Developments in biologicals最新文献

Next generation, foot-and-mouth disease (FMD) molecular vaccines based on replication deficient human adenovirus serotype 5 viral vectored delivery of FMD capsid genes (AdFMD) are being developed by the United States Dept. of Homeland Security and industry partners. The strategic goal of this program is to develop AdFMD licensed vaccines for the USA National Veterinary Stockpile for use, if needed, as emergency response tools during an FMD outbreak. This vaccine platform provides a unique opportunity to develop a set of in vitro analytical parameters to generate an AdFMD vaccine product profile to replace the current lot release test for traditional, inactivated FMD vaccines that requires FMDV challenge in livestock. The possibility of an indirect FMD vaccine potency test based on a serological alternative was initially investigated for a lead vaccine candidate, Adt.A24. Results show that serum virus neutralization (SVN) based serology testing for Adt.A24 vaccine lot release is not feasible, at least not in the context of vaccine potency assessment at one week post-vaccination. Thus, an in vitro infectious titer assay (tissue culture infectious dose 50, TCID50) which measures FMD infectious (protein expression) titer was established. Pre-validation results show acceptable assay variability and linearity and these data support further studies to validate the TCID50 assay as a potential potency release test. In addition, a quantitative physiochemical assay (HPLC) and three immunochemical assays (Fluorescent Focus-Forming Unit (FFU); tissue culture expression dose 50 (TCED50); Western blot) were developed for potential use as in vitro assays to monitor AdFMD vaccine lot-to-lot consistency and other potential applications. These results demonstrate the feasibility of using a traditional modified-live vaccine virus infectivity assay in combination with a set of physiochemical and immunochemical tests to build a vaccine product profile that will ensure the each AdFMD vaccine lot released is similar to a reference vaccine of proven clinical safety and efficacy.

Vaccines play a key role in the control of viral diseases both in humans and in animals. In order to ensure the quality and consistency of vaccines they are extensively tested, including potency control of individual batches. In the case of vaccines against rabies the most widely used test for batch potency control is the National Institutes of Health (NIH) test. The NIH test is performed in mice leading to the consumption of thousands of animals every year. Protection against rabies after vaccination is associated with neutralizing antibodies directed against the viral glycoprotein (G). Therefore the amount of G-protein in vaccine preparations is an important parameter with regard to potency. Additionally the structural integrity of virus particles in vaccine preparations may be crucial for their immunogenicity. The objective of our work is the development of in vitro methods to determine the potency of vaccines against rabies. The result of this ongoing project shall be an assay panel including measurement of the antigenic content as well as parameters of antigen quality in a vaccine preparation allowing a precise prediction of the potency of rabies vaccines without using animal experiments.

Batch potency testing of rabies vaccines could be done by challenge, measurement of serum response or antigen quantification. Here, we show the development of a serological test that was successfully validated for use in batch release. The serological test is based on serum neutralization (SNT). The correlation to the NIH challenge was demonstrated by batches passing respectively failing equivalently in the NIH and SNT. The SNT provides information on immunogenicity and exhibits several advantages to the NIH: 1) SNT uses many fewer animals for batch release. 2) SNT allows quantitative information on the individual serum response, in contrast to the "dead"/"alive" interpretation of the NIH. 3) SNT is quicker than the NIH and needs fewer working hours. 4) SNT avoids the highly disturbing intra-cerebral injection and suffering from rabies for mice and spares the staff the emotional stress of massively harming animals.

The standard requirement for serial release potency testing of Leptospira bacterins in the United States is the hamster vaccination challenge test. It is a test that uses a large number of animals experiencing pain or distress, takes weeks to conduct, can be expensive and requires that laboratory personnel handle a viable zoonotic pathogen. In an effort to address these concerns, the United States Department of Agriculture (USDA) developed an in vitro method for potency testing of four Leptospira serovars. This enzyme-linked immunosorbent assay (ELISA) was subsequently validated in the target species. USDA informed their biologics licensees, permittees and applicants of the availability of reference bacterins and the regulatory acceptance regarding this alternative test method in notices issued in 2007 and 2009. This presentation describes how the initial research and subsequent development and validation work were accomplished.

The relative haemagglutinin-neuraminidase (HN) antigen content of inactivated Newcastle disease virus (NDV) vaccines from different manufacturers was determined by means of an Enzyme-Linked Immunosorbent Assay (ELISA) according to Monograph 870 of the European Pharmacopoeia (Ph. Eur.). Wide ranges of reactivity of the different products were observed. When comparing the antibody responses from chickens vaccinated with vaccines showing either high or low reactivity in the antigen ELISA it was found that approximately the same titres of antibodies were induced in the chickens. One hypothesis is that the inactivation procedures used to inactivate the Newcastle disease antigen may alter the antigenic determinant recognised by the monoclonal antibody used. An alteration of the antigen would influence the binding by the monoclonal antibodies used as catching and detection antibodies in the ELISA which may result in a lower ELISA reactivity. It was also found that HN antigen of two inactivated Paramyxovirus 1 (PMV-1) vaccines for pigeons could not be measured in the ELISA. For these vaccines the antigen-ELISA based on monoclonal antibody IDNDV134.1 cannot be used. Our experience shows that a thorough knowledge of the products tested with the ELISA and their influence on the test method is essential to avoid misinterpretations of the test results. The level of ELISA reactivity should not be used for the comparison of vaccines. Furthermore, prediction of the ability of an unknown vaccine to induce antibodies based on the level of ELISA reactivity is not possible. The results (level of reactivity) of the antigen ELISA for the in vitro potency testing of inactivated Newcastle disease vaccines should therefore be carefully interpreted. However, by knowing the performance characteristics of the NDV antigen ELISA and the characteristics of the vaccines to be tested it becomes a valuable tool for the control of inactivated Newcastle disease vaccines in our laboratory. The implementation of this ELISA method for the batch release testing markedly reduces the number of chickens and the time required for batch release testing.

There is great interest in the veterinary vaccine field to move away from in vivo release tests for vaccines to reduce cost and testing time, improve consistency and of course the 3Rs (reduce, refine, replace). A brief overview of Good Manufacturing Practice (GMP) and the consistency approach is discussed below and an overview of how manufacturers can use the consistency approach and GMP controls along with statistical analysis of processes at each stage of the production process (starting materials, antigen and finished product) to build in quality and reduce the need for in vivo finished product tests. A final summary and outline of some challenges we will face in moving this approach forward is covered in conclusion.

Veterinary vaccines contribute to improved animal and human health and welfare by preventing infectious diseases. However, testing necessary to ensure vaccine effectiveness and safety can involve large numbers of animals and significant pain and distress. NICEATM and ICCVAM recently convened an international workshop to review the state of the science of human and veterinary vaccine potency and safety testing, and to identify priority activities to advance new and improved methods that can further reduce, refine and replace animal use. Rabies, Clostridium sp., and Leptospira sp. vaccines were identified as the highest priorities, while tests requiring live viruses and bacteria hazardous to laboratory workers, livestock, pets, and wildlife were also considered high priorities. Priority research, development and validation activities to address critical knowledge and data gaps were identified, including opportunities to apply new science and technology. Enhanced international harmonization and cooperation and closer collaborations between human and veterinary researchers were recommended to expedite progress. Implementation of the workshop recommendations is expected to advance new methods for vaccine testing that will benefit animal welfare and ensure continued and improved protection of human and animal health.

Vaccines to protect against clostridial diseases are among the most common veterinary biologicals. Each batch of these materials is subjected to a variety of toxicity and antigenicity tests. The potency of the final vaccine is then assessed by Toxin Neutralisation Test (TNT). All of these tests use mice and have lethal endpoints. Development of alternatives for potency testing was based on ELISAs able to measure antibody levels to the specific toxins relative to a standard serum with a defined unitage. These alternative assays were shown to correlate with the relevant TNTs and have been accepted by European Regulatory Authorities as batch release potency tests. Recently we have developed in vitro cell line alternatives for the toxicity and antigenicity tests for Cl. septicum using the VERO cell line. With this cell line it has been possible to develop in vitro assays which, when compared with the in vivo tests, gave correlations of 87% to 100%. Having shown proof of principle, similar cell line assays have been developed for Cl. novyi and Cl. perfringens types C and D.