The safety and efficacy of an experimental, replication-deficient, human adenovirus-vectored foot-and-mouth disease virus (FMDV) serotype A24 Cruzeiro capsid-based subunit vaccine (AdtA24) was examined in eight independent cattle studies. AdtA24 non-adjuvanted vaccine was administered intramuscularly to a total of 150 steers in doses ranging from approximately 1.0×10(8) to 2.1×10(11) particle units per animal. No detectable local or systemic reactions were observed after vaccination. At 7 days post-vaccination (dpv), vaccinated and control animals were challenged with FMDV serotype A24 Cruzeiro via the intradermal lingual route. Vaccine efficacy was measured by FMDV A24 serum neutralizing titers and by protection from clinical disease and viremia after challenge. The results of eight studies demonstrated a strong correlation between AdtA24 vaccine dose and protection from clinical disease (R(2)=0.97) and viremia (R(2)=0.98). There was also a strong correlation between FMDV A24 neutralization titers on day of challenge and protection from clinical disease (R(2)=0.99). Vaccination with AdtA24 enabled differentiation of infected from vaccinated animals (DIVA) as demonstrated by the absence of antibodies to the FMDV nonstructural proteins in vaccinates prior to challenge. Lack of AdtA24 vaccine shedding after vaccination was indicated by the absence of neutralizing antibody titers to both the adenovector and FMDV A24 Cruzeiro in control animals after co-mingling with vaccinated cattle for three to four weeks. In summary, a non-adjuvanted AdtA24 experimental vaccine was shown to be safe, immunogenic, consistently protected cattle at 7 dpv against direct, homologous FMDV challenge, and enabled differentiation of infected from vaccinated cattle prior to challenge.

The safety of a replication-deficient, human adenovirus-vectored foot-and-mouth disease virus (FMDV) serotype A24 Cruzeiro capsid-based subunit vaccine (AdtA24) was evaluated in five independent safety studies. The target animal safety studies were designed in compliance with United States (U.S.) regulatory requirements (Title 9, U.S. Code of Federal Regulation [9CFR]) and international standard guidelines (VICH Topic GL-44) for veterinary live vaccines. The first three studies were conducted in a total of 22 vaccinees and demonstrated that the AdtA24 master seed virus (MSV) was safe, did not revert to virulence and was not shed or spread from vaccinees to susceptible cattle or pigs. The fourth safety study conducted in 10 lactating cows using an AdtA24 vaccine serial showed that the vaccine was completely absent from milk. The fifth safety study was conducted under typical U.S. production field conditions in 500 healthy beef and dairy cattle using two AdtA24 vaccine serials. These results demonstrated that the vaccine was safe when used per the product label recommendations. Additional data collected during these five studies confirmed that AdtA24 vaccinees developed FMDV A24 and the HAd5 vaccine vector serum neutralization antibodies that test negative in a FMDV non-structural protein antibody test, confirming AdtA24 vaccine's capability to differentiate infected from vaccinated animals (DIVA). In conclusion, results from this comprehensive set of cattle studies demonstrated the safety of the replication-deficient AdtA24 vaccine and fulfilled safety-related requirements for U.S. regulatory requirements.

A foot-and-mouth disease (FMD) recombinant subunit vaccine formulated with a lipid/polymer adjuvant was evaluated in two vaccine efficacy challenge studies in steers. The vaccine active ingredient is a replication-deficient human adenovirus serotype 5 vector encoding the FMD virus (FMDV) A24/Cruzeiro/BRA/55 capsid (AdtA24). In the first study, AdtA24 formulated in ENABL® adjuvant was compared to a fourfold higher dose of AdtA24 without adjuvant. Steers vaccinated with AdtA24 + ENABL® adjuvant developed a significantly higher virus neutralizing test (VNT) antibody titer and an improved clinical response following FMDV A24/Cruzeiro/BRA/55 intradermal lingual challenge at 14 days post-vaccination (dpv) than steers vaccinated with the active ingredient alone. In the second study, vaccination with AdtA24 formulated in ENABL® at the same dose used in the first study, followed by FMDV A24/Cruzeiro/BRA/55 challenge on 7 or 14 dpv, prevented clinical FMD in all steers and conferred 90% protection against viremia. In addition, post-challenge FMDV titers in nasal samples from vaccinated steers compared to unvaccinated steers were significantly reduced. In both studies, none of the AdtA24 vaccinated steers developed antibodies to the FMDV non-structural proteins prior to challenge with FMDV, indicative of the capacity to differentiate infected from vaccinated animals (DIVA). These results demonstrate that administration of AdtA24 formulated in ENABL® adjuvant lowered the protective dose and prevented clinical FMD following exposure of vaccinated steers to virulent FMDV at 7 or 14 dpv.

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.

RNA viruses, such as foot-and-mouth disease virus (FMDV), have error-prone replication resulting in the continuous emergence of new viral strains capable of evading current vaccine coverage. Vaccine formulations must be regularly updated, which is both costly and technically challenging for many vaccine platforms. In this report, we describe a plasmid-based virus-like particle (VLP) production platform utilizing transiently transfected mammalian cell cultures that combines both the rapid response adaptability of nucleic-acid-based vaccines with the ability to produce intact capsid epitopes required for immunity. Formulated vaccines which employed this platform conferred complete protection from clinical foot-and-mouth disease in both swine and cattle. This novel platform can be quickly adapted to new viral strains and serotypes through targeted exchanges of only the FMDV capsid polypeptide nucleic acid sequences, from which processed structural capsid proteins are derived. This platform obviates the need for high biocontainment manufacturing facilities to produce inactivated whole-virus vaccines from infected mammalian cell cultures, which requires upstream expansion and downstream concentration of large quantities of live virulent viruses.

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. 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. 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.