Use of ENABL® adjuvant to increase the potency of an adenovirus-vectored foot-and-mouth disease virus serotype A subunit vaccine.

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.

Interaction between FMDV Lpro and transcription factor ADNP is required for optimal viral replication.

The foot-and-mouth disease virus (FMDV) leader protease (Lpro) inhibits host translation and transcription affecting the expression of several factors involved in innate immunity. In this study, we have identified the host transcription factor ADNP (activity dependent neuroprotective protein) as an Lpro interacting protein by mass spectrometry. We show that Lpro can bind to ADNP in vitro and in cell culture. RNAi of ADNP negatively affected virus replication and higher levels of interferon (IFN) and IFN-stimulated gene expression were detected. Importantly, infection with FMDV wild type but not with a virus lacking Lpro (leaderless), induced recruitment of ADNP to IFN-α promoter sites early during infection. Furthermore, we found that Lpro and ADNP are in a protein complex with the ubiquitous chromatin remodeling factor Brg-1. Our results uncover a novel role of FMDV Lpro in targeting ADNP and modulation of its transcription repressive function to decrease the expression of IFN and ISGs.

Adenovirus-vectored foot-and-mouth disease vaccine confers early and full protection against FMDV O1 Manisa in swine.

A human adenovirus (Ad5) vectored foot-and-mouth disease virus (FMDV) O1-Manisa subunit vaccine (Ad5-O1Man) was engineered to deliver FMDV O1-Manisa capsid and capsid-processing proteins. Swine inoculated with Ad5-O1Man developed an FMDV-specific humoral response as compared to animals inoculated with an empty Ad5-vector. Vaccinated animals were completely protected against homologous challenge at 7 or 21 days post-vaccination. Potency studies exhibited a PD50 of about 107 pfu/animal while a dose of 4×107pfu/animal fully protected swine against FMDV intradermal challenge. In-vitro cross-neutralization analysis distinctly predicted that swine vaccinated with Ad5-O1Man would be protected against challenge with homologous FMDV O1Man Middle East-South Asia (ME-SA) topotype and also against recent outbreak strains of Mya-98 South East Asia (SEA) lineage including O1-UK-2001 and O1-South Korea-2010. These results indicate that recombinant Ad5-O1Man is an effective, safe and cross-reacting vaccine that could potentially be used preventively and in outbreak situations, to control FMDV O Mya-98 lineage in swine.

The activation of the IFNß induction/signaling pathway in porcine alveolar macrophages by porcine reproductive and respiratory syndrome virus is variable.

BACKGROUND: It has been recognized that the expression of type I interferon (IFNα/ß) may be suppressed during infection with porcine reproductive, respiratory syndrome virus (PRRSV). This causes profound negative effects on both the innate and adaptive immunity of the host resulting in persistence of infection. OBJECTIVE: Test the effects of PRRSV infection of porcine alveolar macrophages (PAMs), the main target cell, on the expression of interferon beta (IFNß) and downstream signaling events. METHODS: In order to examine those effects, PAMs harvested from lungs of healthy PRRSV-free animals were infected with virulent, attenuated, infectious clone-derived chimeric viruses, or field PRRS virus strains. Culture supernatants from the infected PAMs were tested for IFNß protein expression by means of indirect ELISA and for bioactivity by a vesicular stomatitis virus plaque reduction assay. The expression of the Mx protein was assayed to ascertain signaling events. RESULTS: These experiments demonstrated that PRRSV does induce variably, the expression of bioactive IFNß protein in the natural host cell. To further elucidate the effects of PRRSV infection on IFNß signaling, Mx-1 an interferon stimulated gene (ISG), was also tested for expression. Interestingly, Mx-1 expression by infected PAMs generally correlated with IFNß production. CONCLUSION: The results of this study demonstrate that the induction of IFNß and signaling in PAMs after PRRSV infection is variable.

Combination of Adt-O1Manisa and Ad5-boIFNλ3 induces early protective immunity against foot-and-mouth disease in cattle.

Foot-and-mouth-disease (FMD) remains the most infectious livestock disease worldwide. Although commercially available inactivated or adenovirus-vectored-vaccines (Ad5-FMD) are effective, they require 5-7 days to induce protection. Therefore, new control strategies that stimulate rapid immune responses are needed. Expression of bovine interferon λ3 using the Ad5-vector platform (Ad5-boIFNλ3) is able to delay disease in cattle, but clinical signs appear at 9 days after challenge. We hypothesized that combination of Ad5-boIFNλ3 and Ad5-FMD could induce immediate and lasting protection against FMD. Cattle were vaccinated with an Ad5-FMD, Ad5-boIFNλ3, or the combination of both, followed by challenge at three days post-immunization. All animals treated with Ad5-FMD combined with Ad5-boIFNλ3 were fully protected against FMD, despite the absence of systemic neutralizing antibodies or antiviral activity at the time of challenge. Induction of a strong cell-mediated immune response suggested that Ad5-boIFNλ3 is able to act as an adjuvant of Ad5-FMD vaccine in cattle.

Synonymous Deoptimization of Foot-and-Mouth Disease Virus Causes Attenuation In Vivo while Inducing a Strong Neutralizing Antibody Response.

UNLABELLED: Codon bias deoptimization has been previously used to successfully attenuate human pathogens, including poliovirus, respiratory syncytial virus, and influenza virus. We have applied a similar technology to deoptimize the capsid-coding region (P1) of foot-and-mouth disease virus (FMDV). Despite the introduction of 489 nucleotide changes (19%), synonymous deoptimization of the P1 region rendered a viable FMDV progeny. The resulting strain was stable and reached cell culture titers similar to those obtained for wild-type (WT) virus, but at reduced specific infectivity. Studies in mice showed that 100% of animals inoculated with the FMDV A12 P1 deoptimized mutant (A12-P1 deopt) survived, even when the animals were infected at doses 100 times higher than the dose required to cause death by WT virus. All mice inoculated with the A12-P1 deopt mutant developed a strong antibody response and were protected against subsequent lethal challenge with WT virus at 21 days postinoculation. Remarkably, the vaccine safety margin was at least 1,000-fold higher for A12-P1 deopt than for WT virus. Similar patterns of attenuation were observed in swine, in which animals inoculated with A12-P1 deopt virus did not develop clinical disease until doses reached 1,000 to 10,000 times the dose required to cause severe disease in 2 days with WT A12. Consistently, high levels of antibody titers were induced, even at the lowest dose tested. These results highlight the potential use of synonymous codon pair deoptimization as a strategy to safely attenuate FMDV and further develop live attenuated vaccine candidates to control such a feared livestock disease. IMPORTANCE: Foot-and-mouth disease (FMD) is one of the most feared viral diseases that can affect livestock. Although this disease appeared to be contained in developed nations by the end of the last century, recent outbreaks in Europe, Japan, Taiwan, South Korea, etc., have demonstrated that infection can spread rapidly, causing devastating economic and social consequences. The Global Foot-and-Mouth Disease Research Alliance (GFRA), an international organization launched in 2003, has set as part of their five main goals the development of next-generation control measures and strategies, including improved vaccines and biotherapeutics. Our work demonstrates that newly developed codon pair bias deoptimization technologies can be applied to FMD virus to obtain attenuated strains with potential for further development as novel live attenuated vaccine candidates that may rapidly control disease without reverting to virulence.

Multiple efficacy studies of an adenovirus-vectored foot-and-mouth disease virus serotype A24 subunit vaccine in cattle using homologous challenge.

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.

Evaluation of a Fiber-Modified Adenovirus Vector Vaccine against Foot-and-Mouth Disease in Cattle.

Novel vaccination approaches against foot-and-mouth disease (FMD) include the use of replication-defective human adenovirus type 5 (Ad5) vectors that contain the capsid-encoding regions of FMD virus (FMDV). Ad5 containing serotype A24 capsid sequences (Ad5.A24) has proved to be effective as a vaccine against FMD in livestock species. However, Ad5-vectored FMDV serotype O1 Campos vaccine (Ad5.O1C.2B) provides only partial protection of cattle against homologous challenge. It has been reported that a fiber-modified Ad5 vector expressing Arg-Gly-Asp (RGD) enhances transduction of antigen-presenting cells (APC) in mice. In the current study, we assessed the efficacy of a fiber-modified Ad5 (Adt.O1C.2B.RGD) in cattle. Expression of FMDV capsid proteins was superior in cultured cells infected with the RGD-modified vector. Furthermore, transgene expression of Adt.O1C.2B.RGD was enhanced in cell lines that constitutively express integrin αvß6, a known receptor for FMDV. In contrast, capsid expression in cattle-derived enriched APC populations was not enhanced by infection with this vector. Our data showed that vaccination with the two vectors yielded similar levels of protection against FMD in cattle. Although none of the vaccinated animals had detectable viremia, FMDV RNA was detected in serum samples from animals with clinical signs. Interestingly, CD4(+) and CD8(+) gamma interferon (IFN-γ)(+) cell responses were detected at significantly higher levels in animals vaccinated with Adt.O1C.2B.RGD than in animals vaccinated with Ad5.O1C.2B. Our results suggest that inclusion of an RGD motif in the fiber of Ad5-vectored FMD vaccine improves transgene delivery and cell-mediated immunity but does not significantly enhance vaccine performance in cattle.

A colorimetric bioassay for high-throughput and cost-effectively assessing anti-foot-and-mouth disease virus activity.

Foot-and-mouth disease virus (FMDV) is one of the most contagious animal viruses. This virus is very sensitive to inhibition by type I interferons. Currently, a bioassay based on plaque reduction is used to measure anti-FMDV activity of porcine IFNs. The plaque reduction assay is tedious and difficult to utilize for high-throughput analysis. Using available FMDV susceptible bovine and porcine cells, we developed and tested a colorimetric assay based on cytopathic effect reduction for its ability to quantify FMDV-specific antiviral activity of bovine and porcine type I interferons. Our results show that this new method has significant advantages over other assays in terms of labor intensity, cost, high-throughput capability and/or anti-FMDV specific activity because of simpler procedures and direct measurement of antiviral activity. Several assay conditions were tested to optimize the procedures. The test results show that the assay can be standardized with fixed conditions and a standard or a reference for measuring antiviral activity as units. This is an excellent assay in terms of sensitivity and accuracy based on a statistical evaluation. The results obtained with this assay were highly correlated with a conventional virus titration method.

Poly ICLC increases the potency of a replication-defective human adenovirus vectored foot-and-mouth disease vaccine.

Foot-and-mouth disease virus (FMDV) causes a highly contagious disease of cloven-hoofed animals. We have previously demonstrated that a replication-defective human adenovirus 5 vector carrying the FMDV capsid coding region of serotype A24 Cruzeiro (Ad5-CI-A24-2B) protects swine and cattle against FMDV challenge by 7 days post-vaccination. However, since relatively large amounts of Ad5-CI-A24-2B are required to induce protection this strategy could be costly for livestock production. Poly ICLC is a synthetic double stranded RNA that activates multiple innate and adaptive immune pathways. In this study, we have tested for the first time, the adjuvant effect of poly ICLC in combination with Ad5-CI-A24-2B in swine. We found that the combination resulted in a reduction of the vaccine protective dose by 80-fold. Interestingly, the lowest dose of Ad5-CI-A24-2B plus 1mg of poly ICLC protected animals against challenge even in the absence of detectable FMDV-specific neutralizing antibodies at the time of challenge.

Foot-and-mouth disease: technical and political challenges to eradication.

Foot-and-mouth disease (FMD) is a highly-contagious livestock disease with global socioeconomic ramifications. The disease negatively impacts both individual farmers through reduced herd viability and nations through trade restrictions of animals and animal derivatives. Vaccines for FMD prevention have existed for over 70 years, yet the disease remains enzootic in a large percentage of the globe. FMD persistence is due in part to technical limitations of historic and current vaccine technologies. There also exist many socioeconomic and political barriers to global FMD eradication. Here we highlight the barriers to eradication and discuss potential avenues toward FMD eradication.

Type III interferon protects swine against foot-and-mouth disease.

In recent years, we have developed novel strategies to control foot-and-mouth disease (FMD), including the use of biotherapeutics such as interferons (IFN) delivered by a replication-defective human adenovirus type 5 (Ad5). Swine can be sterilely protected after vaccination with an Ad5 that encodes porcine type I IFN (poIFN-α), and cattle can be similarly protected or develop significantly reduced disease when treated with an Ad5 delivering bovine type III IFN (boIFN-λ3). Here, we have evaluated the efficacy of porcine IFN-λ3 (poIFN-λ3) against FMD virus in vivo. Swine inoculated with different doses of Ad5-poIFN-λ3 were protected against disease in a dose-dependent manner. Despite the absence of systemic antiviral activity, 7 out of 10 Ad5-poIFN-λ3 inoculated animals did not develop disease or viremia, and the other 3 inoculated animals displayed delayed and milder disease by 7 days postchallenge as compared with control animals inoculated with an Ad5 control vector. While analysis of gene expression showed significant induction of IFN and IFN-stimulated genes in Ad5-poIFN-λ3-treated cultured porcine epithelial kidney cells, there was limited gene induction in peripheral blood monocytes isolated from treated swine. These results suggest that treatment with Ad5-poIFN-λ3 is an effective biotherapeutic strategy against FMD in swine.

Characterization of cytotoxic T lymphocyte function after foot-and-mouth disease virus infection and vaccination.

The induction of neutralizing antibodies specific for foot-and-mouth disease virus (FMDV) has been the central goal of vaccination efforts against this economically important disease of cloven-hoofed animals. Although these efforts have yielded much success, challenges remain, including little cross-serotype protection and inadequate duration of immunity. Commonly, viral infections are characterized by induction of cytotoxic T lymphocytes (CTL), yet the function of CTL in FMDV immunity is poorly defined. We developed an assay for detection of CTL specific for FMDV and reported that a modified adenovirus-vectored FMDV vaccine could induce CTL activity. This allowed us to determine whether FMDV-specific CTL responses are induced during infection and to test further whether vaccine-induced CTL could protect against challenge with FMDV. We now show the induction of antigen-specific CTL responses after infection of swine with FMDV strain A24 Cruizero. In addition, we developed a vaccination strategy that induces FMDV-specific CTL in the absence of significant neutralizing antibody. Animals vaccinated using this protocol showed delayed clinical disease and significantly suppressed viremia compared to control animals, suggesting a role for CTLs in the control of virus shedding. These results provide new insights showing induction of CTL responses to FMDV following infection or vaccination, and create the potential for improving vaccine performance by targeting cellular immunity.

Venezuelan equine encephalitis replicon particles can induce rapid protection against foot-and-mouth disease virus.

We have previously shown that delivery of the porcine type I interferon gene (poIFN-α/ß) with a replication-defective human adenovirus vector (adenovirus 5 [Ad5]) can sterilely protect swine challenged with foot-and-mouth disease virus (FMDV) 1 day later. However, the need of relatively high doses of Ad5 limits the applicability of such a control strategy in the livestock industry. Venezuelan equine encephalitis virus (VEE) empty replicon particles (VRPs) can induce rapid protection of mice against either homologous or, in some cases, heterologous virus challenge. As an alternative approach to induce rapid protection against FMDV, we have examined the ability of VRPs containing either the gene for green fluorescent protein (VRP-GFP) or poIFN-α (VRP-poIFN-α) to block FMDV replication in vitro and in vivo. Pretreatment of swine or bovine cell lines with either VRP significantly inhibited subsequent infection with FMDV as early as 6 h after treatment and for at least 120 h posttreatment. Furthermore, mice pretreated with either 10(7) or 10(8) infectious units of VRP-GFP and challenged with a lethal dose of FMDV 24 h later were protected from death. Protection was induced as early as 6 h after treatment and lasted for at least 48 h and correlated with induction of an antiviral response and production of IFN-α. By 6 h after treatment several genes were upregulated, and the number of genes and the level of induction increased at 24 h. Finally, we demonstrated that the chemokine IP-10, which is induced by IFN-α and VRP-GFP, is directly involved in protection against FMDV.

An adenovirus vectored mucosal adjuvant augments protection of mice immunized intranasally with an adenovirus-vectored foot-and-mouth disease virus subunit vaccine.

Foot-and-mouth disease virus (FMDV) is a highly contagious pathogen that causes severe morbidity and economic losses to the livestock industry in many countries. The oral and respiratory mucosae are the main ports of entry of FMDV, so the stimulation of local immunity in these tissues may help prevent initial infection and viral spread. E. coli heat-labile enterotoxin (LT) has been described as one of the few molecules that have adjuvant activity at mucosal surfaces. The objective of this study was to evaluate the efficacy of replication-defective adenovirus 5 (Ad5) vectors encoding either of two LT-based mucosal adjuvants, LTB or LTR72. These vectored adjuvants were delivered intranasally to mice concurrent with an Ad5-FMDV vaccine (Ad5-A24) to assess their ability to augment mucosal and systemic humoral immune responses to Ad5-A24 and protection against FMDV. Mice receiving Ad5-A24 plus Ad5-LTR72 had higher levels of mucosal and systemic neutralizing antibodies than those receiving Ad5-A24 alone or Ad5-A24 plus Ad5-LTB. The vaccine plus Ad5-LTR72 group also demonstrated 100% survival after intradermal challenge with a lethal dose of homologous FMDV serotype A24. These results suggest that Ad5-LTR72 could be used as an important tool to enhance mucosal and systemic immunity against FMDV and potentially other pathogens with a common route of entry.

Novel antiviral therapeutics to control foot-and-mouth disease.

Foot-and-mouth disease virus (FMDV) causes a highly contagious disease of cloven-hoofed animals. Vaccines require ∼7 days to induce protection; thus, before this time, vaccinated animals are still susceptible to the disease. Our group has previously shown that swine inoculated with 1×10(11) focus forming units (FFU) of a replication-defective human adenovirus containing the gene for porcine interferon alpha (Adt-pIFN-α) are sterilely protected from FMDV serotypes A24, O1 Manisa, or Asia 1 when the animals are challenged 1 day postadministration, and protection can last for 3-5 days. Polyriboinosinic-polyribocytidylic acid stabilized with poly-l-lysine and carboxymethyl cellulose (poly ICLC) is a synthetic double-stranded RNA that is a viral mimic and activates multiple innate immune pathways through interaction with toll-like receptor 3 and MDA-5. It is a potent inducer of IFNs. In this study, we initially examined the effect of poly IC and IFN-α on FMDV replication and gene induction in cell culture. Poly ICLC alone or combined with Adt-pIFN-α was then evaluated for its therapeutic efficacy in swine against intradermal challenge with FMDV A24, 1 day post-treatment. Groups of swine were subcutaneously inoculated either with poly ICLC alone (4 or 8 mg) or in combination with different doses of Adt-pIFN-α (2.5×10(9), 1×10(9), or 2.5×10(8) FFU). While different degrees of protection were achieved in all the treated animals, a dose of 8 mg of poly ICLC alone or combined with 1×10(9) FFU of Adt-pIFN-α was sufficient to sterilely protect swine when challenged 24 h later with FMDV A24. IFN-stimulated gene (ISG) expression in peripheral blood mononuclear cells at 1 day post-treatment was broader and higher in protected animals than in nonprotected animals. These data indicate that poly ICLC is a potent stimulator of IFN and ISGs in swine and at an adequate dose is sufficient to induce complete protection against FMD.

Bovine type III interferon significantly delays and reduces the severity of foot-and-mouth disease in cattle.

Interferons (IFNs) are the first line of defense against viral infections. Although type I and II IFNs have proven effective to inhibit foot-and-mouth disease virus (FMDV) replication in swine, a similar approach had only limited efficacy in cattle. Recently, a new family of IFNs, type III IFN or IFN-λ, has been identified in human, mouse, chicken, and swine. We have identified bovine IFN-λ3 (boIFN-λ3), also known as interleukin 28B (IL-28B), and demonstrated that expression of this molecule using a recombinant replication-defective human adenovirus type 5 (Ad5) vector, Ad5-boIFN-λ3, exhibited antiviral activity against FMDV in bovine cell culture. Furthermore, inoculation of cattle with Ad5-boIFN-λ3 induced systemic antiviral activity and upregulation of IFN-stimulated gene expression in the upper respiratory airways and skin. In the present study, we demonstrated that disease could be delayed for at least 6 days when cattle were inoculated with Ad5-boIFN-λ3 and challenged 24 h later by intradermolingual inoculation with FMDV. Furthermore, the delay in the appearance of disease was significantly prolonged when treated cattle were challenged by aerosolization of FMDV, using a method that resembles the natural route of infection. No clinical signs of FMD, viremia, or viral shedding in nasal swabs was found in the Ad5-boIFN-λ3-treated animals for at least 9 days postchallenge. Our results indicate that boIFN-λ3 plays a critical role in the innate immune response of cattle against FMDV. To this end, this work represents the most successful biotherapeutic strategy so far tested to control FMDV in cattle.

The presence of alpha interferon at the time of infection alters the innate and adaptive immune responses to porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most devastating and costly diseases to the swine industry worldwide. Overall, the adaptive immune response to PRRS virus (PRRSV) is weak, which results in delayed elimination of virus from the host and inferior vaccine protection. PRRSV has been shown to induce a meager alpha interferon (IFN-α) response, and we hypothesized that elevated IFN-α levels early in infection would shorten the induction time and increase elements of the adaptive immune response. To test this, we measured both antibody and cell-mediated immunity in pigs after the administration of a nonreplicating human adenovirus type 5 vector expressing porcine IFN-α (Ad5-pIFN-α) at the time of PRRSV infection and compared the results to those for pigs infected with PRRSV alone. Viremia was delayed, and there was a decrease in viral load in the sera of pigs administered the Ad5-pIFN-α. Although seroconversion was slightly delayed in pigs receiving Ad5-pIFN-α, probably due to the early reduction in viral replication, little difference in the overall or neutralizing antibody response was seen. However, there was an increase in the number of virus-specific IFN-γ-secreting cells detected in the pigs receiving Ad5-pIFN-α, as well as an altered cytokine profile in the lung at 14 days postinfection, indicating that the presence of IFN-α at the time of infection can alter innate and adaptive immune responses to PRRSV.

Inoculation of swine with foot-and-mouth disease SAP-mutant virus induces early protection against disease.

Foot-and-mouth disease virus (FMDV) leader proteinase (L(pro)) cleaves itself from the viral polyprotein and cleaves the translation initiation factor eIF4G. As a result, host cell translation is inhibited, affecting the host innate immune response. We have demonstrated that L(pro) is also associated with degradation of nuclear factor κB (NF-κB), a process that requires L(pro) nuclear localization. Additionally, we reported that disruption of a conserved protein domain within the L(pro) coding sequence, SAP mutation, prevented L(pro) nuclear retention and degradation of NF-κB, resulting in in vitro attenuation. Here we report that inoculation of swine with this SAP-mutant virus does not cause clinical signs of disease, viremia, or virus shedding even when inoculated at doses 100-fold higher than those required to cause disease with wild-type (WT) virus. Remarkably, SAP-mutant virus-inoculated animals developed a strong neutralizing antibody response and were completely protected against challenge with WT FMDV as early as 2 days postinoculation and for at least 21 days postinoculation. Early protection correlated with a distinct pattern in the serum levels of proinflammatory cytokines in comparison to the levels detected in animals inoculated with WT FMDV that developed disease. In addition, animals inoculated with the FMDV SAP mutant displayed a memory T cell response that resembled infection with WT virus. Our results suggest that L(pro) plays a pivotal role in modulating several pathways of the immune response. Furthermore, manipulation of the L(pro) coding region may serve as a viable strategy to derive live attenuated strains with potential for development as effective vaccines against foot-and-mouth disease.

Increased efficacy of an adenovirus-vectored foot-and-mouth disease capsid subunit vaccine expressing nonstructural protein 2B is associated with a specific T cell response.

We previously demonstrated that an adenovirus-based foot-and-mouth disease virus (FMDV) serotype A24 capsid subunit vaccine, Ad5-A24, expressed under the control of a cytomegalovirus promoter (CMV) can protect swine and bovines against homologous challenge, but in a similar approach using swine vaccinated with an Ad5-vectored FMDV O1 Campos vaccine, Ad5-O1C, the animals were only partially protected when challenged at 21 days post-vaccination (dpv). Recently, we demonstrated that inclusion of the complete coding region of nonstructural protein 2B in the Ad5-A24 vector resulted in improved immune responses in pigs. We also found that inclusion of a modified CMV promoter (pCI), Ad5-CI-A24-2B, enhanced the efficacy of the vector. To address the limited immunogenicity of Ad5-O1C, we have produced a new set of Ad5 vectors with the complete 2B coding region under the control of either the original or the modified version of the CMV promoter, Ad5-O1C-2B, or Ad5-CI-O1C-2B, respectively. To evaluate the potency and efficacy of the new vectors we performed 2 sets of experiments in cattle. In the first experiment we compared the original vector with vectors containing the pCI promoter and partial or full-length 2B. All groups were challenged, intradermally in the tongue, at 21 dpv with FMDV O1C. We found that in all vaccinated groups 2 of 4 animals were protected from clinical disease. In the second experiment we directly compared the efficacy of vectors with a partial or full-length 2B under the control of the original CMV promoter. While all animals in the control group developed clinical disease, 2 of 4 animals in the group receiving Ad5-O1C vaccine and 3 of 4 animals in the group receiving Ad5-O1C-2B vaccine were completely protected after challenge. We also observed a 100-fold reduction of virus shedding in Ad5-O1C vaccinated animals and the group receiving Ad5-O1C-2B had an additional 10-fold reduction compared with the Ad5-O1C vaccinated group. There was no difference in the level of neutralizing antibodies in the vaccinated groups. However, we detected a significant antigen specific-CD4(+) and CD8(+) T cell response as early as 1 day post-challenge (dpc) in both Ad5-O1C and Ad5-O1C-2B groups. Interestingly, the group receiving Ad5-O1C-2B had a statistically significant higher antigen specific-CD4(+) and CD8(+) T cell response at 5 dpc and 3 and 5 dpc, respectively, as compared to the Ad5-O1C inoculated group. These results indicate that inclusion of the complete 2B coding region improves the efficacy of Ad5 vaccines against FMDV serotype O and induces specific-CD4(+) and CD8(+) T cell responses that correlate with protection.