Extraneous agent detection in vaccines--a review of technical aspects

The quality and safety of commercial vaccines have a profound importance. Contrary to all precautions and efforts the use of biological material in vaccine development and production may lead to potential contamination of the vaccines with known and unknown extraneous agents (EAs). In veterinary field official lists of EAs have been compiled as legal framework to describe the potential agents, which must be tested during manufacture of vaccines. Nevertheless, detection of known and unknown contaminants in vaccines is a common duty for manufacturers and authorities of both veterinary and human field sharing similar needs of special technical approaches. State-of-art molecular methods such as randomly primed PCR combined with massive parallel sequencing (MPS) or microarrays may open new perspectives in extraneous agent testing. The robustness and efficacy of this technical approach in vaccine control was clearly demonstrated on a human vaccine example when porcine circovirus type 1 (PCV1) contamination was revealed in Rotarix, a human rotavirus vaccine. The consequences and implications are reviewed hereby from a veterinary regulatory point of view.     

Evaluation of the analytical sensitivity of a polymerase chain reaction assay for the detection of chicken infectious anemia virus in avian vaccines

Chicken infectious anemia virus (CAV) is a ubiquitous pathogen of chickens causing significant disease in commercial flocks worldwide. During CAV outbreaks, the Center for Veterinary Biologics requires manufacturers of veterinary biologicals to test materials derived from infected flocks for extraneous CAV by polymerase chain reaction (PCR). The analytical sensitivity of a PCR assay for detection of CAV was determined and the applicability of a CAV DNA standard as a positive control for assay validity was evaluated. The analytical sensitivity of the CAV PCR assay was assessed to be 100 copies per reaction for the DNA standard and 1 × 10^1.9 TCID₅₀/reaction for infectious virus. Establishing the analytical sensitivity of this CAV PCR assay and the inclusion of internal and external positive controls for validity provide a basis for determining whether suspect materials are safe for use in the production of veterinary biologics.     

Chicken anemia virus and avian gyrovirus 2 as contaminants in poultry vaccines

This study focuses on the detection of chicken anemia virus (CAV) and avian gyrovirus 2 (AGV2) genomes in commercially available poultry vaccines. A duplex quantitative real-time PCR (dqPCR), capable of identifying genomes of both viruses in a single assay, was employed to determine the viral loads of these agents in commercially available vaccines. Thirty five vaccines from eight manufacturers (32 prepared with live and 3 with inactivated microorganisms) were examined. Genomes of CAV were detected as contaminants in 6/32 live vaccines and in 1/3 inactivated vaccines. The CAV genome loads ranged from 6.4 to 173.4 per 50 ng of vaccine DNA (equivalent to 0.07 to 0.69 genome copies per dose of vaccine). Likewise, AGV2 genomes were detected in 9/32 live vaccines, with viral loads ranging from 93 to 156,187 per 50 ng of vaccine DNA (equivalent to 0.28–9176 genome copies per dose of vaccine). These findings provide evidence for the possibility of contamination of poultry vaccines with CAV and AGV2 and they also emphasize the need of searching for these agents in vaccines in order to ensure the absence of such potential contaminants.     

Phylogenetic analysis of bovine pestiviruses: testing the evolution of clinical symptoms

This study presents a phylogenetic analysis of 115 bovine pestiviruses. A sequence data set from the 5′ untranslated genomic region was analyzed with maximum parsimony, bootstrapping and parsimony jackknifing. We tested for the proposed classifications of the group and analyzed the evolution of the symptoms associated with Pestivirus infections in bovines. Based on the historical framework provided by our phylogenetic trees, we also characterized the extent and importance of contamination caused in biologicals by the virus. Our phylogenetic analyses showed that the previously defined genotypes are monophyletic, except for genotype 1a. Based on our cladograms, we propose the existence of more than 12 monophyletic groups within the species BVDV 1. The mapping of clinical symptoms suggests that the emergence of some genotypes could have been driven by a change in the pathogenic process. Enteric Problems appear to be ancestral, while Reproductive and Respiratory Problems arise with the emergence of genotypes 1b, 1d and the herein‐proposed genotype Arg 1. The distribution of contaminant strains on the cladograms shows that pestiviral contamination is a common process, and also suggests that a contaminated product might be a vehicle for virus dispersion. Implications for virus evolution, virus taxonomy, veterinary medicine and biotechnology are discussed.     

Survey of Pestivirus infection in wild and domestic ungulates from south-western Italian Alps

The transmission of pestiviruses between domestic and wild ruminants has not been documented in communal alpine pastures shared between wildlife and livestock. The aim of this study was to investigate the role of domestic and wild ungulates species from Varaita Valley (SW Italian Alps) in the epidemiology of Pestivirus infections. Sera from free-ranging alpine chamois (Rupicapra rupicapra) and roe deer (Capreolus capreolus) were collected from 1994 to 2009 and 2001 to 2009, respectively. Also, sera from cattle and sheep sampled in 2009 were studied. Sera were tested for the presence of antibodies against pestivirus with an ELISA assay. Sera from positive animals were subsequently tested with a comparative virus neutralisation test using the BVDV-NADL and BDV-137/4 strains. Sera were tested for the presence of pestiviral antigen and the presence of viral RNA with a commercial ELISA assay and RT-PCR. Antibodies against Pestivirus were detected in 132 out of 312 (42%) chamois, in 30 out of 175 (17%) cattle and 6 out of 24 (25%) sheep. No antibodies were found in roe deer. No Pestivirus antigen or RNA was detected in any of the samples. Results indicate circulation of pestiviruses among the studied chamois, cattle and sheep populations. However the role of wild ungulates in the dynamics of Pestivirus infection is still unknown and monitoring the presence of these viruses in wild ungulates would be of importance, especially in the chamois population, where pestiviruses seem to circulate extensively.     

Development,standardization and assessment of PCR systems for purity testing of avian viral vaccines

The European Pharmacopoeia (Ph. Eur.) requires avian viral vaccines to be free of adventitious agents. Purity testing is an essential quality requirement of immunological veterinary medicinal products (IVMPs) and testing for extraneous agents includes monitoring for many different viruses. Conventional virus detection methods include serology or virus culture, however, molecular tests have become a valid alternative testing method.Nucleic acid testing (NAT) is fast, highly sensitive and has a higher degree of discrimination than conventional approaches. These advantages have led to the development and standardization of polymerase chain reaction (PCR) assays for the detection of avian leucosis virus, avian orthoreovirus, infectious bursal disease virus, infectious bronchitis virus, Newcastle disease virus, infectious laryngotracheitis virus, influenza A virus, Marek's disease virus, turkey rhinotracheitis virus, egg drop syndrome virus, chicken anaemia virus, avian adenovirus and avian encephalomyelitis virus. This paper reviews the development, standardization and assessment of PCR for extraneous agent testing in IVMPs with examples from an Official Medicines Control Laboratory (OMCL).     

Viral safety and extraneous agents testing for veterinary vaccines: Rationale for requirements,the European approach

Freedom from extraneous agents is a high priority for any medicinal product. For veterinary vaccines, extraneous agents testing is addressed by different regulations of the European Pharmacopoeia and guidelines issued by the European Medicines Evaluation Agency. This article provides a brief review of the relevant texts.     

9-Aminoacridine-based agents impair the bovine viral diarrhea virus (BVDV) replication targeting the RNA-dependent RNA polymerase (RdRp)

Bovine viral diarrhea virus (BVDV) infection is still a plague that causes important livestock pandemics. Despite the availability of vaccines against BVDV, and the implementation of massive eradication or control programs, this virus still constitutes a serious agronomic burden. Therefore, the alternative approach to combat Pestivirus infections, based on the development of antiviral agents that specifically inhibit the replication of these viruses, is of preeminent actuality and importance.Capitalizing from a long-standing experience in antiviral drug design and development, in this work we present and characterize a series of small molecules based on the 9-aminoacridine scaffold that exhibit potent anti-BVDV activity coupled with low cytotoxicity. The relevant viral protein target – the RNA-dependent RNA polymerase – the binding mode, and the mechanism of action of these new antivirals have been determined by a combination of in vitro (i.e., enzymatic inhibition, isothermal titration calorimetry and site-directed mutagenesis assays) and computational experiments. The overall results obtained confirm that these acridine-based derivatives are promising compounds in the treatment of BVDV infections and, based on the reported structure-activity relationship, can be selected as a starting point for the design of a new generation of improved, safe and selective anti-BVDV agents.     

Comparison of requirements in the European Union and United States of America for pre-clinical viral safety testing of veterinary vaccines

Of paramount importance in ensuring the safety of live and inactivated veterinary vaccines is demonstration of freedom from extraneous agents in biological starting materials used in their production. Both the European Union (EU) and United States of America (US) provide regulations and guidelines on extraneous agent testing of veterinary vaccines including guidance from the Committee for Medicinal Products for Veterinary Use (CVMP), the European Pharmacopoeia (Ph. Eur.) and the USDA Code of Federal Regulations, Title 9 (9CFR). There are distinct requirements prescribed in EU and US regulations and guidelines. The differences in EU and US requirements for extraneous agent testing of starting materials are such that there may be occasions when no one test may satisfy both sets of regulations for a given scenario. For compliance with both, for global licensing purposes it may therefore be necessary to perform additional tests and/or to justify methods chosen from one set of regulations over another, based on a variety of factors.     

Veterinary and human vaccine evaluation methods

Despite the universal importance of vaccines, approaches to human and veterinary vaccine evaluation differ markedly. For human vaccines, vaccine efficacy is the proportion of vaccinated individuals protected by the vaccine against a defined outcome under ideal conditions, whereas for veterinary vaccines the term is used for a range of measures of vaccine protection. The evaluation of vaccine effectiveness, vaccine protection assessed under routine programme conditions, is largely limited to human vaccines. Challenge studies under controlled conditions and sero-conversion studies are widely used when evaluating veterinary vaccines, whereas human vaccines are generally evaluated in terms of protection against natural challenge assessed in trials or post-marketing observational studies. Although challenge studies provide a standardized platform on which to compare different vaccines, they do not capture the variation that occurs under field conditions. Field studies of vaccine effectiveness are needed to assess the performance of a vaccination programme. However, if vaccination is performed without central co-ordination, as is often the case for veterinary vaccines, evaluation will be limited. This paper reviews approaches to veterinary vaccine evaluation in comparison to evaluation methods used for human vaccines. Foot-and-mouth disease has been used to illustrate the veterinary approach. Recommendations are made for standardization of terminology and for rigorous evaluation of veterinary vaccines.     

Identification of a novel GC-rich 113-nucleotide region to complete the circular, single-stranded DNA genome of TT virus, the first human circovirus

The sequence data (H. Okamoto et al., Hepatol. Res. 10:1-16, 1998) of a newly discovered single-stranded DNA virus, TT virus (TTV), showed that it did not have the terminal structure typical of a parvovirus. Elucidation of the complete genome structure was necessary to understand the nature of TTV. We obtained a 1.0-kb amplified product from serum samples of four TTV carriers by an inverted, nested long PCR targeted for nucleotides (nt) 3025 to 3739 and 1 to 216 of TTV. The sequence of a clone obtained from serum sample TA278 was compared with those registered in GenBank. The complete circular TTV genome contained a novel sequence of 113 nt (nt 3740 to 3852 [=0]) in between the known 3'- and 5'-end arms, forming a 117-nt GC-rich stretch (GC content, 90.6% at nt 3736 to 3852). We found a 36-nt stretch (nt 3816 to 3851) with an 80.6% similarity to chicken anemia virus (CAV) (nt 2237 to 2272 of M55918), a vertebrate circovirus. A putative SP-1 site was located at nt 3834 to 3839, followed by a TATA box at nt 85 to 90, the first initiation codon of a putative VP2 at nt 107 to 109, the termination codon of a putative VP1 at nt 2899 to 2901, and a poly(A) signal at nt 3073 to 3078. The arrangement was similar to that of CAV. Furthermore, several AP-2 and ATF/CREB binding sites and an NF-kappaB site were arranged around the GC-rich region in both TTV and CAV. The data suggested that TTV is circular and similar to CAV in its genomic organization, implying that TTV is the first human circovirus.     

Report on the international workshop on alternative methods for human and veterinary rabies vaccine testing: State of the science and planning the way forward

Potency testing of most human and veterinary rabies vaccines requires vaccination of mice followed by a challenge test using an intracerebral injection of live rabies virus. NICEATM, ICCVAM, and their international partners organized a workshop to review the availability and validation status of alternative methods that might reduce, refine, or replace the use of animals for rabies vaccine potency testing, and to identify research and development efforts to further advance alternative methods. Workshop participants agreed that general anesthesia should be used for intracerebral virus injections and that humane endpoints should be used routinely as the basis for euthanizing animals when conducting the mouse rabies challenge test. Workshop participants recommended as a near-term priority replacement of the mouse challenge with a test validated to ensure potency, such as the mouse antibody serum neutralization test for adjuvanted veterinary rabies vaccines for which an international collaborative study was recently completed. The workshop recommended that an in vitro antigen quantification test should be a high priority for product-specific validation of human and non-adjuvanted veterinary rabies vaccines. Finally, workshop participants recommended greater international cooperation to expedite development, validation, regulatory acceptance, and implementation of alternative test methods for rabies vaccine potency testing.     

Production of chicken anemia virus (CAV) VP1 and VP2 protein expressed by recombinant Escherichia coli

Chicken anemia virus (CAV) is an anemia agent of breeder and young chicks. This virus is the cause of economic losses across the chicken industry worldwide as a consequence of severe anemia and immunodeficiency among the birds. Two genes of CAV encoding the VP1 and VP2 proteins were cloned and expressed in Escherichia coli BL21 (DE3). A Western blot assay using His-tag antiserum was used to assess the expression level of the CAV viral proteins in E. coli. The results demonstrated that only full-length VP2 can be successfully expressed in E. coli, but not full-length VP1. A serial of N-terminus deletions of the VP1 protein, VP1Nd30, VP1Nd60 and VP1 Nd129, were created using PCR in order to improve VP1 expression. The results demonstrated that all three of these recombinant VP1 mutant proteins can be expressed in E. coli. VP1Nd129 protein demonstrates the highest expression level compared to the other two proteins. The specificity of Nd129-VP1 and VP2 protein were confirmed by mass spectrometry. By comparing the expression level of VP1Nd129 and VP2 protein after the addition of IPTG, the results indicated that the VP1Nd129 protein gave a higher level of protein expression than VP2. The highest yields of VP1Nd129 and VP2 were 26.2 and 15.5 mg/L, respectively, after IPTG induction with 0.1 mM IPTG for 6 h, respectively. The identification of the optimized conditions for production of the CAV viral proteins VP1 and VP2 will allow them to be used in the future as an antigen for the development of vaccines and diagnostic tests.     

Evaluation of the sensitivity of PCR methods for the detection of extraneous agents and comparison with in vivo testing

In this study the sensitivity of polymerase chain reaction (PCR) methods for the detection of Newcastle disease virus (NDV), avian reovirus (ARV), avian influenza virus (AIV) and avian infectious bronchitis virus (IBV) was compared to the sensitivity of the corresponding serological tests described in the European Pharmacopoeia (Ph. Eur.). For this purpose, serial 10-fold dilutions of the respective inactivated vaccines were prepared and groups of SPF chickens were vaccinated with a double dose of the vaccine dilutions. After a period of 21 days, the animals were revaccinated with a single dose. Two weeks later, serum samples from each animal were tested for antibodies using an Idexx enzyme linked immunosorbent assay (ELISA). In parallel, samples of the diluted vaccines were tested by PCR. It was found that the sensitivity of the four PCR tests is comparable to or even slightly better than that of the corresponding serological tests. Thus these PCR tests fulfil the sensitivity requirements of the Ph. Eur. and could be used as alternative tests for the detection of extraneous agents in final batches of inactivated vaccines.     

Human and animal vaccine contaminations

Vaccination is one of the most important public health accomplishments. However, since vaccine preparation involves the use of materials of biological origin, vaccines are subject to contamination by micro-organisms. In fact, vaccine contamination has occurred; a historical example of vaccine contamination, for example, can be found in the early days of development of the smallpox vaccine. The introduction of new techniques of vaccine virus production on cell cultures has lead to safer vaccines, but has not completely removed the risk of virus contamination. There are several examples of vaccine contamination, for example, contamination of human vaccines against poliomyelitis by SV40 virus from the use of monkey primary renal cells. Several veterinary vaccines have been contaminated by pestiviruses from foetal calf serum.These incidents have lead industry to change certain practices and regulatory authorities to develop more stringent and detailed requirements. But the increasing number of target species for vaccines, the diversity of the origin of biological materials and the extremely high number of known and unknown viruses and their constant evolution represent a challenge to vaccine producers and regulatory authorities.     

Extraneous agents testing for substrates of avian origin and viral vaccines for poultry: Current provisions and proposals for future approaches

In the 1970s the European Pharmacopoeia (Ph. Eur.) established the first requirements for testing starting materials for vaccines and the vaccines themselves. These requirements also cover testing for freedom from extraneous agents of specific pathogen free (SPF) chicken flocks, the embryonated eggs derived from them and viral vaccines for poultry. This was the first common European approach initiated by the Ph. Eur. as an institution of the Council of Europe and it was the beginning of building a scientific basis for vaccine quality. In the following years, the increasingly detailed requirements concerning viral purity also impacted viral vaccines for poultry, SPF chicken flocks and the embryonated eggs derived from them. The core of these requirements is formed by the list of extraneous agents that must be tested for and the accepted test methods. In the early 1990s and in 2004, the next steps were taken towards the harmonisation of quality regulations for the production and testing of veterinary immunological products, this time at the level of the European Community. With the first step, good manufacturing practices (GMP) and good laboratory practices (GLP) were introduced, ensuring more consistent production, validation of production procedures and testing. The next step introduced the risk assessment, which covers the evaluation of the quality of production and control.The intention of these efforts is to contribute to the quality, safety and purity of the products placed on the market. It makes sense that, based on the outcome of the risk-evaluation, a reduction of in-process and final product testing may be called for in certain cases. However, despite the fact that the quality of the starting materials and vaccines has been increased over the years, the provisions of the Ph. Eur. have not been adjusted. Progress made by the manufacturers of starting materials and vaccines with respect to increasing the quality of their products should be recognised. This review gives an analysis of the current provisions of the Ph. Eur. and makes some proposals on how the requirements concerning the testing of extraneous agents could be modified to take into consideration the increase in quality that has been achieved over the past few decades.     

High yield expression in a recombinant <Emphasis Type="Italic">E. coli</Emphasis> of a codon optimized chicken anemia virus capsid protein VP1 useful for vaccine development

Background

Chicken anemia virus (CAV), the causative agent chicken anemia, is the only member of the genus Gyrovirus of the Circoviridae family. CAV is an immune suppressive virus and causes anemia, lymph organ atrophy and immunodeficiency. The production and biochemical characterization of VP1 protein and its use in a subunit vaccine or as part of a diagnostic kit would be useful to CAV infection prevention.

Results

Significantly increased expression of the recombinant full-length VP1 capsid protein from chicken anemia virus was demonstrated using an E. coli expression system. The VP1 gene was cloned into various different expression vectors and then these were expressed in a number of different E. coli strains. The expression of CAV VP1 in E. coli was significantly increased when VP1 was fused with GST protein rather than a His-tag. By optimizing the various rare amino acid codons within the N-terminus of the VP1 protein, the expression level of the VP1 protein in E. coli BL21(DE3)-pLysS was further increased significantly. The highest protein expression level obtained was 17.5 g/L per liter of bacterial culture after induction with 0.1 mM IPTG for 2 h. After purification by GST affinity chromatography, the purified full-length VP1 protein produced in this way was demonstrated to have good antigenicity and was able to be recognized by CAV-positive chicken serum in an ELISA assay.

Conclusions

Purified recombinant VP1 protein with the gene's codons optimized in the N-terminal region has potential as chimeric protein that, when expressed in E. coli, may be useful in the future for the development of subunit vaccines and diagnostic tests.

     

Epidemiology of chicken anemia virus in Central African Republic and Cameroon

ABSTRACT: BACKGROUND: Although chicken anemia virus (CAV) has been detected on all continents, little is known about this virus in sub-Saharan Africa. This study aimed to detect and characterize CAV for the first time in Central African Republic and in Cameroon. RESULTS: An overall flock seroprevalence of 36.7% was found in Central African Republic during the 2008--2010 period. Virus prevalences were 34.2% (2008), 14.3% (2009) and 10.4% (2010) in Central African Republic and 39% (2007) and 34.9% (2009) in Cameroon. CAV DNA was found in cloacal swabs of 76.9% of seropositive chickens, suggesting that these animals excreted the virus despite antibodies. On the basis of VP1 sequences, most of the strains in Central African Republic and Cameroon belonged to 9 distinct phylogenetic clusters at the nucleotide level and were not intermixed with strains from other continent. Several cases of mixed infections in flocks and individual chickens were identified. CONCLUSIONS: Our results suggest multiple introductions of CAV in each country that later spread and diverged locally. Mixed genotype infections together with the observation of CAV DNA in cloacal samples despite antibodies suggest a suboptimal protection by antibodies or virus persistence.