Application of the Nagoya Protocol to veterinary pathogens: concerns for the control of foot-and-mouth disease

The Nagoya Protocol is an international agreement adopted in 2010 (and entered into force in 2014) which governs access to genetic resources and the fair and equitable sharing of benefits from their utilisation. The agreement aims to prevent misappropriation of genetic resources and, through benefit sharing, create incentives for the conservation and sustainable use of biological diversity. While the equitable sharing of the benefits arising from the utilisation of genetic resources is a widely accepted concept, the way in which the provisions of the Nagoya Protocol are currently being implemented through national access and benefit-sharing legislation places significant logistical challenges on the control of transboundary livestock diseases such as foot-and-mouth disease (FMD). Delays to access FMD virus isolates from the field disrupt the production of new FMD vaccines and other tailored tools for research, surveillance and outbreak control. These concerns were raised within the FMD Reference Laboratory Network and were explored at a recent multistakeholder meeting hosted by the European Commission for the Control of FMD. The aim of this paper is to promote wider awareness of the Nagoya Protocol, and to highlight its impacts on the regular exchange and utilisation of biological materials collected from clinical cases which underpin FMD research activities, and work to develop new epidemiologically relevant vaccines and other diagnostic tools to control the disease

Innate Immune Response to Rift Valley Fever Virus in Goats

Rift Valley fever (RVF), a re-emerging mosquito-borne disease of ruminants and man, was endemic in Africa but spread to Saudi Arabia and Yemen, meaning it could spread even further. Little is known about innate and cell-mediated immunity to RVF virus (RVFV) in ruminants, which is knowledge required for adequate vaccine trials. We therefore studied these aspects in experimentally infected goats. We also compared RVFV grown in an insect cell-line and that grown in a mammalian cell-line for differences in the course of infection. Goats developed viremia one day post infection (DPI), which lasted three to four days and some goats had transient fever coinciding with peak viremia. Up to 4% of peripheral blood mononuclear cells (PBMCs) were positive for RVFV. Monocytes and dendritic cells in PBMCs declined possibly from being directly infected with virus as suggested by in vitro exposure. Infected goats produced serum IFN-γ, IL-12 and other proinflammatory cytokines but not IFN-α. Despite the lack of IFN-α, innate immunity via the IL-12 to IFN-γ circuit possibly contributed to early protection against RVFV since neutralising antibodies were detected after viremia had cleared. The course of infection with insect cell-derived RVFV (IN-RVFV) appeared to be different from mammalian cell-derived RVFV (MAM-RVFV), with the former attaining peak viremia faster, inducing fever and profoundly affecting specific immune cell subpopulations. This indicated possible differences in infections of ruminants acquired from mosquito bites relative to those due to contact with infectious material from other animals. These differences need to be considered when testing RVF vaccines in laboratory settings

Immunisation with a Multivalent, Subunit Vaccine Reduces Patent Infection in a Natural Bovine Model of Onchocerciasis during Intense Field Exposure

Human onchocerciasis, caused by the filarial nematode Onchocerca volvulus, is controlled almost exclusively by the drug ivermectin, which prevents pathology by targeting the microfilariae. However, this reliance on a single control tool has led to interest in vaccination as a potentially complementary strategy. Here, we describe the results of a trial in West Africa to evaluate a multivalent, subunit vaccine for onchocerciasis in the naturally evolved host-parasite relationship of Onchocerca ochengi in cattle. Naïve calves, reared in fly-proof accommodation, were immunised with eight recombinant antigens of O. ochengi, administered separately with either Freund's adjuvant or alum. The selected antigens were orthologues of O. volvulus recombinant proteins that had previously been shown to confer protection against filarial larvae in rodent models and, in some cases, were recognised by serum antibodies from putatively immune humans. The vaccine was highly immunogenic, eliciting a mixed IgG isotype response. Four weeks after the final immunisation, vaccinated and adjuvant-treated control calves were exposed to natural parasite transmission by the blackfly vectors in an area of Cameroon hyperendemic for O. ochengi. After 22 months, all the control animals had patent infections (i.e., microfilaridermia), compared with only 58% of vaccinated cattle (P = 0.015). This study indicates that vaccination to prevent patent infection may be an achievable goal in onchocerciasis, reducing both the pathology and transmissibility of the infection. The cattle model has also demonstrated its utility for preclinical vaccine discovery, although much research will be required to achieve the requisite target product profile of a clinical candidate

Accessory-Cell-Mediated Activation of Porcine NK Cells by Toll-Like Receptor 7 (TLR7) and TLR8 Agonists ▿ †

The induction of innate immune responses by toll-like receptor (TLR) agonists is the subject of intense investigation. In large part, this reflects the potential of such compounds to be effective vaccine adjuvants. For that reason, we analyzed the activation of innate cells in swine by TLR7 and TLR8 agonists. These agonists activated porcine NK cells by increasing gamma interferon (IFN-γ) expression and perforin storage. The activation of porcine NK cells was mediated by accessory cells, since their depletion resulted in reduced cytotoxicity toward target cells. Accessory cells were stimulated to produce interleukin 12 (IL-12), IL-15, IL-18, and IFN-α after treatment with TLR7 or TLR8 agonists. Neutralization of these cytokines reduced but did not completely inhibit the induction of NK cell cytotoxicity. Direct stimulation of NK cells with TLR7 or TLR8 agonists resulted in minimal cytotoxicity but levels of IFN-γ equivalent to those detected in the presence of accessory cells. Porcine NK cells express both TLR7 and TLR8 mRNAs, and treatment with these TLR agonists induced higher mRNA expression levels of TRAIL and IL-15Rα, which may contribute to the activity of NK cells. These data indicate that TLR7 and TLR8 agonists indirectly or directly activate porcine NK cells but that optimum levels of activation require cytokine secretion by accessory cells activated by these compounds. Interestingly, NK cells activated by TLR7 or TLR8 agonists were cytotoxic against foot-and-mouth disease virus (FMDV)-infected cells in vitro, indicating that these TLR agonists may be beneficial as adjuvants to stimulate the innate immunity against FMDV

Combining a Universal Capture Ligand and Pan-Serotype Monoclonal Antibody to Develop a Pan-Serotype Lateral Flow Strip Test for Foot-and-Mouth Disease Virus Detection

Foot-and-mouth disease virus (FMDV) causes FMD, a highly contagious disease of cloven-hoofed animals including cattle, goats, pigs and sheep. Rapid detection of FMDV is critical to limit the devastating economic losses due to FMD. Current laboratory methods for FMDV detection such as virus isolation, real-time reverse transcription PCR and antigen detection enzyme-linked immunosorbent assay (AgELISA) are labor-intensive, requiring trained personnel and specialized equipment. We present the development and validation of a pan-serotype lateral flow strip test (LFST) that uses recombinant bovine integrin αvβ6 as a universal capture ligand and a pan-serotype monoclonal antibody (mAb) to detect FMDV. The LFST detected all seven FMDV serotypes, where the diagnostic sensitivity was comparable to the AgELISA, and the diagnostic specificity was 100% without cross-reactivity to other viruses causing vesicular disease in livestock. This rapid test will be useful for on-site FMDV detection, as well as in laboratories in endemic countries where laboratory resources are limited

Combining a Universal Capture Ligand and Pan-Serotype Monoclonal Antibody to Develop a Pan-Serotype Lateral Flow Strip Test for Foot-and-Mouth Disease Virus Detection

Foot-and-mouth disease virus (FMDV) causes FMD, a highly contagious disease of cloven-hoofed animals including cattle, goats, pigs and sheep. Rapid detection of FMDV is critical to limit the devastating economic losses due to FMD. Current laboratory methods for FMDV detection such as virus isolation, real-time reverse transcription PCR and antigen detection enzyme-linked immunosorbent assay (AgELISA) are labor-intensive, requiring trained personnel and specialized equipment. We present the development and validation of a pan-serotype lateral flow strip test (LFST) that uses recombinant bovine integrin αvβ6 as a universal capture ligand and a pan-serotype monoclonal antibody (mAb) to detect FMDV. The LFST detected all seven FMDV serotypes, where the diagnostic sensitivity was comparable to the AgELISA, and the diagnostic specificity was 100% without cross-reactivity to other viruses causing vesicular disease in livestock. This rapid test will be useful for on-site FMDV detection, as well as in laboratories in endemic countries where laboratory resources are limited

Immunopathogenesis of severe acute respiratory disease in Zaire ebolavirus-infected pigs.

Ebola viruses (EBOV) are filamentous single-stranded RNA viruses of the family Filoviridae. Zaire ebolavirus (ZEBOV) causes severe haemorrhagic fever in humans, great apes and non-human primates (NHPs) with high fatality rates. In contrast, Reston ebolavirus (REBOV), the only species found outside Africa, is lethal to some NHPs but has never been linked to clinical disease in humans despite documented exposure. REBOV was isolated from pigs in the Philippines and subsequent experiments confirmed the susceptibility of pigs to both REBOV and ZEBOV with predilection for the lungs. However, only ZEBOV caused severe lung pathology in 5-6 weeks old pigs leading to respiratory distress. To further elucidate the mechanisms for lung pathology, microarray analysis of changes in gene expression was performed on lung tissue from ZEBOV-infected pigs. Furthermore, systemic effects were monitored by looking at changes in peripheral blood leukocyte subsets and systemic cytokine responses. Following oro-nasal challenge, ZEBOV replicated mainly in the respiratory tract, causing severe inflammation of the lungs and consequently rapid and difficult breathing. Neutrophils and macrophages infiltrated the lungs but only the latter were positive for ZEBOV antigen. Genes for proinflammatory cytokines, chemokines and acute phase proteins, known to attract immune cells to sites of infection, were upregulated in the lungs, causing the heavy influx of cells into this site. Systemic effects included a decline in the proportion of monocyte/dendritic and B cells and a mild proinflammatory cytokine response. Serum IgM was detected on day 5 and 6 post infection. In conclusion, a dysregulation/over-activation of the pulmonary proinflammatory response may play a crucial role in the pathogenesis of ZEBOV infection in 5-6 weeks old pigs by attracting inflammatory cells to the lungs

Virus yield from goat monocyte-derived dendritic cells (MoDCs) inoculated with RVFV.

<p>MoDCs were infected with insect cell-derived RVFV (IN-RVFV) or mammalian cell –derived RVFV (MAM-RVFV) and after 24 h, the virus in supernatants was quantified by plaque assay. Histograms represent means + standard deviation.</p

Changes in cell population frequencies in peripheral blood mononuclear cells in RVFV-infected goats.

<p>Column A: PBMCs from IN-RVFV-infected goats; Column B: PBMCs from MAM-RVFV-infected goats; Column C: Cell frequencies expressed as a percentage of pre-infection value for —▪— IN-RVFV and —♦—MAM RVFV- infected goats (n = 4 goats each). Data points in column A and B represent individual animals and the line represents the means. In column C data points represent means + standard deviation (error bars). MAM-RVFV = RVFV produced in the mammalian cell line Vero E6, IN-RVFV = RVFV produced in the insect cell line C6/36.</p

Adaptive cell mediated immunity in RVFV-infected goats.

<p>Means of PBMC IFN-γ response from IN-RVFV-infected goats are represented by the open histograms and MAM-RVFV infected goats by the filled histograms. Error bars represent standard deviation of means (n = 4 goats each). MAM-RVFV = RVFV produced in the mammalian cell line Vero E6, IN-RVFV = RVFV produced in the insect cell line C6/36.</p