Production Of Veterinary Vaccines Research Articles

Veterinary and biological industry on the guard of animal health: modernity and prospects

Ensuring epizootic well-being on the territory of the Russian Federation is an integral part of the concept of national security of the country. In order to implement plans to improve the epizootic situation in the Russian Federation, the National Association of Veterinary and Biological Industry Organizations (Association "Vetbioprom") was established.The strategic goal of the enterprises belonging to the Association "Vetbioprom" is economic stability, the main goals and objectives are the development, improvement and introduction of promising domestic technologies in the field of veterinary medicine and immunobiotechnology, and the creation of new domestic immunobiological drugs for veterinary use.The main goals and objectives of the enterprises that are members of the Vetbioprom Association are the development, improvement and implementation of promising domestic technologies in the field of veterinary medicine and immunobiotechnology, and the creation of new domestic immunobiological drugs for veterinary use.The enterprises belonging to the Vetbioprom Association produce medicines for veterinary use, immunobiological preparations for diagnosing animal diseases, as well as disinfectants for veterinary use.The potential of domestic enterprises, the available capacities today allow to increase production volumes and output for veterinary use by 1.5 2 times. In addition, all enterprises carry out innovative developments in the field of veterinary vaccines and are ready, subject to state support, to bring them into production and provide farmers with replacements for imported analogues.The development of the domestic production of veterinary vaccines will contribute to the achievement of the national development goals of the Russian Federation and the implementation of a set of measures aimed at creating and implementing competitive domestic technologies based on the latest scientific achievements and ensuring the production of highquality medicines and diagnostics for veterinary use.

Detection and genotyping of bovine viral diarrhea virus found contaminating commercial veterinary vaccines, cell lines, and fetal bovine serum lots originating in Mexico.

In this communication, we report the presence of RNA of bovine viral diarrhea virus (BVDV) as a contaminant of different biological products used in Mexico for veterinary vaccine production. For this purpose, six batches of monovalent vaccines, eight cell line batches used for vaccine production, and 10 fetal bovine serum lots (FBS) commercially available in Mexico from different suppliers were tested by reverse transcription polymerase chain reaction (RT-PCR). Viral RNA was detected in 62.5% of the samples analyzed. Phylogenetic analysis revealed the presence of the subgenotypes BVDV-1a, 1b, and BVDV-2a in the tested samples. Collectively, these findings indicate that contamination by BVDV RNA occurs in commercial vaccines and reagents used in research and production of biological products. The ramifications of this contamination are discussed.

Quantitative Assessment of the Physical Virus Titer and Purity by Ultrasensitive Flow Virometry

AbstractRapid quantification of viruses is vital for basic research on viral diseases as well as biomedical application of virus‐based products. Here, we report the development of a high‐throughput single‐particle method to enumerate intact viral particles by ultrasensitive flow virometry, which detects single viruses as small as 27 nm in diameter. The nucleic acid dye SYTO 82 was used to stain the viral (or vector) genome, and a laboratory‐built nano‐flow cytometer (nFCM) was employed to simultaneously detect the side‐scatter and fluorescence signals of individual viral particles. Using the bacteriophage T7 as a model system, intact virions were completely discriminated from empty capsids and naked viral genomes. Successful measurement of the physical virus titer and purity was demonstrated for recombinant adenoviruses, which could be used for gene delivery, therapeutic products derived from phage cocktails, and infected cell supernatants for veterinary vaccine production.

Quantitative Assessment of the Physical Virus Titer and Purity by Ultrasensitive Flow Virometry.

Rapid quantification of viruses is vital for basic research on viral diseases as well as biomedical application of virus‐based products. Here, we report the development of a high‐throughput single‐particle method to enumerate intact viral particles by ultrasensitive flow virometry, which detects single viruses as small as 27 nm in diameter. The nucleic acid dye SYTO 82 was used to stain the viral (or vector) genome, and a laboratory‐built nano‐flow cytometer (nFCM) was employed to simultaneously detect the side‐scatter and fluorescence signals of individual viral particles. Using the bacteriophage T7 as a model system, intact virions were completely discriminated from empty capsids and naked viral genomes. Successful measurement of the physical virus titer and purity was demonstrated for recombinant adenoviruses, which could be used for gene delivery, therapeutic products derived from phage cocktails, and infected cell supernatants for veterinary vaccine production.

Requirements for standard viral vaccine samples for veterinary medicine

One of the branches of domestic biotechnology is the production of immunobiological drugs, including vaccines for medical and veterinary purposes. Despite the general decline observed in the global economy in recent years, the pharmaceutical market is developing. In 2020, the biological drug market could exceed $250 billion. In the Russian Federation and the countries of the Eurasian Economic Union, serious actions are being taken to harmonize national regulatory requirements with international rules and foreign control systems in the field of the production of immunobiological drugs and quality control. It should be noted the price availability of domestic vaccines, their high adaptation to the Russian epizootic situation, since they were created on the basis of strains characteristic of the territory of the country and the Eurasian continent. For the prevention of diseases such as foot-and-mouth disease, anthrax, trichophytia, microsporia, brucellosis, domestic vaccines are used in Russia. The main component of the strategy for the production of veterinary vaccines to increase competition of products is ensuring their quality. Products of Russian companies that do not have the practice of applying international standards cannot be competitive. This leads to the loss of the domestic market. Currently, regulatory documents of the Russian Federation and the Eurasian Economic Union have established uniform requirements for the organization of the production and quality control of medicines for medical and veterinary use. Within the framework of these documents, areas related to the development and certification of standard samples of various levels are actively developing, the purpose of which is to quantify the specific activity of drugs and the metrological characteristics of the methods used to evaluate quality, stability indicators, as well as control of auxiliary substances. The use of auxiliary substances and pharmaceutical substances is regulated by regulatory documentation: GF, FFS, FS, FSP or industry standards. This is necessary, since heterogeneity of drug quality parameters is possible due to various factors, including uncontrolled and uncontrolled.

Livestock and Poultry Production in Nepal and Current Status of Vaccine Development.

The livestock and poultry sectors are an integral part of Nepalese economy and lifestyle. Livestock and poultry populations have continuously been increasing in the last decade in Nepal and are likely to follow that trend as the interests in this field is growing. Infectious diseases such as Foot and Mouth Disease (FMD), Peste des Petits Ruminants (PPR), hemorrhagic septicemia (HS), black quarter (BQ), swine fever, avian influenza, and Newcastle disease (ND) constitute one of the major health challenges to the Nepalese livestock and poultry industry. Vaccinations are an efficient means of preventing the occurrence and spread of several diseases in animals and birds. Considering this fact, the government of Nepal began the production of veterinary vaccines in the 1960s. Nepal is self-reliant in producing several vaccines for cattle and buffaloes, sheep and goats, pigs, and poultry. Despite these efforts, the demand for vaccines is not met, especially in the commercial poultry sector, as Nepal spends billions of rupees in vaccine imports each year. There is a need of strengthening laboratory facilities for the isolation and characterization of field strains of pathogens and capacity building for the production of different types of vaccines using the latest technologies to be self-reliant in veterinary vaccine production in the future in Nepal.

Advanced applications of nanotechnology in veterinary medicine.

The invention of new techniques to manipulate materials at their nanoscale had an evolutionary effect on various medical sciences. At the time, there are thousands of nanomaterials which can be divided according to their shape, origin, or their application. The nanotechnology provided new solutions for old problems. In medical sciences, they are used for diagnostic or therapeutic purposes. They can also be applied in the preparation of nanovaccines and nanoadjuvants. Their use in the treatment of cancer and in gene therapy opened the door for a new era in medicine. Recently, various applications of nanotechnology started to find their way in the veterinary sector. They increasingly invade animal therapeutics, diagnostics, production of veterinary vaccines, farm disinfectants, for animal breeding and reproduction, and even the field of animal nutrition. Their replacement of commonly used antibiotics directly reflects on the public health. By so doing, they minimize the problem of drug resistance in both human and veterinary medicine, and the problem of drug residues in milk and meat. In addition, they have a great economic impact, by minimizing the amounts of discarded milk and the number of culled calves in dairy herds. Nanotechnology was also applied to develop pet care products and hygienic articles. The present review discusses the advantage of using nanomaterials compared to their counterparts, the various classes of nanoparticles, and illustrates the applications and the role of nanotechnology in the field of veterinary medicine.

Application of Chitosan in Veterinary Vaccine Production

Probability-based surveys of the application of chitosan succinate as an adjuvant added to the vaccine against animal necrobacteriosis have been performed. The addition of 3.7% chitosan succinate (MM 330 kDa) to the standard-dose vaccine formulation could contribute to improvement of the vaccine immunogenicity, as measured by the hemagglutination test (HA): 1 : 512 as compared to 1 : 128 in the control range. The use of 0.5-% chitosan succinate solution as a protective medium for drying in the production of a dried inactivated vaccine against infectious bovine rhinotracheitis could ensure the preservation of the biopreparation’s high immunogenic activity. Chitosan succinate was shown to have quite good solubility in water, allowing the possible use of saline solution to reconstitute the vaccine into the suspension for injection and to abandon expensive solvents.

Designing Two Individual AcMNPV Polyhedrin-Plus Bac-to-Bac Expression System in order to Express GFP and CPV-VP2 in Insect Cells.

Background: The importance of viral protein-2 (VP2) of canine parvovirus (CPV) in binding to human cancer cells, production of veterinary vaccines and diagnostic kits has motivated several researches on producing this protein. Objectives: Our purpose was to construct recombinant bacmid shuttle vectors expressing VP2 of CPV using Bac-to-Bac baculoviral expression system. Materials and Methods: Mini-Tn7 transposones engineered in pFastBac1 donor vectors were used to construct expression cassettes of GFP and CPV-VP2. The plasmids were transferred into E. coli DH10Bac competent cells. Site-specific transposition of the genes into bacmid was accomplished using helper plasmid. Occurrence of Transposition was confirmed via PCR using specific primers and PUC/M13 universal primers. The recombinant bacmid DNAs were transfected into Sf9 cells using cationic lipids to generate new recombinant baculoviruses expressing GFP and CPV-VP2. GFP and VP2 expressions were evaluated by fluorescence microscopy and western analysis, respectively. Results: Cloning, subcloning and recombination processes of both GFP and VP2 were accomplished and verified. Accuracy of transfection process was confirmed by GFP fluorescence microscopy.VP2 expression was verified by SDS-PAGE and western analysis. Conclusions: Two Bac-to-Bac expression systems were designed to produce recombinant VP2 and GFP in insect cells.

Veterinary vaccine production using bioreactors: scale up study from laboratory and pd up to commercial production

Background Vaccination has proven itself as the most effective tool to control and prevent the disease and to facilitate the safe trade of live animals. Viral vaccine manufacturing processes present some specific constraints as compared to other biotech products linked to the cell substrate used and to the viral production. Multiple cell lines are used for productions such as VERO, MDCK, MRC5, BHK, and CHO cells. BHK-21 cells were selected because they are the most commonly used cells for vaccine production. As the feasibility of transferring the BHK-21 cells growth in microcarriers from a conventional bioreactor to single-use bioreactor (SU) was demonstrated, actually was time to scale-up and verify the flexibility and ease of use of these SU bioreactors enable rapid scale-up without any loss in product quality. This document describes a scaled-up production of a bovine viral vaccine. Identification of optimal parameters values, testing at intermediary bioreactors scales (2 liters 50 liters) and application of the new process settings at industrial bioreactor scale of 200 liters were performed. Taking into account that a critical feature of microcarrier-based cell culture is the homogeneity of cell adhesion to the beads, mixing and aeration performance evaluation remain mandatory to ensure robust scale-up of cell culture processes. The goal of this study was to evaluate the possibility to produce the antigens for animal vaccines manufacturing in a bioreactor SU as alternative to roller bottles without any loss in product quality. Evaluation of COG reduction was done.

Exploring the transcriptome space of a recombinant BHK cell line through next generation sequencing

Baby Hamster Kidney (BHK) cell lines are used in the production of veterinary vaccines and recombinant proteins. To facilitate transcriptome analysis of BHK cell lines, we embarked on an effort to sequence, assemble, and annotate transcript sequences from a recombinant BHK cell line and Syrian hamster liver and brain. RNA-seq data were supplemented with 6,170 Sanger ESTs from parental and recombinant BHK lines to generate 221,583 contigs. Annotation by homology to other species, primarily mouse, yielded more than 15,000 unique Ensembl mouse gene IDs with high coverage of KEGG canonical pathways. High coverage of enzymes and isoforms was seen for cell metabolism and N-glycosylation pathways, areas of highest interest for biopharmaceutical production. With the high sequencing depth in RNA-seq data, we set out to identify single-nucleotide variants in the transcripts. A majority of the high-confidence variants detected in both hamster tissue libraries occurred at a frequency of 50%, indicating their origin as heterozygous germline variants. In contrast, the cell line libraries' variants showed a wide range of occurrence frequency, indicating the presence of a heterogeneous population in cultured cells. The extremely high coverage of transcripts of highly abundant genes in RNA-seq enabled us to identify low-frequency variants. Experimental verification through Sanger sequencing confirmed the presence of two variants in the cDNA of a highly expressed gene in the BHK cell line. Furthermore, we detected seven potential missense mutations in the genes of the growth signaling pathways that may have arisen during the cell line derivation process. The development and characterization of a BHK reference transcriptome will facilitate future efforts to understand, monitor, and manipulate BHK cells. Our study on sequencing variants is crucial for improved understanding of the errors inherent in high-throughput sequencing and to increase the accuracy of variant calling in BHK or other systems.

Comparative study on three locally developed live orf virus vaccines for sheep in Saudi Arabia

The epidemiology of orf virus infection in Saudi Arabia (SA) has been researched since 1990. The results obtained during this period indicate that the disease is widespread, has great economic impact and that no vaccine has been used against it. The present study compares the immunogenicity and protective efficacy of three locally developed live orf virus vaccines. Two of them differ in their passage history in Vero cell culture and the third was used as a virulent virus in glycerine buffer. To the best of the authors' knowledge, no similar comparative study has been conducted in the Middle East utilising three types of vaccines prepared from the same virus strain. Selection of the candidate seed orf virus and performance of the quality control tests were as laid out by the OIE for veterinary vaccine production. The vaccine seed virus was a field orf virus isolated from a previous orf outbreak in Saudi Arabia. A simple novel formula was developed to calculate the rate of reduction in the healing time (RHT %) in the challenged sheep. This allowed direct comparison of the efficacy of the three types of vaccines employed in the present study. The efficacy of each vaccine was tested on a cohort of local Noemi sheep.

Expression behaviour of the fusion antigen CTB::VP60, a promising RHD-vaccine, in pea

The production of veterinary vaccines in transgenic plants appears to be an upcoming strategy. Since 15 years transgenic plants express a number of viral and bacterial antigens -amongst others VP60 (1, 2), the only structural capsid protein of the rabbit haemorrhagic disease virus (RHDV). However plantderived VP60 demonstrated very low immunogenicity compared to other production systems like baculovirus. In order to enhance the immunogenicity of plant-derived VP60 we fused the protein to the well known adjuvant CTB, the non-toxic B subunit of Cholera toxin (CTA-CTB5). The chimaric gene ctbvp60SEKDEL, regulated by the CaMV 35S promoter, was integrated into pea (Pisum sativum) by Agrobacterium-mediated transformation. Around 60 independent transgenic plant lines of the F1 generation were analysed and the protein content of CTB::VP60 was estimated using CTB-, GM1-gangliosideand VP60-dependent ELISA. Four transgenic F1 lines demonstrated detectable amount of the two complex components CTB and VP60. The heterogeneous pentamer complex could be detected by a newly developed combined GM1-VP60-ELISA Test. Additionally, we attempted to increase expression levels by analysing transgenic plant lines in the F2 and F3 generation after self pollination of the two highest expression lines. Up to 100 independent transgenic F3-lines with significant enhancement of the expression level up to 0.7 % TSP were selected to produce CTB::VP60-seeds for immunisation experiments with rabbits using mucosal and parenteral vaccination strategies.

Highly protective E2–CSFV vaccine candidate produced in the mammary gland of adenoviral transduced goats

Classical swine fever virus is the etiological agent of the most economically important highly contagious disease of swine worldwide. E2 is the major envelope glycoprotein present as a homodimer on the outer surface of the virus and represents an important target for the induction of neutralizing immune response against the viral infection. The E2 extracellular domain was expressed in the milk of adenoviral transduced goats at the highest level about 1.2 g/L. The recombinant glycoprotein was purified from clarified serum milk by a single metal chelate affinity chromatography step, as a homodimer of approximately 100 kDa and purity over 98%. Glycosylation analysis showed the presence of oligomannoside, hybrid and complex type N-glycans, attached to the recombinant E2. The capacity of goat milk-derived E2 antigen to protect pigs from both classical swine fever clinical signs and viral infection was assessed in a vaccination and challenge trial. The immunized pigs became protected after challenge with 10 5 LD 50 of a highly pathogenic CSFV strain. In the context of veterinary vaccines, this expression system has the advantages that the recombinant antigen could be harvested in about 48 h after adenoviral transduction with expression levels in the range of g/L. This approach may turn into a scalable expression system for the assessment and production of veterinary vaccines.

Transgenic plants for the production of veterinary vaccines

The expression of antigens in transgenic plants has been increasingly used in the development of experimental vaccines, particularly oriented to the development of edible vaccines. Hence, this technology becomes highly suitable to express immunogenic proteins from pathogens. Foot and mouth disease virus, bovine rotavirus and bovine viral diarrhoea virus are considered to be the most important causative agents of economic loss of cattle production in Argentina, and they are thus optimal candidates for alternative means of immunization. Here, we present a review of our results corresponding to the expression of immunogenic proteins from these three viruses in alfalfa transgenic plants, and we discuss the possibility of using them for the development of plant-based vaccines.

Characterization of intermediate phenotypes induced by chemically undefined laboratory media in virulent Bordetella bronchiseptica strains.

The expression of many virulence factors of Bordetella bronchiseptica is regulated by the bvgAS locus and reduced in response to environmental signals called modulators. Virulent strains can alternate between virulent (Bvg(+)), intermediate (Bvg(i)), and modulated (Bvg(+)mod) phenotypes. Potential vaccine antigens can be expressed by Bvg1 strains grown only in the absence of modulators. In the present study we evaluated filamentous hemagglutinin (FHA) and outer membrane protein (OMP) expression in Bvg(+) B. bronchiseptica strains grown in chemically undefined media: nutrient agar (NA), tryptic soy agar (TSA), tryptose phosphate broth (TPB), and brain-heart infusion (BHI). Our results suggest that TSA and TPB usually induce semimodulation, since Bvg(+) strains cultured in these media retained the expression of FHA and virulence-associated OMPs in the 30 kDa region, but failed to express other virulence markers such as OMPs in the regions of 90 and 200 kDa, though they expressed flagellin (avirulence marker). On the other hand, NA and BHI usually induce modulation. Thus the assayed chemically undefined media should not be used in vaccine production. Semimodulation induced by TSA and TPB can be accurately detected by SDS-PAGE Sarkosyl-insoluble OMP-enriched profiles. The reduction or absence of OMPs in the regions of 90 and 200 kDa is the most sensitive marker, and in some cases the presence of flagellin in intermediate profiles is another trait of the Bvg(i) phenotypes. Therefore these markers could be useful for selecting media for vaccine production. We also characterized the phenotype of Bvg(+) strains grown in Stainer-Scholte broth, an expensive medium, with and without glutathione, and we have detected no differences; this is the first attempt to reduce the cost of a Bordetella growth medium for veterinary vaccine production.

Real continuous biomass production ofBradyrhizobium japonicum in a veterinary vaccine production unit

In a real continuous culture,Bradyrhizobium japonicum strain 1755 was maintained in the 'Göttingen bioreactor unit' developed to produce veterinary bacterial vaccines for a period of 110 days without contamination or mutations. The average yield of viable bacteria over the whole period was more than 10(10)/ml.

Biotechnology and veterinary science : production of veterinary vaccines

The fundamental principles of genetic manipulation are explained, as are the methods of production of vaccines of veterinary importance. Specific attenuation of micro-organisms may result from genetically-engineered mutations which produce metabolic blocks, from the development of effective bacterial toxoids and from viral gene segment reassortment. Recombinant DNA techniques have been used to produce multi-component vaccines by the insertion of genes into carrier organisms, such as pox viruses and attenuated salmonella, and also to produce subunit vaccines. The use of synthetic peptide vaccines and anti-idiotype vaccines, together with problems of parasite vaccines, are described. Biotechnology would appear to have a major role to play in the future prevention of many animal diseases.