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Chairs: Professor Sandra Adams (University of Stirling, United Kingdom) and Dr Thao Ngo (Biotechnology Centre of Ho Chi Minh City, Vietnam)

Expression systems for Bacillus subitilis and its potential applications as vaccine delivery vector

Dr Nguyen Duc Hoang

Vietnam National University, Vietnam

Bacillus species are essential hosts for production of proteins at industrial levels, 60% of industrial enzymes produced from these species. This model Gram-positive bacterium is considered as GRAS, endotoxin-free, allowing high amounts of protein secretion. In this study, we want to introduce the expression systems for Bacillus subtilis and their potential use as vaccine vectors.

We used molecular biology methods, focusing on promoters to develop expression systems for B. subtilis to produce proteins in the cytoplasm. We also explored the sortase/sorting mechanism to anchor proteins covalently on the surface of B. subtilis vegetative cells. The authors generated B. subtilis strains with a model antigen to express LTB antigen in the cytoplasm, in the culture medium, on the cell surface of the vegetative cells. The LTB-expressing B. subtilis strains were introduced orally into mice and the immune responses, IgG levels were measured. Besides, we also tested the use of B. subtilis expressing virus coat protein of Betanoda virus for immunization and challenge study of sea bass fish.

We developed expression vectors for overexpression of recombinant proteins in the cytoplasm and in the culture medium, and on the cell surface of B. subtilis cells. The expression systems could be checked by using different reporters that could be expressed in the inducible and inducer-free manners. For applications as a vaccine delivery vector, the immune response study in mice showed that the LTB-expressing B. subtilis strains could be used to deliver the antigen orally and induce the immune response. Also, the specific IgM antibodies in the fishes against the Betanoda-coat protein increased when introducing the B. subtilis expressing the coat protein. Conclusion: The results infer that the available genetics tools would be potentially exploited to produce recombinant proteins and generate antigen-expressing B. subtilis strains for a multivalent vaccine.

How adjuvants work: progress and challenges for use in fish

Dr Carolina Tafalla

Animal Health Research Center (CISA-INIA), Madrid, Spain

From all points of view, vaccination is the most adequate method to control infectious diseases that threaten the expansion of the aquaculture industry worldwide. Unfortunately, in fish, most vaccines provide insufficient protection on their own and require the use of adjuvants to increase their efficacy. An added problem in aquaculture is derived from the fact that most commercialized vaccines have to be delivered by injection, usually along with an oleic adjuvant. Despite their relative efficacy, these adjuvants often generate important side effects in vaccinated fish. Furthermore, vaccinating fish by injection is a labour-intensive activity that provokes a great amount of stress, known to often condition the immune response mounted to the antigen and handling mortalities. In this context, in aquaculture, the search for novel adjuvants that increase the efficacy of the vaccine without generating unwanted side effects, should go along with a search for adjuvants suitable for mass delivery strategies (such as oral or immersion). In this talk, we will review the work undertaken to date in fish concerning both traditional and new generation adjuvants, focusing in the work that our group has been carrying in the past years to identify adjuvants that are suitable for oral vaccination.

Future of vaccination in warm water aquaculture

Mr Lee Yeng Sheng

MSD Animal Health Innovation Pte Ltd, Singapore

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Chairs: Professor Bryan Charleston (The Pirbright Institute, United Kingdom) & Dr. Yanmin Li (Lanzhou Veterinary Research Institute, China)

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Dr Vivek Kapur

Pennsylvania State University, State College, PA, United States

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High expression of an echinococcosis vaccine antigen EG95 in yeasts and its application

Professor Wanzhong Jia

Lanzhou Veterinary Research Institute, China

Cystic echinococcosis (CE), one of the parasitic zoonoses, which is caused by the larval stage of Echinococcus granulosus, may cause not only morbidity, disability and death in humans, but also substantial economic losses in animal husbandry. Breakthroughs have been made towards the development of practical and recombinant vaccines in sheep and cattle to interrupt the transmission of disease from dogs to animals and humans. For example, experimental studies and trials or field trials with the EG95 vaccine, performed in sheep and cattle against experimental challenge infection and natural infection with E. granulosus respectively in New Zealand, Australia, Argentina, China and other countries, demonstrated that EG95 vaccination conferred a high protection against E. granulosus infections or echinococcosis. Up to date, EG95 antigen has been expressed in Escherichia coli as glutathione S-transferase (GST) or His-tag fusion proteins.  However, another taeniid protective antigen, designed TSOL18, was highly produced in Pastoris pichia in a secreted and glycosylated form described by CAI XP et al (2008), which closely resembles the native TSOL18 antigen in Taenia solium oncospheres.

In our work, production of EG95 antigen using P. pichia and its antigenicity were explored. The results were as following: (1) Genetically modified P. pichia strains were constructed and screened with high level of secreted expression EG95 antigen. Under high density fermentation (a 5L fermenter) conditions, total protein content per litre of culture supernatant reached over 4 grams (BCA method) with a purity of approximate 90% EG95 antigen. (2) N-glycosylation of recombinant EG95 antigen could be changed or controlled through carbon sources etc. (3) EG95 antigen from yeasts performed good immunogenicity, which could stimulate higher production of antibodies than GST-EG95 antigen. (4) Indirect ELISA kits based on purified P. pichia origin of EG95 antigen had much better performance in detecting anti-EG95 antibodies in vaccinated animals than those based on purified E. coli origin of EG95-His fusion antigen.       

In conclusion, a high level of EG95 expression based-on yeast system has been successfully developed. Such antigen expression technology based on yeast cells has potential to produce taeniid protective glycoproteins such as TSOL18 and EG95. The EG95 antigen is being used for the development of novel engineered vaccines against ovine and bovine echinococcosis and ELISA detection kits for the vaccinated animals. Also, purified EG95 antigen without tags has potential to develop vaccines for human usage.

Why is control of brucellosis in low- and middle-income countries making so little progress?

Professor Jacques Godfroid

University of Tromsø, Norway

Brucellosis is not only a threat to livestock but also a global public health issue. Vaccination of livestock is a critical strategy for brucellosis control. Making use of safe and efficient vaccines, and implementing sound programs endorsed by all stakeholders, are equally important for protecting livestock and humans.

Currently, there are three commercially available vaccines recommended by the World Organisation for Animal Health (OIE). Brucella abortus strain 19 and Brucella melitensis strain Rev.1 remain the reference vaccines for the control of Brucella infections in cattle and in sheep and goats, respectively. The rough B. abortus strain RB51 vaccine has also become an official vaccine for prevention of B. abortus infection in cattle in some countries. There are no licensed human, swine, canine and wildlife anti-brucellosis vaccines available.

When all categories of new experimental anti-Brucella vaccines are analysed together, the trend line clearly demonstrates that, in the mouse model of infection, there is no improvement of the protection indexes over the past 30 years.

In low- and middle-income countries (LMICs), most brucellosis studies report seroprevalence surveys and risk factors analysis. Only a handful of studies report interventions in some LMICs (ie mass Rev. 1 vaccination of sheep and goats), with preliminary success. While strategies for controlling abortion induced by Rev. 1 in pregnant sheep and goats have been implemented, S19 mass vaccination in cattle (and water buffalo) is often not accepted by farmers because it induces abortion in pregnant animals. Vaccination of pregnant animals with RB51 or S19 (reduced dose) is safer than S19 (full dose), but may still induce abortion.

In order to make progress, it is necessary to better understand the mucosal immune response induced by Brucella infection to prevent horizontal transmission and to assess curative vaccination in its ability to reduce vertical transmission. Simulation modelling of different interventions (vaccination, serological testing), may help deciding the best strategy to be implemented and for how long.

Stakeholders should be informed that commitment needs to last for decades. Haphazard vaccination is always counterproductive. If vaccination is discontinued, return to the pre-vaccination situation will happen very rapidly, potentially ruining decades of interventions and investments.

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Dr Yu Qiu

OIE Southeast Asia and China Foot-and-Mouth Disease Campaign, Thailand

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AgResults foot-and-mouth disease (FMD) vaccine challenge project

Dr Jeremy Salt

GALVmed, United Kingdom

The AgResults Foot and Mouth Disease (FMD) Vaccine Challenge Project is an eight-year, US$15.8 million prize competition that supports the development and uptake of high-quality FMD vaccines tailored to meet the needs of Eastern Africa. The prize is structured as a cost-share that reduces the cost-per-dose for buyers, enabling public and private sector actors to better combat FMD through more consistent purchases of the new vaccines.

In order to meet the needs of Eastern Africa, AgResults is looking for animal health companies, with experience in the development and commercialisation of FMD vaccines, to develop and register vaccines that are safe, efficacious and viable for use against the currently circulating strains in the region. Vaccines registered in at least two of the AgResults target countries that meet all the requirements set out in the Official Competition Rules will be eligible to participate in this cost-share initiative.

To support the adoption of new high-quality vaccines in the region, AgResults has established a cost-share mechanism which will reduce the purchase cost-per-dose. Each year, AgResults commits to funding a portion of the purchase price of the vaccine for a specified volume of vaccine doses. AgResults will provide funding directly to vaccine manufacturers, on behalf of the buyers. The cost-share will be available for 4.5 years after the first eligible vaccine is approved by the Project’s Judging Panel. The volume of doses available for the cost-share benefit will gradually increase over the four-year period, from 2 million doses in Year 1 to 5 million in Year 4, while the level of cost-share support will gradually decrease, to help buyers prepare for price adjustments that will occur after the project has closed.

Co-hosted by One Health Poultry Hub

Chaired by Professor Fiona Tomley (Royal Veterinary College, United Kingdom) & Professor Hualan Chen (Harbin Veterinary Research Institute, China)

New approaches for the development of recombinant glycoconjugate vaccines for poultry, pigs and ruminants

Professor Brendan Wren

London School of Hygiene & Tropical Medicine, United Kingdom

This lecture will describe the origin of the production of recombinant glycoconjugate vaccines developed through the functional characterization of an N-linked OTase general glycosylation system from the enteric pathogen Campylobacter jejuni. It will describe how these basic studies were used to re-constitute the glycosylation system in E. coli, which allowed for the first time the production of recombinant glycoproteins through a process termed Protein Glycan Coupling Technology (PGCT). The major application of PGCT is in the construction of affordable recombinant glycoconjugate vaccines. To date, the technology has been used to produce novel recombinant Campylobacter, Shigella, Streptococcus pneumoniae, Francisella, Burkholderia pseudomallei, E. coli and MRSA glycoconjugate vaccines. These have been shown to be protective and some are in clinical trials.

I will describe the applications and limitations of PCGT for the construction of low-cost recombinant glycoconjugate vaccines in three areas; (i) designing novel vaccines against pathogens where no current vaccine exists (eg Burkholderia pseudomallei), (ii) improving existing glycoconjugate vaccines (eg pneumococcal vaccine), and (iii) affordable glycoconjugate vaccines for the veterinary market (eg poultry, pigs and ruminants).

From a One Health perspective, low-cost glycoconjugate vaccines for animals not only adds to economic prosperity but can reduce animal suffering and the zoonotic transmission of disease to humans. Furthermore, it will reduce usage of antibiotics in the livestock industry and the subsequent spread of antimicrobial resistance.

Successful control of H7N9 influenza in China

Professor Hualan Chen

Harbin Veterinary Research Institute, Chinese Academy Agricultural Sciences, China

The H7N9 viruses that emerged in China in 2013 were nonpathogenic in chickens but mutated to a highly pathogenic form in early 2017 and caused severe disease outbreaks in chickens. The H7N9 influenza viruses have caused five waves of human infection, with almost half of the total number of human cases (766 of 1,567) being reported in the fifth wave, raising concerns that even more human infections could occur in the sixth wave. In September 2017, an H5/H7 bivalent inactivated vaccine for chickens was introduced, and the H7N9 virus prevalence in poultry after vaccination was successfully controlled based on the large-scale virus surveillance results. More importantly, since the H7N9 vaccine was applied in poultry, only four H7N9 human cases were reported in over two years (three H7N9 human cases were reported between October 1, 2017 and September 30, 2018, and one human case was reported between October 1, 2018 and September 30, 2019), indicating that vaccination of poultry successfully eliminated human infection with H7N9 virus. These facts emphasize that active control of animal disease is extremely important for zoonosis control and human health protection.

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Chairs: Dr Simon Graham (The Pirbright Institute, United Kingdom) & Dr Filip Claes (Emergency Center for Transboundary Animal Diseases (ECTAD), Thailand)

Architecture of African swine fever virus and implications for viral assembly

Professor Xiangxi Wang

Chinese Academy of Sciences, China

African swine fever virus (ASFV) is a giant and complex DNA virus that causes a highly contagious and often lethal swine disease without vaccine available. Using an optimized image reconstruction strategy, we solved the ASFV capsid structure up to 4.1-angstroms, which is built from 17,280 proteins, including one major (p72) and four minor capsid proteins (M1249L, p17, p49 and H240R), organized into pentasymmetrons and trisymmetrons. The atomic structure of the p72 informs putative conformational epitopes, distinguishing ASFV from other nucleocytoplasmic large DNA viruses (NCLDV). The minor capsid proteins form a complicated network below the outer capsid shell, stabilizing the capsid by holding adjacent capsomers together. Acting as core organizers, 100-nm long M1249L proteins run along each edge of trisymmetrons bridging two neighboring pentasymmtrons and form extensive intermolecular networks with other capsid proteins, driving the formation of the capsid framework. These structural details unveil the basis of capsid stability and assembly, opening up new avenues for ASF vaccine development.

Towards an ASFV subunit vaccine: identification and characterisation of protective antigens

Dr Christopher Netherton

The Pirbright Institute, United Kingdom

African swine fever virus (ASFV) causes a lethal haemorrhagic disease of domestic pigs that causes a significant disease burden in endemic areas of sub-Saharan Africa. Since its introduction into Georgia in 2007 it has spread across most Eurasia killing millions of animals. The lack of an effective vaccine against the disease is a major impediment to effective control of the virus in the field and may be required to eradicate the disease from wild boar. Attenuated strains of ASFV derived by repeated passage through tissue culture do protect against lethal challenge, but have unacceptable side effects including a chronic form of ASF. An alternative route to a vaccine is to identify immunogenic and protective proteins out of the more than 150 that the virus encodes. We have used two strategies to identify antigenic ASFV proteins and incorporated these into replication deficient adenovirus and modified vaccinia Ankara vectors. The results of immunisation and challenge experiments with pools of these viral vectors is also presented.

Building capacity for sustainable vaccine production in Thailand: Porcine Circovirus 2 vaccine as a model

Dr Peera Jaru-Ampornpan

National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand

Thailand produces almost 11 million pigs each year, most of which for domestic consumption. In an effort to protect the pigs against myriads of swine diseases and maintain their health until finishing, pig farmers rely on a strict vaccination schedule. In 2017, the market value for swine vaccines was almost 2,000 M THB (51 M GBP). However, all swine vaccines currently used in Thailand have to be imported at high cost, creating a possible liability in the regional pork supply chain and inequality between large conglomerates and independent farmers. To close this gap, this GCRF project aims to build capacity to locally produce veterinary therapeutics or vaccines by bringing together expertise from the UK and the Thai groups. We selected porcine circovirus 2 (PCV2) as our first model veterinary subunit vaccine, as the commercially available PCV2 vaccines are all imported (valued at 590 M THB in 2017) and based on obsolete strains. Working collaboratively, our team designed new PCV2 vaccines based on epidemiological data gathered in Thailand to best match the current, locally-circulating viruses. We then selected the most cost-efficient protein expression platform and developed downstream purification processes that could be adapted for large-scale production. Currently, we are planning to evaluate the safety and efficacy of the vaccine. In the final phase of the project, we aim to establish the capacity for production at a commercial scale in the South East Asia region and partner with a leading veterinary product distributor for providing more affordable PCV2 vaccines to local farmers.

Developing a ‘one health’ Nipah virus vaccine to protect animal and public health

Rebecca McLean¹, Nazia Thakur¹, Miriam Pedrera¹, Sue Lowther², Tristan Reid², Shawn Todd², Brenton Rowe², Jemma Bergfeld², Lee Trinidad², Sarah Riddell², Sarah Edwards², Jean Payne², Jennifer Barr², Nick Rye², Matt Bruce², Tim Poole², Sheree Brown²,Toni Dalziel², Gough Au², Megan Fisher², Rachel Layton², Teresa Lambe³, Keith Chappell⁴, Ariel Isaacs⁴, Daniel Watterson⁴, Francesca Macchi⁵, Valentina Franceschi⁵, Mercedes Mourino⁶, Jean-Christophe Audonnet⁷, Gaetano Donofrio⁵, Norbert Pardi⁸, Drew Weissman⁸, Li-Yen Chang⁹, Mustafizur Rahman¹⁰, Elma Tchilian¹, Sarah Gilbert³, Paul Young⁴, Dalan Bailey¹, Glenn Marsh², Simon Graham¹

1 The Pirbright Institute, Pirbright, United Kingdom
2 CSIRO Health and Biosecurity, Geelong, Australia
3 Jenner Institute, University of Oxford, Oxford, United Kingdom
4 University of Queensland, Brisbane, Australia
5 University of Parma, Parma, Italy
6 Zoetis, Girona, Spain
7 Boehringer Ingelheim Animal Health, Saint Priest, France
8 University of Pennsylvania, Philadelphia, United States
9 University of Malaya, Kuala Lumpur, Malaysia
10 International Centre for Diarrhoeal Disease Research, Bangladesh, Dhaka, Bangladesh

Pig-to-human transmission was responsible for the first and most severe Nipah virus (NiV) outbreak in Malaysia and Singapore in 1998-99. This outbreak caused severe and lasting economic costs to the local pig industry. Despite the threat NiV poses to some of the most pig dense regions of the world, no vaccines are currently available. To address this gap, we are developing a vaccine for use in pigs that would reduce the risk that NiV poses to the pig industry, livestock keepers and public health. We evaluated the immunogenicity of NiV G or F-based vaccine candidates delivered as recombinant protein subunits, viral or mRNA vectors in pigs. Bovine herpesvirus-4 (BoHV-4) vectored NiV G induced the strongest specific T-cell response. BoHV-4 NiV G, BoHV-4 NiV F, replication-deficient adenoviral vectored (ChAdOx1) NiV G, canarypox (ALVAC) vectored NiV G and synthetic nucleoside-modified mRNA expressing NiV G (mRNA NiV G) all stimulated measurable CD8⁺ T-cell responses. mRNA NiV G and recombinant soluble NiV G (sG) protein in adjuvant induced the greatest NiV-neutralising antibody titres, whereas, recombinant F protein stabilised in the prefusion conformation (mcsF) and BoHV-4 NiV F induced antibodies capable of neutralising the fusion of cells expressing NiV G and F. Despite these differences in immunogenicity, prime-boost immunisation with NiV sG protein, NiV mcsF protein, or ChAdOx1 NiV G all conferred a comparable high degree of protection against challenge infection, with significant reduction in NiV RNA and no isolation of live virus from vaccinated pigs. Vaccine candidates will now be evaluated for efficacy after a single immunisation before selection of prototype vaccine, which will be evaluated in pigs under field conditions in Malaysia and Bangladesh. In addition to providing a platform for the development of a NiV vaccine for pigs, these studies will also support ongoing human vaccine development efforts.

Comparative efficacy of CIRCOQ™ PCV2 subunit vaccinated one- versus two-dose in high MDA-derived antibody piglets against disease caused by PCV2-type 2D naturally exposed in a Vietnamese swine farm

Do Tien Duy¹, Quach Tuyet Anh¹, David Lee², Frank CJ Chang², Carol PY Wu², Le Thanh Hien¹, Nguyen Thi Thu Nam¹, Nguyen Phuoc Ninh¹, Nguyen Tat Toan¹

1 Faculty of Animal Science and Veterinary Medicine, Nong Lam University, Ho Chi Minh City, Vietnam
2 Reber Genetics, Taipei, Taiwan

The aim of this study is to identify the protective efficacy of CIRCOQ™ PCV2 subunit vaccine in high MDA-derived antibody piglets against disease caused by PCV2d naturally exposed in field condition. 130 weaned 21-day-old (± 1 day) healthy pigs were used in this study then allocated into 3 trial groups. The signs of respiratory disorder increased gradually in non-vaccinated pigs and were different higher from vaccinated pigs (1-dosed Vac and 2-dosed Vac) at 13-17 weeks of age (P < 0.05), 18-22 weeks of age (P <0.001) and 23-27 weeks of age (P <0.01). The non-vaccinated pigs had an early rate of dermatitis at 8-12 weeks of age (10.0%), increasing gradually at 13-17 weeks of age (30.0%), 18-22 weeks of age (46.7%) and 23-27 weeks of age (33.3%); while there was no case of dermatitis on both vaccinated pigs. 3/30 pigs had stunted signs in non-vaccinated group, 4/50 stunted pigs appeared in 1-dosed Vac group and only 1 per 50 pigs were stunted in 2-dosed Vac group. There was a significant difference (P <0.05) in mortality in pigs of the non-vaccinated group (6/30) compared to the 2-dosed Vac group (2/50). Blood PCV2 viremia was detected and peaked at 105 days of age in non-Vac pigs (Ct-adj = 3.40; Ct-adj = 40-sample Ct value realtime PCR) and 1-dosed Vac group (Ct-adj = 1.37) ; while no detectable PCV2 virus in pigs’ blood (Ct-adj = 0.00) of the 2-dose Vac group. At 77 and 105 days of age, PCV2 viremia Ct-adj of non-Vac pigs was significantly higher (P <0.05) than the two groups of vaccinated pigs. In the 2-dosed Vac pigs, the S/P ratio increased forward after the second dosed vaccination, which was higher (P <0.05) than the 1-dosed Vac and non-Vac pigs at 77 (S/P = 1.23) and 105 (S/P = 1.31) days of age. The 2-dosed Vac pigs that reached BW (90.5 ± 12.7 kg), AWG (84.7 ± 12.4 kg) and ADG (526.5 ± 74.4 g/pig/day) were significantly higher (P <0.05) than pigs of non-Vac group respectively of BW (80.9 ± 24.7 kg), AWG (75.0 ± 24.8 kg) and ADG (476.8 ± 132.0 g/pig/day). In conclusion, vaccinated pigs of CIRCOQ™ PCV2 subunit has a significant efficacy on protecting pigs against clinical symptoms, blood viral load and mortality and then improvement of productivity compared to non-vaccinated pigs.

The emergence of African swine fever as a threat to the global pig industry

Professor Dirk Pfeiffer

City University of Hong Kong, Hong Kong

Since the most recent introduction of African swine fever virus (ASFv) into Europe in 2007, the virus has been spreading slowly but steadily from Eastern to Central Europe. In those regions, it is now present in domestic pig and wild boar populations, with variation between countries in the relative importance of these two species in terms of their role in local transmission dynamics. ASFv introduction into China was reported in mid 2018, and it has since spread to all of China’s provinces. The vast spatial expanse of China (about twice the land area of the European Union) together with its enormously large pig population (keeping about 50% of the global pig population) concentrated in the eastern half of the country together with the predominance of low biosecurity pig farms and intensive short to long-distance trade of pigs, pork and pork products means that control of the virus in this country is extremely challenging. And it will become even more difficult if the virus was to become established in wild boar populations. As indicated by recent virus spread to various other countries in the East and South-East Asian region, it is unlikely that further spread of ASFv across the region and beyond can be stopped. The emergence of the global ASFv threat has been facilitated by the evolution of a global pork food system driven by trade opportunities and increasing meat consumption demand arising from globalisation, fast economic development and rapid urbanisation.

Toward the development of a West Nile virus vaccine: lineage 8 as a vaccine strain against virulent lineages from Africa

André P Bédékélabou¹, Moussa Dia², Rianatou Bada Alambedji¹ and Yakhya Dièye²

1 Ecole Inter-États Des Sciences et de Médecine Vétérinaires, Dakar, Senegal
2 Institut Pasteur de Dakar, Dakar, Senegal

West Nile fever is an emerging disease caused by the West Nile virus (WNV), a neurotropic arbovirus (arthropod-borne virus) belonging to the family of Flaviviridae. WNV infects, is maintained and is transmitted from different vertebrates, humans and horses being dead-end hosts. WNV infection is mostly asymptomatic, but it can range from a flu-like disease to severe neurological cases. Currently, there is no specific therapy or universally accepted vaccine against WNV. The objective of this project was to develop a live vaccine against WNV. For this purpose, we evaluated the ability of the benign lineage 8 (WNV8) to confer a protection against lineages 1 and (WNV1) and 7, also called Koutango, the two most virulent lineages circulating in Africa. Groups of mice were administered with a single (at day 0) or two (at days 0 and 14) doses of WNV8 and challenged four weeks after the first exposure with lethal doses of WNV1 or Koutango. Mouse survival were monitored for three weeks post challenge. Subgroups of mice were sacrificed at different time point after administration of WNV8 for measurement of viral loads in different organs and for analysis of the production of antibodies against WNV. After administration to mice, WNV8 cleared within a week, with a tropism for the brain being the most noticeable among the tested organs. Mouse exposure to WNV8 conferred a significant protection against WNV1 and Koutango. Interestingly, administration of high doses of WNV8 completely protected mice both virulent lineages. This protection correlated well with the induction of neutralizing antibodies. Lineage 8 can serve as a basis for the development of a live vaccine against virulent WNV lineages.

Enhanced antimicrobial activity of lectin conjugated with platinum nanoparticles and its possible use in aquaculture as nanovaccine

Abdul Salam Rubeena¹ and Preetham Elumalai^1,2

1 School of Ocean Science and Technology, Kerala University of Fisheries and Ocean Studies, Panangad, Kerala, India
2 Department of Processing Technology (Biochemistry), Kerala University of Fisheries and Ocean Studies, Panangad, Kerala, India

Lectins are glycoproteins which are involved in innate immune response in invertebrates by helping in neutralizing pathogens by the recognition of Pathogen associated molecular patterns. However, it alone is insufficient to curtail severe infections. The over dose and unethical use of antibiotics led to the evolution of several dreadful antibiotic resistant bacteria. Hence, there is an increasing demand for an alternative strategy to treat bacterial infections with no or minimal side effects along with the lack of resistance development on the part of the pathogen. Developments in nanotechnology have revealed metal nanoparticles as promising new generation antibacterial agents. However, the mechanism of action of nanoparticles remains unclear. It is proposed that the NPs can disrupt bacterial membrane integrity, generate reactive oxygen species and result in bacterial cell damage. But the overdosage of nanoparticles may lead to bioaccumulation which is another serious issue. Thus lectins, having antibacterial property, which are isolated from shellfish when conjugated with nanoparticles will enhance its antibacterial activity and in turn can help in combating bacterial infections at a minimum concentration of nanoparticles which are safe. In the present study we have synthesized lectin coated nanoparticles and characterized their functional properties including, antibacterial activity by zone of inhibition assay, lipid peroxidase assay, membrane integrity study etc. Thus after proper further research and findings this conjugate can be used as a nanovaccine which can be incorporated in feed or by immersion for mass application in aquaculture.

Characterization of potential vaccine antigens from Mycoplasma mycoides subsp. mycoides based on homology with Mycoplasma capricolum subsp. capripneumoniae

Moses Orwe¹, Johnson Kinyua², Jane Ngaira², Martina Kyalo³, Nimmo Gicheru⁴, Harrison Lutta⁴, Univer Chepleting¹, Maureen Luvanda⁴, Hezron Wesonga⁴, Roger Pelle³ and Jan Naessens⁴

1 Kenya Veterinary Vaccines Production Institute, Nairobi, Kenya
2 Jomo Kenyatta University of Agriculture and Technology, Nairobi, Kenya
3 Biosciences Eastern and Central Africa – International Livestock Research Institute Hub, Nairobi, Kenya
4 Veterinary Science Research Institute – Kenya Agricultural and Livestock Research Organisation, Muguga, Kenya

Mycoplasma mycoides ssp mycoides (Mmm) and Mycoplasma capricolum ssp capripneumoniae (Mccp) are causative agents of two lethal respiratory diseases: contagious bovine pleuropneumonia (CBPP) in cattle and contagious caprine pleuropneumonia (CCPP) in goats respectively. The two pathogens belong to the mycoides cluster of livestock Mycoplasma and are phylogenetically related. Some success was achieved in identification of potential vaccine antigens in CBPP. In silico identification of high score homologues of Mmm in the Mccp genome was carried out from a group of thirteen potential Mmm vaccine candidate genes encoding proteins by BLAST analysis. Heterologous expression in E. coli and purification by metal-chelate affinity chromatography of five out of six of the highest score recombinant His-tagged proteins was done, followed by western blot with Mccp-positive sera. Genome alignment of Mmm (strain PG1) and Mccp (strain F38) using Mauve software gave similar locally collinear blocks (LCBs). With cut off points set at 0.0 for E-value, 80% for query coverage and 60% for identity, six proteins, MSC_0160 (elongation factor Tu), MSC_0265 (pyruvate dehydrogenase E1), MSC_0499 (prolipoprotein), MSC_0610 (molecular chaperone DnaK), MSC_0052 (hypothetical protein) and MSC_0775 (prolipoprotein) gave the best homology scores on tBLASTn. Western blot using Mccp-positive sera showed cross-reactivity with MSC_0160, MSC_0499, MSC_0052 and MSC_0775 but not MSC_0610. All proteins were negative when treated with commercial Mccp-negative naïve goat sera, except for MSC_0499. Western blot using sera from Mccp-positive goats gave a reaction with four out of the five recombinant Mccp proteins tested. Therefore, this clears the way for scaling up, inoculation and challenge of goats with four of the proteins that reacted with Mccp-positive sera. MSC_0499 which had a reaction with Mccp-negative sera from the field in Kenya is still under investigation to determine presence of other Mycoplasma species in the field.

Intranasal vaccination with PPRV vaccine in goats using natural mucoadhesive polymer as vaccine delivery system: haematological and humoral immune responses. 

CK Ezeasor¹, BO Emikpe², SVO Shoyinka¹ and SM Anika¹

1 Department of Veterinary Pathology and Microbiology, University of Nigeria, Nsukka, Nigeria
2 Department of Veterinary Pathology, University of Ibadan, Nigeria

Peste des petits ruminants (PPR) is a highly contagious and economically important transboundary viral disease of sheep and goats. There is need for immunopotentiation of the current PPR vaccination and strategy, using cheap and affordable materials.  This study evaluates the immunomodulatory potential of Irvingia gabonensis gum polymer extract as mucoadhesive vaccine delivery system against PPR in goats. Twenty-five (25) male West African dwarf goats (6-9months) were divided into 5 groups (n=5). Attenuated 75/1 PPR lineage 1 strain PPR vaccine was used. Group 1 was vaccinated via the intranasal route using mucoadhesive polymer as vehicle; Group 2 was vaccinated intranasaly with reconstituted vaccine only; Group 3 were vaccinated via subcutaneous injection while Groups 4 and 5 served as the non-vaccinated controls administered intranasal mucoadhesive polymer and water respectively. Haematology and H-based PPR blocking ELISA was done weekly for 4 weeks post vaccination to evaluate the immune response. H-PPR bELISA detected antibodies against PPR by 7 day post vaccination (dpv), reaching a peak by 21dpv with mean Percentage inhibition (PI) of 78.2% ; 69.6% and 87% in Groups 1, 2 and 3, respectively. The unvaccinated control animals remained negative. The haematocrit, haemoglobin, Red cell count and total leucocyte counts did not differ significantly between groups. However, low neutrophil to lymphocyte ratios (NLR) which differed significantly (P<0.05) from the controls were observed by 14dpv to 28dpv in the vaccinated groups. The findings of this study suggest that use of mucoadhesive polymers in mucosal vaccine delivery have an immunomodulatory effect on intranasal PPRV vaccination.

Comparison of the efficacy of three vaccination programs in prevention of infectious bronchitis (IB) for broilers

Vo Thi Tra An and Nguyen Thi Kim Yen

Faculty of Animal Science and Veterinary Medicine, Nong Lam University, Ho Chi Minh City, Vietnam

The objective of this study aimed at evaluating the efficacy of three different vaccination programs for broiler in prevention of IB. 4080 broilers at one day old were inoculated with the vaccine IB H120 and then divided into three experimental groups. Only the broilers in the group 1 and group 2 at 14 days old were re-vaccinated with vaccines IB 4/91 and IB 88, respectively. The antibody titre of the broilers at 35 days old in the group 1, group 2 and the broilers in the group 3 were not re-vaccinated (used for control) was examined by ELISA technique. These antibody titres were 663, 1184 and 1125, respectively. Respiratory score of the broilers in the group 1 and group 2 reduced 25 points compared to the control broilers. The mortality rate of the broilers in the group 1, 2 and 3 was 2.5%, 2.4% and 9.6% respectively. The final average weights of the broilers in the group 1, 2 and 3 were 3.4 kg, 3.5 kg and 2.9 kg, respectively. This experimental result indicated that the vaccination procedures for the broilers in the group 1 and 2 prevented IB better and gained higher economical effect compared to the vaccination schedule for the broilers in the group 3.

An overview of 15 years’ experience with fish vaccination in Iran

Nastaran Shahbazian

Department of Fish Health and Diseases, Iran Veterinary Organization, Tehran, Iran

Iran has a diverse range of water bodies that are ideal for aquaculture, including the Caspian Sea in the north, composed of brackish water, the Persian Gulf in the south, composed of salt water, and a number of freshwater lakes throughout the country. There has in fact been a rapid increase in aquaculture production in Iran in recent years, with aquaculture production increasing from 124,560 to 349,365 tons between 2004 and 2014. Rainbow trout (Oncorhynchus mykiss), cyprinid species (Cyprinus carpio, Hypophthalmichthys molitrix, Hypophthalmichthys nobilis, Ctenopharyngodon idella) and sturgeon species (Huso huso, Acipenser baerii, Acipenser persicus, Acipenser ruthenus, Acipenser stellatus) are the main cultured fish species that are farmed, but as the aquaculture expands and diversifies a number of new finfish species are being piloted, eg cobia (Rachycentron canadum), silver pomfret (Pampus argenteus), fourfinger threadfin (Eleutheronema tetradactylum), Asian sea bass (Lates calcarifer), sobaity seabream (Sparidentex hasta), grouper (Epinephelus coioides) and rabbit fish (Siganus canaliculatus). The incidence of disease has increased as the aquaculture industry in Iran has expanded and the farming systems have intensified. There is increasing concern about the use of antibiotics to control bacterial disease outbreaks and attention has therefore focused on vaccination as a method for controlling these. The first experience of fish vaccination in Iran come back to around 15 years ago when the oral vaccine for streptococcosis in rainbow trout was used. After 15 years, streptococcosis is still the first bacterial disease not only in rainbow trout but also in marine fishes such as sea bass and sea bream that are the main species in the future development of aquaculture in Iran. In this review we are trying to explain the causes of unsuccessful experience in fish vaccination in Iran.

Developing an effective and economical bacterial expression system for the production of animal vaccines in Thailand and South East Asia.

Amber Peswani¹, Benjamas Liwnaree², Katanchalee Muensaen², Jaraspim Narkpuk², Peera Jaru-Ampornpan², Anan Jongkaewwattana² and Colin Robinson¹

University of Kent; University College London; Imperial College London; London School of Hygiene and Tropical Medicine; BIOTEC; National Biopharmaceutical Facility; King Mongkut’s University of Technology Thonburi

Pork consumption in South East Asia has doubled over the past 20 years. However, many pig farms in the region are vulnerable to porcine circovirus (PCV). This virus devastates pig farms by reducing farrowing rates to below 60% and can cause up to 30% fatality in piglets, creating strains on pork supply and increasing prices for ordinary people. Currently, Thai and South East Asian farmers rely on imported PCV2 commercial vaccines which come with high cost, valued at £10.5M (GBP) in 2013. The imported vaccine risks reduced effectiveness due to mismatches with the local strains. Additionally, a new species, PCV3 has been recently identified for which no commercial vaccines are available. The goal of this research is to create a new PCV vaccine that can be produced regionally in Thailand and South East Asian countries, is better matched to local PCV2 strains, and protects against PCV3. In this project, a pilot process has been developed by recombinantly expressing truncated PCV constructs using fed-batch fermentation in Escherichia coli then performing a single-step purification process to produce a high purity, high yield product that is capable of generating neutralising antibodies in PK15 cells.

Immunostimulatory effect of Phoenix dactylifera fruit-supplemented diet in Aeromonas hydrophila-infected Clarias gariepinus

Funmilayo ID Afolayan¹, Oriade Tajudeen¹ and Alao S Omoniyi²

1 Department of Zoology, University of Ibadan, Ibadan, Nigeria
2 Department of Microbiology, University of Ibadan, Ibadan, Nigeria

The health of an organism depends on the level of its immunity. Phoenix dactylifera fruit has been widely reported for its effective anti-inflammatory properties which can possibly contribute to immune enhancement. Thus, the effect of P. dactylifera on immune response in  Aeromonas hydrophila infected Clarias gariepinus was evaluated. P. dactylifera fruit powder was compounded with fish meal at different concentrations  0 (control diet), 0.5, 1.0 and 2.0%. The experiment was conducted with sixty fish, and determined antibody titres using haemagglutination assay, evaluate phagocytic activity and genetic damaging effect of Aeromonas hydrophila pathogenusing micronucleus assay. In addition, heamatological and organ indices were accessed using standard methods. The results revealed that the groups fed with 0.5 and 2.0% date showed significant elevated level of immune responses with antibody titres of 231.49±77.06 and 257.18±51.37, and phagocytic activity values of 0.07±0.01 and 0.04±0.00, respectively (p˂0.05).The DNA damaging effect of A. hydrophila on the infected fish was observed, this was significantly reduced in 2% P. dactylifera fed group. In addition, increased white blood cell count was recorded for C. gariepinus group fed with 0.5% date supplement. Higher lymphoid organ index was also observed in fish fed with supplemented diet, when compared to the control. These results indicate that P. dactylifera fruit powder enriched diet showed immunostimulatory activity in C. gariepinus, in a dose dependent manner.

Development of a plant-made diagnostic immunoassay to detect porcine circovirus type 2 infection in South African swine herds

Aune Angobe, Alta van Zyl, Cornelius Gunter, Edward Rybicki and Inga Hitzeroth

Biopharming Research Unit, University of Cape Town, Cape Town, South Africa

Porcine circovirus type 2 (PCV-2) is considered the major cause of porcine circovirus associated diseases and is one of the major pathogens in swine producing countries. PCV-2 is a non-enveloped virus with a single stranded circular DNA genome of about 1.8 kb. This encodes the single capsid protein (CP) which is highly immunogenic, as well as a replication associated protein. Recombinantly expressed CP can self-assemble into virus-like particles (VLPs) that are structurally and immunogenically very similar to native virions. Current commercially available diagnostic kits are VLP-based and are effective at detecting PCV-2 antibodies in sera. However, these diagnostic assays are expensive, therefore limiting their use in developing countries. Plant-based transient expression systems have recently been investigated to express PCV-2 CP for a cheaper diagnostic reagent. The aim of this study is to develop an inexpensive lateral flow device to be able to test for PCV infection in pig herds. For this we optimised the expression of PCV-2 CP in Nicotiana benthamiana by comparing pEAQ-HT and pCBP2 expression vectors, and to use VLPs produced in plants to set up a lateral flow device. The CP was successfully expressed in N. benthamina with both pEAQ-HT and pCBP2, and VLPs subsequently purified on discontinuous sucrose gradients by ultracentrifugation. The assembly of the CP was assessed by transmission electron microscopy, which showed the presence of assembled VLPs.  Purified VLPs were successfully used as coating antigen in indirect enzyme-linked immunosorbent assays (ELISAs), which were able to detect PCV-2 CP antibodies in CP-immunised rabbit sera.

Understanding the role of the ubiquitin-proteasome system in inducing cytotoxic T-cell lymphocytes responses to recombinant adenovirus vectored bovine viral pathogens.

Stephen Chiweshe¹, Janice Gilray¹, Scott Hamilton¹, Pawana Tospitakkul², Dan Price¹, George Russell1, William Golde¹

1 Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik EH26 0PZ
2 Bureau of Beterinary Biologics, Department of Livestock Development, Pakchong, Nakhon Ratchasima, Thailand 30130

Where they have been appropriately and successfully used, vaccines have been a key measure in controlling several infectious diseases. Effective vaccines elicit strong cellular and antibody responses and often confer responses that lasts for several decades, with even a single immunisation1.  However, the exact immunological mechanism by which they induce protective immunity is not fully understood. By understanding these mechanisms, vaccine design can be tailored to augment their efficiency. Using bovine viral pathogens, BHV-1, FMDV and RSV, we manipulated previously reported antigens, on a gene level, preventing protein maturation resulting in degradation by the proteasome machinery. The proteasome degrades proteins into amino acids but leave a level of inefficiency resulting in small peptides which, having the necessary compatibility, can be bound by newly synthesised class I MHC molecules and presented on the cell surface. If peptides derive from a foreign source, like a virus that infected the cell, this leads to presentation of a potential T cell epitope and induction of a cellular immune response, specifically, induction of cytotoxic T-cell lymphocytes (CTLs). By altering the protein antigens of interest, we anticipate these will be ubiquitinated and targeted to the proteasome degradation system. Thus, peptide pools from intracellular pathogen would be available for class I MHC binding. Using an adenovirus delivery system, we demonstrated the targeted processing of pathogen derived proteins in vitro. We now aim to test these in vivo and study the resulting immune response in comparison to the unaltered proteins. If these mechanisms are unravelled, antigens can be designed and primed to effectively elicit both cellular and antibody responses, facilitating a more rational and effective approach to vaccine development.

The neutralizing antibody response against 3A truncated negative marker foot-and-mouth disease virus serotype A vaccine in cattle

Dhanesh VV¹, H Madhusudan¹, HB Suresh¹, P Saravanan¹, KB Jitendra², RP Tamil Selvan¹, S Aniket¹ and BP Sreenivasa¹.

1 ICAR Indian Veterinary Research Institute, Bengaluru, India
2 ICAR Directorate of Foot and Mouth Disease, Mukteswar, India

Foot-and-mouth disease (FMD) is a highly contagious, economically important disease of cloven-hoofed animals caused by FMD Virus (FMDV) of Picornaviridae family. The use of an inactivated vaccine can efficiently minimize the disease burden among the susceptible animals in endemic areas. An effective disease control programme warrants the requirement of a precise sero-surveillance tool to differentiate infected from vaccinated animals (DIVA). The presences of traces of non-structural proteins (NSPs) in the conventional inactivated vaccines evoke detectable levels of NSP immune response among repeatedly vaccinated animals leading to flaws in the current DIVA strategy. In such a scenario, the use of a negative marker vaccine will be beneficial to rule out false positive results in a disease-free zone. Here, we report the characterisation and neutralisation antibody response of a recombinant FMDV serotype ‘A’ Indian vaccine strain lacking 58 amino acid residues (87-144 amino acid position) in the carboxyl terminal region of the viral 3A protein. The in vitro characterisation of recombinant deletion mutant virus exhibited similar replication and antigenic characteristics as that of parental strain. Immunisation of cattle with inactivated vaccine from the recombinant deletion mutant virus induced potent neutralizing antibody response to protective levels on par with the parent virus vaccine. Taken together, recombinant deletion mutant virus vaccine could be useful in the implementation of a precise DIVA policy in conjunction with a diagnostic tool targeting the deleted portion of 3A protein in FMD endemic countries where vaccinate to live policy is being followed.

Characterization of the bovine adaptive immune repertoire responses to a candidate vaccine for East Coast fever

Sonal Henson, Samuel O Oyola, Elizabeth Kibwana, Benjamin Nzau and Vish Nene

International Livestock Research Institute, Nairobi, Kenya

In order to develop effective vaccines against pathogens, there is a need to assess the immunogenicity, not only by quantifying antigen-specific antibodies, but also by characterizing the diversity of the activated B-cells that generate functional antibodies, through B-cell receptor characterization. High-throughput sequencing (HTS) technologies provide the ability to study antigen receptor repertoires at both single cell and single nucleotide resolution. We are using HTS to qualitatively and quantitatively study bovine immune responses upon immunization with vaccine candidates against Theileria parva, the causative agent of East Coast Fever in cattle, with the aim to identify antigen-specific antibodies. We describe a novel protocol for capturing ultra-long antibodies through sequencing. We compare the characteristics of naïve and immunized bovine B cell repertoires focusing on VH genes with ultra-long CDRH3. We observe clonality, preferential germline gene usage, skewed CDRH3 amino acid properties and public immunoglobulins in the immunized repertoires. We also identify putative antigen-specific B cell sequences with ultra-long CDHR3.

Development of ELISA against African swine fever

Dr Alexander S Jones¹, Dr Hoang Vu Dang² and Professor Colin Robinson¹

1 School of Biosciences, University of Kent, Canterbury, England
2 Department of Biochemistry and Immunology, National Institute of Veterinary Research, Hanoi, Vietnam

African swine fever is a virus that is devastating the South East and East Asian pig industry. It is rapid onset, killing infected animals within 10 days. The complex nature of the virus (two protein ‘coats’ and multiple ways to enter cells), has meant that there is currently no vaccine or antiviral strategy available. As such, in 2019 ASF has resulted in the death or culling of one-quarter of the global domesticated swine population and the death of 5,000,000 pigs across all 69 provinces of Vietnam. The only way to stop ASF is to quarantine and cull infected herds. Diagnosis usually requires lengthy laboratory protocols (PCR), thus allowing the virus to spread to otherwise healthy pigs. Here we have recombinantly expressed (in E. coli) the CD2v haemagglutinin-like protein of African Swine Fever Virus (ASFV). Purification of this protein is underway, and we will raise antibodies against it that will coat an ELISA plate to detect ASF in pig blood samples. This will provide a simple, rapid screening method to identify infected pigs greatly reducing the number of pigs culled and helping to limit the spread of the disease.

Incorporating mutations in the NS4 protein to develop alternative vaccine candidates for African horse sickness virus

S Boughan¹, AC Potgieter^2,3 and V van Staden¹

1 Department of Biochemistry, Genetics and Microbiology, University of Pretoria, South Africa
2 Deltamune (Pty) Ltd, Pretoria, South Africa
3 Department of Biochemistry, Centre for Human Metabolomics, North-West University, Potchefstroom, South Africa

African horse sickness (AHS) is a severe disease of equids caused by African horse sickness virus (AHSV). AHS is endemic to southern Africa and has a mortality rate of over 90% in infected horses. Strict regulations govern the import and export of horses, as AHS is listed as a notifiable disease by the OIE. AHSV is transmitted via Culicoides biting midges which thrive in warmer areas, this poses an increase risk in Europe and other countries due to global warming. A live attenuated vaccine is currently utilised in South Africa, however it is not licensed elsewhere due to its potential for reversion back to virulence and reassortment with field strains. We therefore require a better vaccine for worldwide usage. AHSV falls in the Orbivirus genus and the Reoviridae family, for which the prototype virus is bluetongue virus (BTV) which infects ruminants. The dsRNA genome of AHSV encodes 7 structural proteins (VP1-VP7) and 4 non-structural proteins (NS1-NS4). AHSV NS4 localises to the nucleus of infected cells and binds to dsDNA. BTV NS4 is known to act as an interferon antagonist, making it a candidate gene involved in virus virulence. The aim of this study was to introduce mutations into NS4 by targeting domains potentially involved in its nuclear translocation and DNA binding and assaying the effect of these on virus virulence. The recombinant AHSV strains harbouring either mutant NS4 proteins or NS4 knockouts were rescued by reverse genetics and compared to the wild-type virulent AHSV. Confocal laser scanning microscopy and western blot was used to detect the expression and intracellular localisation of NS4. We performed comparative growth kinetics assays and proceeded to an in ovo model to assess virus attenuation. We found that the AHSV NS4 mutants showed reduced virulence in a chicken embryo model and can potentially be developed further as vaccine candidate for AHSV.

Reverse vaccinology for the development of universal subunit vaccines against motile Aeromonas septicaemia

Chayanit Soontara, Pattanapon Kayansamruaj

Department of Aquaculture, Kasetsart University, Bangkok, Thailand

Aeromonas spp is the major cause of infection in motile Aeromonas septicaemia, a fatal disease in freshwater fish. In this study, we identified potential vaccine candidates against three species of Aeromonas, including A. hydrophila, A. jandaei and A. veronii by reverse vaccinology (RV). In this study, we applied a novel reverse vaccinology negative strategy and pan-genome analysis for screening possible surface exposed proteins (PSEs) from 16 strains of three species of Aeromonas isolated from freshwater fish and aquatic habitats. From a total of 1285 core genes as predicted by Anvio pan-genome analysis, a series of bioinformatic analyses indicated that 66 genes were PSEs. Among these, 79.45% were including outer membrane proteins (OMPs) and extracellular proteins (ECPs) and other is unknown protein. Antigenicity of these PSEs was predicted in silico using Vaxijen program and four proteins, namely putative lipoprotein (PuL), penicillin-binding protein activator (LpoB), K-transporting ATPase C chain (KdpC) and MSHA biogenesis protein (MSHA), possessed the highest antigenicity score were selected. The recombinant proteins were constructed using E. coli overexpression system based on the full-length gene sequences derived from A. veronii genome. western blot analysis of these recombinant proteins against antiserum of Nile tilapia experimentally infected with either A. hydrophila, A. jandaei and A. veronii indicated cross reactivity of these proteins toward all three aeromonad species. Thus, the recombinant proteins generated in this study possessed high potential as efficient universal vaccine candidates against motile Aeromonas septicaemia which required to be investigated in vivo further.

Evaluating the effectiveness of vaccines in commercial poultry against Newcastle disease viruses of genotype VI and VII

Muhammad Zubair Shabbir¹, Sameera Akhtar¹, Yi Tang², Tahir Yaqub¹, Arfan Ahmad¹, Ghulam Mustafa¹, Muhammad Azhar Alam¹, Diwakar Santhakumar³, Venugopal Nair⁴ and Muhammad Munir³

1 University of Veterinary and Animal Sciences, Lahore, Pakistan
2 Pennsylvania State University, State College, PA, United States
3 Department of Biomedical and Life Sciences, Lancaster University, Lancaster, United Kingdom
4 The Pirbright Institute, Woking, United Kingdom

Newcastle disease virus causes economically devastating disease in avian species around the world. Newcastle disease is enzootic in Pakistan and recurrent outbreaks are frequent in multiple avian species even after continuous and extensive use of vaccines. A number of APMV-1 and pigeon paramyxovirus serotype 1 (PPMV-1) strains have been isolated and genetically characterized in recent years. However, the impact of recently characterized wild bird-origin APMVs in domestic poultry, and the potency of routinely used vaccines against these novel and genetically diverse viruses remained elusive. Here, we applied next-generation sequencing for unbiased complete genome characterization of APMV-1 and PPMV-1 strains isolated from clinically diseased peacocks (Pavo cristatus) and pigeons (Columba livia), respectively. Global phylodynamics and evolutionary analysis demonstrates Pigeon/ MZS-UVAS-Pak/2014 is clustered into lineage 4 (or genotype VI) and Peacock/MZS-UVAS-Pak/ 2014 into lineage 5 (or genotype VII). The genomes of both isolates encoded for polybasic residues at the fusion protein cleavage motif (¹¹²RRQKR↓F¹¹⁷) along with a number of important substitutions in the surface glycoproteins compared with the vaccine strains. Clinico-pathological and vaccine-driven immunological investigations in domesticated chickens indicate that these isolates can potentially transmit between tested avian species, can cause systemic infections, and can induce antibodies that are unable to prevent virus shedding. A noticeable antigenic variation between vaccine strain and wild-originated strain (R-value = 0.26) was also observed while performing cross-HI assay. Collectively, the data from these genomic and biological assessments highlight the potential of wild birds in transmitting APMVs to domesticated chickens. The study also demonstrates that the current vaccine regimens are incapable of providing complete protection against wild bird-origin APMVs and PPMVs.

Epidemiology of brucellosis on livestock, wildlife and human interface in Virunga and Kahuzi-Biega National Parks for safe and efficient Brucella vaccines and suitable vaccination program

Kambere Kavulikirwa Olivier¹ and Bwihangane Birindwa²

1 Université Catholique du Graben, Butembo, Democratic Republic of the Congo
2 Université Evangélique en Afrique, Bukavu, Democratic Republic of the Congo

Brucellosis has a tremendous impact on multiple animal species and humans thus making it a high priority disease both in sub- Saharan Africa and other regions of the developing world. The prevalence and risk factors for infections for brucellosis in small ruminants seems to be poorly understood since a lot of attention is most paid to bovine brucellosis. In the Democratic Republic of the Congo, the epidemiology of brucellosis is not clearly highlighted as there is no relevant data on the status of the diseases in different places of the country while suspect cases are being reported in animals as well as in humans. As a result, there is a real lack of an official neither vaccine nor vaccination program against brucellosis diseases in the whole country. In the interest of contributing to the improvement of public health and animal production, an epidemiological study will be conducted within and around two protected areas including Virunga and Kahuzi-Biega National parks. The activities consisting in improving the diagnostic capacities as well as the farmer’s awareness of brucellosis disease, enhancing the control strategies, will make a special emphasis on the characterization of the circulating Brucella species to create a relevant databank for future use of suitable and safe brucella vaccines, as well as a sustainable vaccination program. For the capacity bulding before the research starts, a training on brucellosis diagnosis and control by experts from a famous universities will be conducted to train 20 scientists in laboratory techniques and data analysis. The trained scientists will then participate in an integrated effort to create awareness to over 1000 farmers living in close proximity to Virunga and Kahuzi Biega wildlife conservation areas. Furthermore, the study will include an investigation about brucellosis epidemiology in goats, cattle and sheep in relation to the humans and wildlife. Once this epidemiological information is obtained, strategies for a suitable vaccination program with suitable safe vaccines in goats, sheep and cattle will be proposed to the public authorities for implementation.

Identification of ixodid tick-specific aquaporin-1 potential anti-tick vaccine epitopes: an in silico analysis

Christian Ndekezi¹, Joseph Nkamwesiga¹, Sylvester Ochwo¹, Magambo P Kimuda^1,2,3, Frank N Mwiine¹, Robert Tweyongyere⁴, Wilson Amanyire¹ and Dennis Muhanguzi¹

1 School of Biosecurity, Biotechnical and Laboratory Sciences, College of Veterinary Medicine, Animal Resources and BioSecurity, Makerere University, Kampala, Uganda
2 Research Unit in Bioinformatics, Department of Biochemistry and Microbiology, Rhodes University, Grahamstown, South Africa
3 Member of the Trypanogen Consortium
4 School of Veterinary Medicine and Animal Resources, College of Veterinary Medicine, Animal Resources and Biosecurity, Makerere University, Kampala, Uganda

Ticks are arthropod vectors of pathogens of both Veterinary and Public health importance. Acaricide application which is currently the mainstay of tick control is hampered by high cost, the need for regular application and selection of multi-acaricide resistant tick populations. In light of this, future tick control approaches are poised to rely on integration of rational acaricide application and other methods such as vaccination. To contribute to systematic research-guided efforts to produce anti-tick vaccines, we carried out an in-silico analysis of tick aquaporin-1 (AQP1) protein in order to identify tick-specific AQP1 peptide motifs that can be used in future peptide anti-tick vaccine development. We carried out multiple sequence alignment (MSA), motif analysis, homology modeling, and structural analysis to identify tick-specific AQP1 peptide motifs. BepiPred, Chou & Fasman-Turn, Karplus and Schulz Flexibility, and Parker-Hydrophilicity prediction models were used to predict these motifs’ potential to induce B cell mediated immune responses. Tick AQP1 (GenBank ID: QDO67142.1) protein was largely similar to the bovine AQP1 (PDB:1J4N) (23 % sequence similarity; Structural superimposition of the homology model and 14JN homotetramers gave an RMSD=3.269 while superimposition of a single chain gave an RMSD=1.475). Tick and bovine AQP1 transmembrane domains were largely similar while their extracellular and cytoplasmic domain loops showed variation. Two tick-specific AQP1 peptide motifs; M7 (residues 106-125, p=5.4e-25), and M8 (residues 85-104, p=3.3e-24) were identified. These two motifs are located on the extracellular AQP1 domain. Motifs; M7 and M8 showed the highest Parker-Hydrophilicity prediction immunogenicity scores of 1.784 and 1.536 respectively. These two motifs can be a good starting point for development of potential tick AQP1 peptide-based anti-tick vaccines. Further analyses such as molecular dynamics, in vitro assays, and in vivo immunisation assays are required to validate these findings.

Development of an enzyme linked immunosorbent assay based on CD150/SLAM for the detection of peste des petits ruminants virus

Meera Prajapati^1,2,3, Dou Yongxi¹, Xueliang Zhu¹, Alfred Niyokwishimira¹, Yanmin Li¹ and Zhidong Zhang^1,2

1 State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China
2 CAAS-ILRI Joint Laboratory for Ruminant Disease Control, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China
3 Animal Health Research Division, Nepal Agricultural Research Council, Khumaltar, Lalitpur, Nepal

Peste des petits ruminants (PPR) is an economically important severe viral disease of sheep and goats that affects primarily respiratory and digestive tract of animals. Specific detection of PPR virus (PPRV) antigen plays an important role in the disease control and eradication program. In this study, an indirect enzyme-linked immunosorbent assay based-on recombinant goat SLAM as an antigen capture ligand was successfully developed for detection of PPRV antigen (PPRV SLAM-iELISA). The results showed that the optimal working concentration of the recombinant goat SLAM was 0.4ng/well and the cut-off value of the assay was established as twice the mean OD value of the negative control. The assay was highly specific for PPRV with no cross-reactions among foot-and-mouth disease virus, Orf virus, sheep pox and goat pox virus, and a sensitivity with a detection limit of 1.56*101 TCID50/reaction(50µL). Assessment with 136 samples showed that the developed PPRV SLAM-iELISA was well correlated with real-time RT-qPCR assays and commercially available sandwich ELISA for detection of PPRV, with 94.73% sensitivity and 100.83% specificity, respectively. These results suggest that the developed PPRV SLAM-iELISA is suitable for specific detection of PPRV antigen.

The development of autogenous vaccines against amoebic gill disease in the Atlantic salmon (Salmo salar)

Sophie Fridman¹, James Bron¹, David Haydon², Tim Wallis² and Alexandra Adams¹

1 Institute of Aquaculture, University of Stirling, Stirling, United Kingdom
2 Ridgeway Biologicals Ltd., Old Station Business Park, Compton, United Kingdom

Amoebic gill disease (AGD) has emerged as an increasing threat to the Atlantic salmon aquaculture industry. Originally reported in Tasmania in the mid-1980s, it has since spread to become a global problem and currently costs the salmon farming industry millions of pounds every year in treatment costs and fish losses. Amoebic gill disease is caused by the free-living marine amoeba Paramoeba perurans, which is widely distributed in the environment, only becoming a concern when it attaches to the gills, causing lesions and respiratory distress to the fish. Unless treated, it can ultimately lead to fish morbidity and mortality. Currently control measures are restricted to treating sea cages with fresh water or hydrogen peroxide however this approach is expensive and logistically difficult and can have detrimental impacts on fish health and welfare. This project aims to develop autogenous vaccines based on farm-specific seed isolates of P. perurans that can be developed and deployed rapidly in the face of emerging disease threats and offer a novel control strategy for this costly emerging pathogen.  Amoebae, collected from gills of salmon infected with AGD from farm sites on Scotland’s west coast have been cultured, isolated and characterised and vaccines developed based on the results. A number of autogenous vaccine have been efficacy tested in Atlantic salmon under experimental conditions, using a cohabitation challenge, and some promising results have been seen. An AGD vaccine would have a profound impact on the sustainability of the wider salmon industry, increasing productivity and improving animal health and welfare without recourse to medication and associated potential threats to the environment.

Flagellin A toll-like receptor 5 agonist as an adjuvant in chicken vaccines

Dr Javed Ahmed Ujan

Department of Zoology, Shah Abdul Latif University, Khairpur, Pakistan

Chicken grown commercially is sensitive to environmental exposure to a number of pathogens. Therefore, regular vaccination of flocks is an absolute prerequisite to prevent the onset of infectious diseases. To fight infectious against the disease, vaccines require the inclusion of effective adjuvants that promote enhanced protection and do not cause side effects a side effect if given to birds along with the vaccine. With that in mind, there is an increased need for more effective vaccine adjuvants Efforts are being made to increase the effectiveness of the vaccine by using appropriate adjuvants, in particular toll-based receptor-based supplements (TLRs). TLRs are among the types of pattern recognition receptors (PRRs) they recognize pathogen molecules are preserved. Numerous studies have documented the effectiveness of flagellin as an adjunct as well as its medicinal substances the ability to promote cytokine production by a series of innate immune cells. This mini review summarizes our current understanding the action of flagellin, its role in stimulating responses to cytokines in chicken cells, and the potential use of flagellin as well as its combination with other TLR ligands as adjuvants in chicken vaccines.

Response of triploid Atlantic salmon to vaccination

Lynn Chalmers¹, Herve Migaud¹, Alexandra Adams¹, Luisa M Vera¹, Elsbeth McStay¹, Ben North², Chris Mitchell², John F Taylor¹

1 Institute of Aquaculture, University of Stirling, Stirling, United Kingdom
2 Pharmaq (part of Zoetis), Fordingbridge, United Kingdom

Atlantic salmon production has increased ten-fold since the 1990’s, with expansion of production expected to continue over the next decades. Despite this success, sexual maturation continues to represent a serious issue for Atlantic salmon aquaculture. The diversion of energy from muscle growth to gonad development can negatively impact productivity & profitability through reduced growth and flesh quality as well as increased disease susceptibility. Sterile triploid Atlantic salmon represent a practical and affordable solution to these issues but empirical evidence suggests they can be more susceptible to diseases and vaccine side-effects than diploids.

With increasing reports of pathogens developing resistance to antibiotics and chemotherapeutants, vaccination has become an essential tool in aquaculture husbandry practices. However, vaccines can cause unwanted side-effects such as adhesions and reduced growth. Thus, characterising the response of triploid Atlantic salmon to vaccines is crucial for determining their suitability for commercial production. To this end, the study assessed the response of triploid and diploid Atlantic salmon to vaccination with commercially available vaccines. Triploid and diploid siblings were injected with one of three vaccines (or a sham vaccine) and monitored throughout a commercial production cycle. Sampling at smolt and harvest was undertaken along with performance assessments through the cycle. Antibody response to Aeromonas salmonicida vaccination was similar between ploidy, with a positive response in vaccine-injected fish. Adhesion scores increased according to the anticipated vaccine severity. Ploidy did not significantly affect adhesion score at smolt or harvest. Triploids had higher body weights during freshwater and early seawater, with diploids significantly heavier at harvest. Thermal growth coefficient (TGC) decreased in both ploidy as adhesion severity increased. Vertebral deformity prevalence was consistently higher in triploids (smolt 12.3 ± 4.5 %; harvest 34.9 ± 5.9 %) than diploids (smolt 0.8 ± 0.5 %; harvest 15.9 ± 1.9 %), with no significant effect of vaccine group in each ploidy. This study demonstrates that triploids respond as well to vaccination as diploids, thus providing further evidence of triploid robustness and refuting anecdotal reports that triploid Atlantic salmon are more susceptible to vaccine side-effects.

Process development of inactivated Rift Valley fever vaccine adjuvanted on Nano-aluminium hydroxide

Shourok Essam Aly

Veterinary Medicine Directorate. Cairo, Egypt

Recently, there is an arising interest in the Nano-materials as for their unique characters and their ability to enhance the drug delivery and to enhance the immune response, herein we applied a protocol for dispersion of aluminium hydroxide to have a vaccine formula in the nano-scale.

Molecular and antigenic relatedness of highly pathogenic avian influenza H5N8 for evaluating current commercial vaccines in Egypt.

MT Taher¹, A Arafa¹, Hesham A Sultan²

1 Animal Health Research Institute, Dokki, Egypt
2 Faculty of Veterinary Medicine, University of Sadat City, Sadat City, Egypt

The introduction of avian influenza H5N8 to Egypt caused a dramatic change for the epidemiological situation of AI. Current inactivated vaccines commonly utilize H5N1 or H5N2 subtypes. This could increase the occurrence of escape mutants due to HA and NA differences. In the current study samples were collected from Egyptian farms during 2018/2019 and characterized as HPAI H5N8. Full HA and NA genes were sequenced followed by phylogenetic analysis for 11 samples. A phylogenetic tree revealed that H5N8 field isolates clustering in clade 2.3.4.4, with amino acid identity ranged from (96.8% - 99.3%) between each other, while The amino acid identity between H5N8 field isolates and vaccinal strains ranged from (87.8% 89.6%). In addition, sera from chicken immunized by three commonly used commercial vaccines were evaluated for their potential to cross neutralize against the antigens of H5N8 field isolates using cross HI test. Results indicate a significant difference between the H5N8 isolates and the three commercial vaccines, showing low cross neutralizing capacity toward the Egyptian strains. Moreover, experimental study was done evaluating the three commercial vaccines challenged with HPAI H5N8. Results showed that Re-5 commercial vaccine was able to protect chicken and reduce virus shedding but the two other vaccines were ineffective due to the seed viruses in these vaccines are genetically distinct from the H5N8 viruses currently circulating in Egypt. Altogether, our results support that, HPAI H5N8 virus is genetically and antigenically distinct from HPAI H5N1 virus. In conclusion, there is an imperative need to re-consider AI policy in Egypt by vaccine manufactures and government authorities.

Cross-reactive CD8 T cell responses to Japanese encephalitis virus and dengue virus recognise Zika virus sequences and have anti-viral function

Shona Moore¹, Krishanthi Subramaniam¹, Lynsey Goodwin¹, Filippo Tatullo¹, Raquel Medialdea Carrera¹, Ayako Kurioka², Laura Scott¹, Daniela Weiskopf³, Sutee Yoksan⁴, Paul Klenerman², Tom Solomon¹, Lance Turtle^1,5

1 Institute of Infection and Global Health, University of Liverpool, Liverpool, United Kingdom
2 Peter Medawar Building for Pathogen Research, University of Oxford, Oxford, United Kingdom
3 Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA, United States
4 Center for Emerging and Neglected Infectious Diseases, Mahidol University, Salaya, Thailand
5 Tropical and Infectious Diseases Unit, Royal Liverpool University Hospital, Liverpool, United Kingdom

Flaviviruses are closely related and exhibit immunological cross-reactivity. Previous studies suggest a role for CD8 T cells in protection and pathology in dengue, and a protective role in Zika and JE. CD8 T cell responses are generated by live flavivirus vaccines; T cell responses against Flaviviruses are also cross-reactive and therefore a vaccine that stimulates cross-reactive CD8 T cell responses may be broadly protective. We examined whether CD8 T cell responses against DENV and JEV could recognise ZIKV, and whether they have anti-viral function. Twenty-six of 61 subjects studied (43%) had dengue exposure, either diagnosed clinical illness or residence in a dengue endemic area and detectable neutralising antibody. Two subjects had recovered from JE, fifteen had received flavivirus vaccines and eighteen were negative controls without flavivirus exposure. Only one subject had ZIKV neutralising antibody, indicating that the majority of subjects were not ZIKV exposed. We separated PBMC and screened for T cell responses against JEV and DENV peptide libraries by IFNγ-ELISpot assay. Of the 61 subjects, 27 had positive IFNγ-ELISpot assays. In total, 9 minimal CD8 T cell epitopes either cross-react with ZIKV variants in ex-vivo IFNγ assays or were identical in sequence with ZIKV. Short-term T cell cultures expanded to DENV/JEV peptides could recognise ZIKV variant peptides equally well, further demonstrating that these responses are part of a single cross-reactive pool of CD8 T cells, and do not represent a distinct population of responding cells.Short-term T cell cultures expanded to DENV/JEV peptides lysed autologous target cells labelled with ZIKV peptides, indicating that CD8 T cells primed to DENV or JEV could recognise and kill ZIKV infected cells, mediating anti-viral function. Our findings imply that inclusion of CD8 T cell antigenic determinants into novel flavivirus vaccines may be beneficial and is worthy of consideration in vaccine design.