Immunopotentiation of a foot-and-mouth disease virus subunit vaccine by interferon alpha

The adjuvant effect of porcine interferon alpha (pIFN-alpha) was examined in swine vaccinated with a replication-defective adenovirus containing foot-and-mouth disease virus (FMDV) A24 capsid and 3C proteinase coding regions (Ad5-A24). Groups of swine were inoculated with either high or low doses of Ad5-A24 in the presence or absence of Ad5-pIFNalpha or with a control Ad5 and challenged by intradermal inoculation in the heel bulb with FMDV at 42 days post-vaccination. After challenge all control animals developed viremia and lesions. Animals receiving low-dose Ad5-A24 had similar clinical disease, but only three of five animals developed viremia, while addition of IFN resulted in a delayed onset of lesions in three animals and only one animal had detectable viremia. Animals vaccinated with high-dose Ad5-A24 had no viremia, significantly fewer lesions and delayed onset of disease compared to the control and low-dose vaccine groups. Four of five pigs vaccinated with high-dose Ad5-A24 plus IFN were completely protected from disease and only one animal had a lesion which was restricted to the site of challenge. Thus, pIFN-alpha enhances the long-term level of protection induced by the Ad5-FMD vaccine, supporting its use as a potential adjuvant in FMD vaccination strategies.     

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.     

Some guidelines for determining foot-and-mouth disease vaccine strain matching by serology

The selection of matching strains for use in outbreaks of foot-and-mouth disease (FMD) virus can be assessed in vivo or by serological r-value determination. Sera from animals involved in vaccine potency and cross-protection trials performed using the “Protection against Podal Generalization” (PPG) test for two serotype A strains were collected and analyzed by the virus neutralization test (VNT) and liquid-phase ELISA (lpELISA) in three laboratories. The average VNT r-values for medium and high serum titer classes from the A₂₄ Cruzeiro vaccinated animals were in line with the A/Arg/01 heterologous PPG outcome for all testing laboratories, suggesting that the vaccine strain A₂₄ Cruzeiro is unlikely to protect against the field isolate A/Arg/01. The corresponding lpELISA r-values were slightly higher and indicate a closer relationship between both strains. Pooling of serum samples significantly reduced the inter-animal and inter-trial variation. The results suggest that a suitable reference serum for vaccine matching r-value experiments might be a pool or a medium to high VNT or lpELISA titer serum. Furthermore, the VNT seems to produce the most reproducible inter-laboratory results. More work is, however, needed in order to substantiate these claims.     

Indirect foot-and-mouth disease vaccine potency testing based on a serological alternative

Foot-and-mouth disease (FMD) vaccine potency testing has historically been performed by experimentally infecting vaccinated cattle. A few alternative approaches to the in vivo challenge test based on the correlation between serum titres of primo-vaccinated cattle and protection against infection have been proposed, but none have been accepted by the European Pharmacopoeia (Ph.Eur.) due to the lack of statistical power and the pooling of data over time. The present study addresses these issues and presents data of 150 cattle vaccinated according to Ph.Eur. standards. Four laboratories took part in the serological testing and different serological assays were used, including virus neutralisation assays and ELISA formats. Models correlating specific anti-FMD virus antibody titres to protection were built using logistic regression followed by Receiver Operating Characteristic (ROC) analysis. The best models accurately predicted the in vivo protection status in 80.0% of the cases. Although differences were observed between laboratories and assays used, the majority of antibody pass-levels, determined using ROC analysis, corresponded to at least 75.0% probability of protection. The indirect potency assessment procedure proposed is at least as precise (repeatability=65.8%, reproducibility=60.7%) as the in vivo test, can be standardised and results in a quantitative PD50 value. The validity of the procedure was also demonstrated.     

Immediate protection of swine from foot-and-mouth disease: a combination of adenoviruses expressing interferon alpha and a foot-and-mouth disease virus subunit vaccine

We have previously shown that swine inoculated with recombinant, replication-defective human adenovirus type 5 containing the porcine interferon alpha gene (Ad5-pIFNalpha) are completely protected when challenged 1 day later with virulent foot-and-mouth disease virus (FMDV). In the current study, we examined the duration of protection afforded swine by Ad5-pIFNalpha and the ability of a combination of Ad5-pIFNalpha and a FMDV subunit vaccine delivered by Ad5-A24 (an Ad5 vector containing the capsid coding region of FMDV serotype A24 Cruzeiro and the 3C proteinase coding region of FMDV serotype A12) to induce immediate as well as long-lasting protection against homologous FMDV challenge. Groups of swine were inoculated with Ad5-pIFNalpha and challenged with virulent FMDV A24 1, 3, 5, and 7 days postinoculation (dpi) or 1 day preinoculation. All animals challenged 1 and 3dpi were completely protected from disease. The animals in the remaining groups had either no clinical signs of disease or clinical signs were delayed and less severe compared to the control group. Swine inoculated with a combination of Ad5-pIFNalpha and Ad5-A24 and challenged 5dpi were all completely protected from disease and developed a significant FMDV-specific neutralizing antibody response.     

Development and serology based efficacy assessment of a trivalent foot-and-mouth disease vaccine

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals throughout the world. The endemicity of this disease in Bangladesh has been causing high economic loss and an impediment to the full potential surge of livestock industries. In Bangladesh, vaccination using imported or locally produced FMD vaccines is the existing practice of controlling the disease, although vaccine failure cases are very common. Hence, to address the problem, the present study was envisaged to develop an effective FMD vaccine tailored to the circulating indigenous foot-and-mouth disease virus (FMDV) strains. Three local circulating FMDVs O/BAN/TA/Dh-301/2016 (MK088170.1), A/BAN/CH/Sa-304/2016 (MK088171.1) and Asia1/BAN/DH/Sa-318/2018 (MH457186.1) isolates were selected as vaccine strains based on recent epidemiology, genetic and antigenic analyses. These serotype O, A and Asia1 vaccine strains showed strong antigenic relationship (r1 > 0.3) with 100% to 75% of the respective circulating viruses. The candidate viruses were successfully inactivated by 3.0 mM binary ethylenimine within 7–10 h after the onset of inactivation. Extrapolation of inactivation kinetics confirmed < 1 log₁₀ TCID₅₀ in a 10000-liter batch liquid preparation after 24 h inactivation cycle. The inactivated virus particles were significantly (p < 0.05) concentrated and the trivalent vaccine was formulated using 6 µg per dose per serotype antigen payload. The trivalent vaccine was administered in divided doses in different groups of cattle. All doses of the vaccine elicited significantly (p < 0.05) higher levels of antibodies as early as 14-day post-vaccination (dpv) and peak antibody titers were achieved in 28 dpv. The ‘full dose’ (6.0 µg per dose per serotype) vaccine elicited antibody titers expected to confer protection in 100% cattle of the respective group and maintained such level of antibodies beyond 180 dpv. Thus, the trivalent FMD vaccine prepared with 6.0 µg antigen per dose per serotype of the selected candidate viruses will confer protection against circulating FMDVs of Bangladesh and its neighboring countries.     

Retrospective evaluation of foot-and-mouth disease vaccine effectiveness in Turkey

Foot-and-mouth disease (FMD) is present in much of Turkey and its control is largely based on vaccination. The arrival of the FMD Asia-1 serotype in Turkey in 2011 caused particular concern, spreading rapidly westwards across the country towards the FMD free European Union. With no prior natural immunity, control of spread would rely heavily on vaccination.     

Reducing animal experimentation in foot-and-mouth disease vaccine potency tests

The World Organisation for Animal Health (OIE) Terrestrial Manual and the European Pharmacopoeia (EP) still prescribe live challenge experiments for foot-and-mouth disease virus (FMDV) immunogenicity and vaccine potency tests. However, the EP allows for other validated tests for the latter, and specifically in vitro tests if a “satisfactory pass level” has been determined; serological replacements are also currently in use in South America. Much research has therefore focused on validating both ex vivo and in vitro tests to replace live challenge. However, insufficient attention has been given to the sensitivity and specificity of the “gold standard” in vivo test being replaced, despite this information being critical to determining what should be required of its replacement. This paper aims to redress this imbalance by examining the current live challenge tests and their associated statistics and determining the confidence that we can have in them, thereby setting a standard for candidate replacements. It determines that the statistics associated with the current EP PD₅₀ test are inappropriate given our domain knowledge, but that the OIE test statistics are satisfactory. However, it has also identified a new set of live animal challenge test regimes that provide similar sensitivity and specificity to all of the currently used OIE tests using fewer animals (16 including controls), and can also provide further savings in live animal experiments in exchange for small reductions in sensitivity and specificity.     

Effective synthetic peptide vaccine for foot-and-mouth disease in swine

We have designed a peptide-based vaccine for foot-and-mouth disease (FMD) effective in swine. The peptide immunogen has a G-H loop domain from the VP1 capsid protein of foot-and-mouth disease virus (FMDV) and a novel promiscuous T helper (Th) site for broad immunogenicity in multiple species. The G-H loop VP1 site was optimised for cross-reactivity to FMDV by the inclusion into the peptide of cyclic constraint and adjoining sequences. The incorporation of consensus residues into the hypervariable positions of the VP1 site provided for broad immunogenicity. The vaccine protected 20 out of 21 immunised pigs from infectious challenge by FMDV O1 Taiwan using peptide doses as low as 12.5 microg, and a mild adjuvant that caused no lesions. A safe chemically-defined product would have considerable advantages for vaccination against FMD.     

Characteristics of serology-based vaccine potency models for foot-and-mouth disease virus

Background

Foot-and-mouth disease (FMD) vaccine potency testing involves hundreds of animals each year. Despite considerable efforts during the past decades, a challenge-free alternative vaccine potency test to replace the European protective dose 50% test (PD₅₀) has not been implemented yet. The aim of the present study was to further characterize the properties of serological vaccine potency models.

Methods

Logistic regression models were built for 5 serological assays from 3 different laboratories. The serum samples originated from 5 repeated PD₅₀ vaccine potency trials with a highly potent A/IRN/11/96 vaccine. Receiver Operating Characteristic analysis was used to determine a serological pass mark for predicting in vivo protected animals. Subsequently, an estimated PD₅₀ was calculated and the serotype dependency of the logistic models was investigated.

Results

Although differences were observed between the laboratories and the serological assays used, the logistic models accurately predicted the in vivo protection status of the animals in 74–93% of the cases and the antibody pass levels corresponded to 84–97% of protection, depending on the serological assay used. For logistic models that combine different serotypes, the model fit can be increased by inclusion of a serotype factor in the logistic regression function.

Conclusions

The in vitro estimated PD₅₀ method may be at least as precise as the in vivo PD₅₀ test and may accurately predict the PD₅₀ content of a vaccine. However, the laboratory-effect and the serotype-dependency should be further investigated.     

A ferritin nanoparticle vaccine for foot-and-mouth disease virus elicited partial protection in mice

Foot-and-mouth disease (FMD) is an acute, febrile, and highly contagious infectious disease common in cloven-hoofed animals. Outbreaks and epidemics of FMD can result in major economic losses of livestock. Using ferritin nanoparticles as the scaffold for an antigen can enhance the immunogenicity of the subunit vaccine and provide possible protection against FMD. We used a baculovirus expression system to express four recombinant proteins (VP1, VP1-Ft, G-H loop-Ft, and ferritin) and the protective immunity of the FMD ferritin nanoparticle vaccines was evaluated in mice. The recombinant subunit vaccines containing VP1, VP1-Ft, and G-H loop-Ft proteins significantly increased FMDV-specific IgG and IgG subclass antibody titers compared with the PBS group, as well as enhancing splenocyte proliferation and the expression of IL-4 and IFN-γ. The VP1 and VP1-Ft vaccines provided survival rates of 55.6% and 66.7%, respectively. The G-H loop-Ft vaccine provided a 77.8% survival rate compared with 100% survival in the inactivated vaccine group. The partial survival provided by the ferritin nanoparticle vaccines indicated that further study of the effects of the fused ferritin nanoparticle FMDV vaccines in animals is warranted.     

Application of mouse model for effective evaluation of foot-and-mouth disease vaccine

Efficacy evaluation of foot-and-mouth disease (FMD) vaccines has been conducted in target animals such as cows and pigs. In particular, handling FMD virus requires a high level of biosafety management and facilities to contain the virulent viruses. The lack of a laboratory animal model has resulted in inconvenience when it comes to using target animals for vaccine evaluation, bringing about increased cost, time and labor for the experiments. The FMD mouse model has been studied, but most FMD virus (FMDV) strains are not known to cause disease in adult mice. In the present study, we created a series of challenge viruses that are lethal to adult C57BL/6 mice. FMDV types O, A, and Asia1, which are related to frequent FMD outbreaks, were adapted for mice and the pathogenesis of each virus was evaluated in the mouse model. Challenge experiments after vaccination using in-house and commercial vaccines demonstrated vaccine-mediated protection in a dose-dependent manner. In conclusion, we propose that FMD vaccine evaluation should be carried out using mouse-adapted challenge viruses as a swift, effective efficacy test of experimental or commercial vaccines.     

Efficacy of foot-and-mouth disease vaccine in pigs with single dose immunization

Two experiments were conducted to demonstrate the efficacy of a commercial foot-and-mouth disease (FMD) vaccine in pigs born to well-vaccinated sows at various ages with a single injection under field conditions. The first experiment showed that single dose vaccination of pigs could be conducted at an age younger than 10 weeks. Second experiment demonstrated that pigs vaccinated once at the age of 8 weeks had mean serum neutralization (SN) titer of 1.89+/-0.95 log(10)SN(50) with full protection by challenge test at the age of 24 weeks. Results indicate that the most appropriate age for single dose FMD vaccination in pigs born to well-vaccinated sows would be at 8 weeks.     

Marker vaccine potential of a foot-and-mouth disease virus with a partial VP1 G-H loop deletion

Previous work in cattle and pigs demonstrated that protection against foot-and-mouth disease (FMD) could be achieved following vaccination with chimeric foot-and-mouth disease virus (FMDV) vaccines, in which the VP1 G-H loop had been substituted with that from another serotype. This indicated that the VP1 G-H loop may not be essential for the protection of natural hosts against FMDV. If this could be substantiated there would be potential to develop FMD marker vaccines, characterised by the absence of this region. Here, we investigate the serological responses to vaccination with a virus with a partial VP1 G-H loop deletion in order to determine the likelihood of achieving protection and the potential of this virus as a marker vaccine. Inactivated, oil adjuvanted, vaccines, consisting of chemically inactivated virus with or without a partially deleted VP1 G-H loop, were used to immunise cattle. Serum was collected on days 0, 7, 14 and 21 and antibody titres calculated using the virus neutralisation test (VNT) to estimate the likelihood of protection.     

Novel low-molecular-weight synthetic vaccine against foot-and-mouth disease containing a potent B-cell and macrophage activator

Most synthetic peptide vaccines described to date are effective only in combination with proteins and Freund's adjuvant. The work describes a novel completely synthetic virus peptide vaccine, which consists of a synthetic activator of B cells and macrophages, covalently linked to an amphiphilic alpha-helical T-cell epitope. The low-molecular-weight vaccine of 3.4 kDa developed against foot-and-mouth disease virus (FMDV) is composed of a synthetic VP1 (135-154) with a sequence homologous to an FMDV protein and the adjuvant tripalmitoyl-S-glyceryl-cysteinylserylserine (P3CSS). P3CSS is the synthetic analogue of the N-terminal part of the lipoprotein from Gram-negative bacteria. The antigenic determinant VP1 (135-154) is an alpha-helix as shown by circular dichroism. The resulting novel type of vaccine tripalmitoyl-S-glyceryl-cysteinylserylseryl-FMDV-VP1 (135-154) induces a long-lasting high protection against foot-and-mouth disease and serotype-specific virus-neutralizing antibodies in guinea-pigs after a single administration without any additional adjuvant or carrier. In contrast to other simple fatty acid conjugates this new type of vaccine contains a built-in adjuvant with high affinity to both B and T lymphocytes.     

A simple and rapid assay to evaluate purity of foot-and-mouth disease vaccine before animal experimentation

Currently, foot-and-mouth disease (FMD) vaccine purity is tested in cattle to detect antibodies against the non-structural protein (NSP) after repeated immunization with the final vaccine product. In case of vaccine failure, the manufacturing company would suffer significant economic loss. To prevent such unfortunate losses with the final vaccine product, in vitro testing is required to quantitate an NSP antigen during the manufacturing process prior to animal experiments. A novel lateral-flow assay device was developed using a monoclonal antibody (MAb) against the 3B NSP. To determine the minimal amount of NSP required to elicit antibodies in livestock, goats were immunized several times with various concentrations of either the recombinant 3AB (rec.3AB) protein or FMD virus culture supernatant. Antibodies against 3AB were elicited after a second immunization with 10.6 ng to 42.5 ng of rec.3AB and a third immunization with a 10-fold diluted FMD virus culture supernatant in goats. The lateral-flow assay device detected the minimal amount of rec.3AB and native NSP in FMD virus culture supernatant required to induce NSP antibodies in goats. The in vitro assay device is simple and economical, provides rapid results, and should be useful for FMD vaccine-manufacturing companies prior to conducting animal experiments to test the vaccine purity.     

A scale-down model of 4000-L cell culture process for inactivated foot-and-mouth disease vaccine production

The anticipated increasing demand for inactivated foot-and-mouth (FMD) disease vaccine calls for its larger production capacity, while development of a large-scale process typically requires high running cost and has very limited experimental throughput at manufacturing scale. Thus, an economic scale-down model of representing a large-scale process becomes necessary and essential. In this study, we used a systematic approach to establish a scale-down model representing a 4000-L culture process for FMD vaccine production by suspension BHK-21 cells. In detail, we firstly compared hydrodynamic properties of three bioreactors (14-L, 800-L and 4000-L) under three different conditions (equivalent mixing time, equivalent shear stress and equivalent volumetric power). We figured out equivalent volumetric power (P/V) potentially as an appropriate scale-down strategy, since it resulted in comparable calculated hydrodynamic parameters among three bioreactors. Next, we used computational fluid dynamics (CFD) simulation to provide more details about hydrodynamic environments inside the bioreactors, which supports the reliability of this scale-down strategy. Finally, we compared cell growth, metabolites, vaccine productivity and product quality attributes during FMD vaccine production by BHK-21 cells and observed very close performances among three bioreactors, which once again demonstrates the robustness of this scale-down model. This scale-down strategy can be applied to study variations and critical quality attributes (CQAs) in the resultant production process based on quality by design (QbD) principles, aiming at further more efficient optimization of vaccine production.     

Prototype Alzheimer's disease epitope vaccine induced strong Th2-type anti-Abeta antibody response with Alum to Quil A adjuvant switch

Beta-amyloid (Abeta) peptide has been proposed to be a causal factor in Alzheimer's disease (AD). Currently being investigated, active and passive Abeta-immunotherapy significantly reduce Abeta plaque deposition, neuritic dystrophy, and astrogliosis in the brains of APP transgenic (APP/Tg) mice. Immunization with Abeta42 formulated in the Th1-type adjuvant QS21 was beneficial for AD patients with significant titers of anti-Abeta antibodies, however, 6% of participants developed meningoencephalitis, likely due to anti-Abeta-specific autoimmune Th1 cells. Thus, successful Abeta vaccination requires the development of strong antibody responses without Th1-type cellular immunity. In this study, we compared the induction of humoral immune responses with Th1-type (Quil A) and Th2-type (Alum) adjuvants singly and in combination, using our novel epitope vaccine composed of self B cell epitope Abeta(1-15) and foreign T cell epitope PADRE (PADRE-Abeta(1-15)-MAP). Formulated in Quil A, this vaccine resulted in significantly higher anti-Abeta antibody responses in both BALB/c (H-2d) and C57BL/6 (H-2b) mice, compared with Alum. Anti-Abeta antibodies induced by Alum were predominantly IgG1 type accompanied by lower levels of IgG2a and IgG2b. Quil A induced robust and almost equal titers of anti-Abeta antibodies of IgG1 and IgG2a isotypes and slightly lower levels of IgG2b. Switching adjuvants from Alum to Quil A induced higher concentrations of antibodies than injections with Alum only, however slightly lower than Quil A only. Switching both adjuvants did not change the profile of antibody responses generated by the initial adjuvant injected. These results suggest that switching from Alum to Quil A would be beneficial for AD patients because anti-Abeta antibody production was enhanced without changing the initially generated and likely beneficial Th2-type humoral response.