmids, each encoding a single open reading frame, with the foot-and-mouth disease virus 2A sequence placed either N-terminal or C-terminal to the Gaussia princeps luciferase gene. Two plasmids included novel Gaussia princeps luciferase variants with the position 1 methionine deleted. Placing a foot-and-mouth disease virus 2A translational interrupter sequence on either the N- or C-terminus of the Gaussia princeps luciferase gene did not prevent the secretion or luminescence of resulting chimeric luciferase proteins. We also measured the ability of another polycistronic plasmid vector with a 2A-luciferase sequence placed downstream of the foot-and-mouth disease virus P1 and 3C protease genes to produce of foot-and-mouth disease virus-like particles and luciferase activity from transfected cells. Incorporation of the 2A-luciferase sequence into a transgene encoding foot-and-mouth disease virus structural proteins retained luciferase activity and the ability to form virus-like particles. CONCLUSIONS: We demonstrated a mechanism for the near real-time, sequential, non-destructive quantitative monitoring of transcriptionally-linked recombinant proteins and a valuable method for monitoring transgene expression in recombinant vaccine constructs.

Multiplexed molecular assay for rapid exclusion of foot-and-mouth disease.

A nucleic acid-based multiplexed assay was developed that combines detection of foot-and-mouth disease virus (FMDV) with rule-out assays for two other foreign animal diseases and four domestic animal diseases that cause vesicular or ulcerative lesions indistinguishable from FMDV infection in cattle, sheep and swine. The FMDV "look-alike" diagnostic assay panel contains 5 PCR and 12 reverse transcriptase PCR (RT-PCR) signatures for a total of 17 simultaneous PCR amplifications for 7 diseases plus incorporating 4 internal assay controls. It was developed and optimized to amplify both DNA and RNA viruses simultaneously in a single tube and employs Luminex liquid array technology. Assay development including selection of appropriate controls, a comparison of signature performance in single and multiplex testing against target nucleic acids, as well of limits of detection for each of the individual signatures is presented. While this assay is a prototype and by no means a comprehensive test for FMDV "look-alike" viruses, an assay of this type is envisioned to have benefit to a laboratory network in routine surveillance and possibly for post-outbreak proof of freedom from foot-and-mouth disease.

Gamma irradiation can be used to inactivate Bacillus anthracis spores without compromising the sensitivity of diagnostic assays.

The use of Bacillus anthracis as a biological weapon in 2001 heightened awareness of the need for validated methods for the inactivation of B. anthracis spores. This study determined the gamma irradiation dose for inactivating virulent B. anthracis spores in suspension and its effects on real-time PCR and antigen detection assays. Strains representing eight genetic groups of B. anthracis were exposed to gamma radiation, and it was found that subjecting spores at a concentration of 10(7) CFU/ml to a dose of 2.5 x 10(6) rads resulted in a 6-log-unit reduction of spore viability. TaqMan real-time PCR analysis of untreated versus irradiated Ames strain (K1694) spores showed that treatment significantly enhanced the detection of B. anthracis chromosomal DNA targets but had no significant effect on the ability to detect targets on the pXO1 and pXO2 plasmids of B. anthracis. When analyzed by an enzyme-linked immunosorbent assay (ELISA), irradiation affected the detection of B. anthracis spores in a direct ELISA but had no effect on the limit of detection in a sandwich ELISA. The results of this study showed that gamma irradiation-inactivated spores can be tested by real-time PCR or sandwich ELISA without decreasing the sensitivity of either type of assay. Furthermore, the results suggest that clinical and public health laboratories which test specimens for B. anthracis could potentially incorporate gamma irradiation into sample processing protocols without compromising the sensitivity of the B. anthracis assays.