Virus-like particles: potential veterinary vaccine immunogens

Virus-like particle (VLP) composed of outer shell but no genome of virus mimics the natural configuration of authentic virion and has no characteristics of self-replication. A close resemblance to native viruses in molecular scaffolds and an absence of genomes make VLPs effectively elicit both humoral and cell-mediated immune responses even with no requirement of adjuvant for vaccines. As effective immunogens, characterized by high immunogenicity and safety, VLPs have been employed in production of human vaccines, such as the licensed vaccines of hepatitis B virus and human papillomavirus. However, there has been no report of licensed veterinary VLP vaccine worldwide as yet. Despite the wide application in vaccination, both the conventional inactivated and live attenuated vaccines for animals are subject to potential limitations due to incomplete inactivation and reversion to virulence. Therefore, those conventional vaccines may, to some extent, be replaced with the VLP-based vaccines conferring higher protection and safety to vaccinated animals. Here, we review the current status of VLPs as veterinary vaccines, and discuss the characteristics and problems associated with generating VLPs for different animal viruses.     

犬瘟热的诊断及其预防免疫的研究进展

本文对犬瘟热（CD）的诊断、预防免疫和免疫失败的影响因素及犬瘟热病毒（CDV）的宿主范围进行了综述。CDV不仅感染陆生食肉动物，而且也感染水生食肉动物，并且其宿主范围还在不断扩大。CDV感染主要采用病毒分离、特异性病毒抗原或特异性核酸检测等方法确诊。疫苗包括灭活的CDV疫苗、麻疹病毒（MV）异源苗及CDV弱毒活苗。疫苗接种犬的免疫反应主要取决于毒株特性及犬的应答能力，只有弱毒活苗能诱导产生持久而坚强的保护力。尽管多年来CDV弱毒活苗的使用控制了CD的发生，但最近免疫过的犬发生CD的病例并不少见。分析免疫失败的原因，主要是母源抗体干扰、疫苗质量差、其它病毒的免疫抑制以及CDV流行株可能发生了变异等因素的影响。     

Current perspectives on control of equine influenza

Influenza A viruses of the H3N8 subtype are a major cause of respiratory disease in horses. Subclinical infection with virus shedding can occur in vaccinated horses, particularly where there is a mismatch between the vaccine strains and the virus strains circulating in the field. Such infections contribute to the spread of the disease. Rapid diagnostic techniques are available for detection of virus antigen and can be used as an aid in control programmes. Improvements have been made to methods of standardising inactivated virus vaccines, and a direct relationship between vaccine potency measured by single radial diffusion and vaccine-induced antibody measured by single radial haemolysis has been demonstrated. Improved adjuvants and antigenic presentation systems extend the duration of immunity induced by inactivated virus vaccines, but high levels of antibody are required for protection against field infection. In addition to circulating antibody, infection with influenza virus stimulates mucosal and cellular immunity; unlike immunity to inactivated virus vaccines, infection-induced immunity is not dependent on the presence of circulating antibody to HA. Live attenuated or vectored equine influenza vaccines, which may better mimic the immunity generated by influenza infection than inactivated virus vaccines, are now available. Mathematical modelling based upon experimental and field data has been applied to examine issues relating to vaccine efficacy at the population level. A vaccine strain selection system has been implemented and a more global approach to the surveillance of equine influenza is being developed.     

动物细胞规模化培养及其在兽用疫苗生产中的应用

哺乳动物细胞大规模培养技术已经广泛应用于生产各种生物制品,也广泛应用于各种兽用疫苗的生产.细胞培养生产兽用疫苗不但能够降低成本,而且提高产品质量.细胞培养生产兽用疫苗已经成为各个生物制品公司关注的焦点.凡是影响细胞大规模培养的因素,都直接或者间接的影响疫苗质量.论文针对哺乳动物细胞大规模培养相关影响因素、相关技术和在兽用疫苗中的应用进行概述.     

三个厂家猪瘟活疫苗免疫效果比较试验

在同一条件下对3个厂家生产的猪瘟细胞源活疫苗进行了免疫效果评价试验,并与猪瘟传代细胞苗免疫效果进行比较。结果发现3个厂家生产的猪瘟细胞源活疫苗存在一定差异,2个厂家的免疫效果较好,1个厂家的免疫效果较差。猪瘟传代细胞苗免疫效果优于猪瘟细胞源活疫苗,猪瘟传代细胞苗免疫1次,抗体合格率高,持续时间长,猪瘟细胞源活疫苗要免疫两次才能获得比较好的效果。同时,我们发现高致病性猪蓝耳病活疫苗（JXA1-R株）对猪瘟抗体产生有一定影响。     

Viruses as vectors

Traditional vaccines against diseases caused by viruses are based on live attenuated viruses or killed virus preparations. Through the application of molecular biology it is now possible to consider several new approaches to making vaccines, which may combine increased efficacy with greater safety. One of these approaches is to manipulate genetically a virus so that it carries and expresses a foreign gene (or part of a gene) which codes for a protective antigen for another disease. Adeno-, polio- and herpesviruses have been engineered to act as vectors in this way but vaccinia virus remains the main candidate for a recombinant virus vector for vaccine use. The broad host-range of vaccinia virus has made it an effective vector for the analysis of expression of "foreign" antigens as well as a tool for the dissection of the host animal's immune system. For practical purposes in veterinary vaccines, recombinant viruses based on other poxviruses, with more restricted host-ranges, may have certain advantages. Work on the development of recombinant avipoxviruses and capripoxviruses as prototype vaccines for use in poultry and ruminants, respectively, is discussed and illustrated.     

Fundamental safety requirements in the use of live vaccine in food animals]

Live vaccines have a number of advantages over inactivated ones--above all in respect of the stimulation of cell-mediated immune reactions. Various live vaccines, based on viruses, bacteria, fungi or parasites, have been approved for use in Germany in animals used as a source of food. Safety requirements obviously play a more important role for live vaccines, both in vaccine development and in batch testing, than with inactivated vaccines. Vaccine strains isolated from tissue samples must be clearly distinguishable from field strains. The safety of overdoses and the spread of the vaccine strain in the immunized animals have to be investigated, as well as shedding of the vaccine strain and its safety in non-target species. Any impact of a live vaccine strain on humans and the environment must be assessed. Live vaccines will remain an important research field in the long term, with efforts focused on developing deletion mutants and vector vaccines.     

外源病毒检验用抗鸡传染性法氏囊病病毒特异性血清的制备与检定

为制备外源病毒检验用抗鸡传染性法氏囊病病毒(Infectious Bursal Disease Virus,IBDV)的特异性血清,对300只3~4周龄的SPF鸡进行了基础免疫和加强免疫。最后一次免疫后21d采血并分离血清,血清经混合、分装、冷冻真空干燥后,对其进行了无菌检验、外源病毒检验、剩余水分测定、中和效价测定和特异性检验。结果表明,本研究制备的抗IBDV特异性血清无菌检验、外源病毒检验和剩余水分测定均符合《中华人民共和国兽药典》三部(二〇一〇年版)规定;血清中和效价为1:5747,与IBDV B87株毒种中和指数为104.3,与鸡新城疫病毒La Sota株、鸡传染性支气管炎病毒H120株、鸡传染性支气管炎病毒H52株、鸡传染性喉气管炎病毒、鸡痘病毒、禽呼肠孤病毒S1133株的中和指数均不大于10,通过AGP、ELISA、HI等试验确定血清中不含其他禽源外源病毒抗体。本研究为鸡传染性法氏囊病活疫苗或毒种的外源病毒检验提供了具有良好特异性和高效价的中和用血清。     

Safety and efficacy studies of live- and killed-feline leukemia virus vaccines.

The safety and the efficacy of several feline leukemia virus (FeLV) vaccines for 16-week-old kittens were determined. Vaccines were derived from an FL74 lymphoblastoid cell line that has been in continuous tissue culture passage for about 4 years. The vaccines were made from living virus, formaldehyde-inactivated whole FL74 cells, and formaldehyde-inactivated whole virus. The efficacy of each produced vaccine was determined by challenge exposure of vaccinated cats with virulent FeLV. The two formaldehyde-inactivated vaccines were found to be safe for use in kittens. Neither vaccine produce a significant feline oncornavirus-associated cell membrane antigen or virus-neutralizing antibody response, nor did they prevent infection with virulent FeLV. The inactivated whole-virus vaccine, however, did substantially decrease the proportion of kittens infected with virulent FeLV that became persistently viremic. In contrast, the whole FL74 cell vaccine did not reduce the number of infected kittens that became persistently viremic. The live-virus vaccine was found to be both safe and efficacious. About a half of the kittens vaccinated with live virus had transient bone marrow infection that lasted from 2 to 4 weeks. Viral antigen was not detected in peripheral blood, and infective virus was not shed in saliva, urine, or feces during the period that the vaccinal virus could be recovered from the bone marrow. In addition, there was no horizontal spread of vaccinal virus from vaccinated to non-vaccinated cagemates. Within several weeks, vaccinated kittens demonstrated no clinical or hematologic abnormalities and had high serum levels of feline oncornavirus-associated cell membrane antigen and virus-neutralizing antibody. Kittens vaccinated with living FeLV were resistant to infection with virulent virus.     

Stimulation of serum antibody formation in pigs by oral, subcutaneous or combined administration of live porcine rotaviruses or intramuscular inoculation of inactivated porcine rotaviruses in an oil adjuvant]

Only live vaccines prepared from attenuated strains have been used for the specific prophylaxis of rotavirus infections in pigs. These vaccines are administered to sows per os or parenterally to increase the content of antibodies in the blood serum, colostrum and milk, and in this way to provide for the better passive protection of suckling piglets through the maternal antibodies, or to induce the active immunity by pig vaccination. The data on the efficiency of live vaccines administered in both ways differ as to their ability to stimulate significantly increases in the actual levels of antibodies in sows and also as to the possibility of inducing the protection of vaccinated pigs from virulent virus infection. The objective of our trials was to compare the intensity of antibody response evoked by pig vaccination with live virus if the virus was implanted in different ways, and by vaccination with inactivated virus emulsified in oil adjuvant. The live vaccine consisted of a suspension of porcine rotavirus, strain OSU/6, cultivated in MA-104 culture medium with the content of 10(7) TKID50.ml-1, the inactivated vaccine was the identical virus suspension inactivated by an addition of 0.2% formaldehyde during 24 hours at a temperature of 37 degrees C, emulsified in oil adjuvant by means of an ULTRATURAX equipment at a 4:1 ratio.(ABSTRACT TRUNCATED AT 250 WORDS)     

口蹄疫灭活疫苗研究进展

灭活疫苗仍然是当前口蹄疫免疫控制的主要疫苗,研制口蹄疫灭活疫苗具有重要的现实意义,其研制方法在不断丰富和完善。文章详细描述了口蹄疫灭活疫苗研制的重要步骤及其进展,包括毒株筛选,病毒生产、灭活、抗原浓缩、纯化和配苗及疫苗的质量控制。制苗毒株的选择是研制疫苗的首要问题,直接关系到疫苗的免疫效果;病毒灭活和随后的安全试验是在制备灭活FMD疫苗中最关键的步骤;抗原浓缩纯化和配苗是保证疫苗良好免疫效果的必需。最后阐述了口蹄疫灭活疫苗在口蹄疫防控中的作用。     

Protection of chickens after live and inactivated virus vaccination against challenge with nephropathogenic infectious bronchitis virus PA/Wolgemuth/98

Protection provided by live and inactivated virus vaccination against challenge with the virulent nephropathogenic infectious bronchitis virus (NIBV) strain PA/Wolgemuth/98 was assessed. Vaccinations with combinations of live attenuated strains Massachusetts (Mass) + Connecticut (Conn) or Mass + Arkansas (Ark) were given by eyedrop to 2-wk-old specific-pathogen-free leghorn chickens. After live infectious bronchitis virus (IBV) vaccination, some chickens at 6 wk of age received an injection of either an oil emulsion vaccine containing inactivated IBV strains Mass + Ark or an autogenous vaccine prepared from NIBV PA/Wolgemuth/98. Challenge with PA/Wolgemuth/98 was given via eyedrop at 10 wk of age. Serum IBV enzyme-linked immunosorbent assay antibody geometric mean titers (GMTs) after vaccination with the combinations of live attenuated strains were low, ranging from 184 to 1,354, prior to NIBV challenge at 10 wk of age. Both inactivated vaccines induced an anamnestic response of similar magnitudes with serum GMTs of 6,232-12,241. Assessment of protection following NIBV challenge was based on several criteria virus reisolation from trachea and kidney and renal microscopic pathology and IBV-specific antigen immunohistochemistry (IHC). Live attenuated virus vaccination alone with combinations of strains Mass + Conn or Mass + Ark did not protect the respiratory tract and kidney of chickens after PA/Wolgemuth/98 challenge. Chickens given a live combination vaccination of Mass + Conn and boosted with an inactivated Mass + Ark vaccine were also susceptible to NIBV challenge on the basis of virus isolation from trachea and kidney butshowed protection on the basis of renal microscopic pathology and IHC. Live IBV-primed chickens vaccinated with an autogenous inactivated PA/Wolgemuth/98 vaccine had the highest protection against homologous virulent NIBV challenge on the basis of virus isolation.     

DNA vaccination against influenza viruses: a review with emphasis on equine and swine influenza

The influenza virus vaccines that are commercially-available for humans, horses and pigs in the United States are inactivated, whole-virus or subunit vaccines. While these vaccines may decrease the incidence and severity of clinical disease, they do not consistently provide complete protection from virus infection. DNA vaccines are a novel alternative to conventional vaccination strategies, and offer many of the potential benefits of live virus vaccines without their risks. In particular, because immunogens are synthesized de novo within DNA transfected cells, antigen can be presented by MHC class I and II molecules, resulting in stimulation of both humoral and cellular immune responses. Influenza virus has been used extensively as a model pathogen in DNA vaccine studies in mice, chickens, ferrets, pigs, horses and non-human primates, and clinical trials of DNA-based influenza virus vaccines are underway in humans. Our studies have focused on gene gun delivery of DNA vaccines against equine and swine influenza viruses in mice, ponies and pigs, including studies employing co-administration of interleukin-6 DNA as an approach for modulating and adjuvanting influenza virus hemagglutinin-specific immune responses. The results indicate that gene gun administration of plasmids encoding hemagglutinin genes from influenza viruses is an effective method for priming and/or inducing virus-specific immune responses, and for providing partial to complete protection from challenge infection in mice, horses and pigs. In addition, studies of interleukin-6 DNA co-administration in mice clearly demonstrate the potential for this approach to enhance vaccine efficacy and protection.     

动物细胞悬浮培养技术在兽用疫苗生产领域中的应用

近年来,动物细胞悬浮培养技术备受关注,该技术已广泛应用于各类生物制品及兽用疫苗的研究和生产过程中。细胞悬浮培养生产兽用疫苗既能降低成本, 也能提高产品质量。以生物反应器技术为基础的细胞悬浮培养技术平台正逐步被建立起来且日趋成熟,成为推动兽用疫苗生产快速发展的主要动力。文章介绍了细胞悬浮培养技术,并就该技术在兽用疫苗生产中的应用进行了论述。     

非洲猪瘟疫苗研制方向及进展

非洲猪瘟(African swine fever,ASF)给全球养殖业造成了巨大的威胁和挑战.非洲猪瘟病毒(African swine fever virus,ASFV)具有比寻常病毒更加巨大的基因组、迅速的变异能力和更加复杂多变的免疫逃逸机制,目前市场上仍无有效预防疫苗.科研人员几十年来,从灭活疫苗、减毒活疫苗、亚单...     

Effect of cell-culture passage on the pathogenicity and immunogenicity of two variant strains of infectious bursal disease virus.

Two variant strains of infectious bursal disease virus, IN and E, were adapted and passaged in an established cell line (BGM-70) 30 times and 40 times, respectively. Passage in cell culture resulted in loss of pathogenicity. However, both viruses maintained their antigenicity and immunogenicity, as demonstrated by the immunofluorescence and virus-neutralization tests and by the satisfactory protection induced by vaccinating specific-pathogen-free (SPF) chickens with inactivated preparations of both passaged viruses. No protection was induced when the passaged viruses were given to SPF chickens as live vaccines. It is speculated that the passaged viruses might have lost some ability to replicate in their natural host, resulting in lack of protection.     

Bluetongue vaccines in Europe

The article reviews the history, present status and the future of BT vaccines in Europe. So far, an attenuated (modified live viruses, MLV) and inactivated virus vaccines against BT were developed and used in the field. Moreover, the virus-like particles (VLPs) produced from recombinant baculovirus, and live recombinant vaccinia or canarypox virus-vectored vaccines were tested in the laboratory. The main aims of BT vaccination strategy are: to prevent clinical disease, to reduce the spread of the BTV in the environment and to protect movement of susceptible animals between affected and free zones. Actually, all of the most recent European BT vaccination campaigns have used exclusively inactivated vaccines. The use of inactivated vaccines avoid risk associated with the use of live-attenuated vaccines, such as reversion to virulence, reassortment of genes with field strain, teratogenicity and insufficient attenuation leading to clinical disease. The mass vaccinations of all susceptible animals are the most efficient veterinary method to fight against BT and successful control of disease. The vaccination of livestock has had a major role in reducing BTV circulation and even in eradicating the virus from most areas of Europe.     

两株狂犬病病毒强毒株糖蛋白基因的克隆及潜在抗原表位分析

通过RT-PCR分别获得了狂犬病病毒强毒CVS株、DRV82株糖蛋白基因,进行克隆及测序,并推导出氨基酸序列,与犬用疫苗弱毒株ERA、SRV9、犬源性街毒株CGX及人用疫苗株PG的糖蛋白序列进行比较。结果表明,以上狂犬病病毒毒株间的核苷酸同源性为83.1%～99.2%,氨基酸序列同源性为87.0%～98.5%。经Jameson-Wolf抗原表位优势图分析,CVS株与其他各株相比发现在304位、372位抗原表位优势升高;而DRV82株与其它各株差异不明显。抗原优势变化可能导致狂犬病病毒糖蛋白出现新的潜在抗原位点,为下一步构建不同毒株的狂犬病病毒糖蛋白重组疫苗奠定了基础。     

Lessons from the cat: development of vaccines against lentiviruses

Feline immunodeficiency virus (FIV) is a natural infection of domestic cats, which produces a disease with many similarities to human immunodeficiency virus (HIV) infection in man. The virus is an important cause of morbidity and mortality in pet cats worldwide. As such an effective vaccine is desirable both for its use in veterinary medicine and also as a model for the development of an HIV vaccine. A large number of candidate vaccines have been tested against feline immunodeficiency virus. These include inactivated virus and infected cell vaccines, DNA and viral vectored vaccines, subunit and peptide vaccines and vaccines using bacterial vectors. Ultimately, the development of inactivated virus and infected cell vaccines led to the release of the first licensed vaccine against FIV, in 2002. This review highlights some of the difficulties associated with the development of lentiviral vaccines and some of the lessons that have been learned in the FIV model that are of particular relevance to the development of HIV vaccines.     

A novel M2e-multiple antigenic peptide providing heterologous protection in mice

Swine influenza viruses (SwIVs) cause considerable morbidity and mortality in domestic pigs, resulting in a significant economic burden. Moreover, pigs have been considered to be a possible mixing vessel in which novel strains loom. Here, we developed and evaluated a novel M2e-multiple antigenic peptide (M2e-MAP) as a supplemental antigen for inactivated H3N2 vaccine to provide cross-protection against two main subtypes of SwIVs, H1N1 and H3N2. The novel tetra-branched MAP was constructed by fusing four copies of M2e to one copy of foreign T helper cell epitopes. A high-yield reassortant H3N2 virus was generated by plasmid based reverse genetics. The efficacy of the novel H3N2 inactivated vaccines with or without M2e-MAP supplementation was evaluated in a mouse model. M2e-MAP conjugated vaccine induced strong antibody responses in mice. Complete protection against the heterologous swine H1N1 virus was observed in mice vaccinated with M2e-MAP combined vaccine. Moreover, this novel peptide confers protection against lethal challenge of A/Puerto Rico/8/34 (H1N1). Taken together, our results suggest the combined immunization of reassortant inactivated H3N2 vaccine and the novel M2e-MAP provided cross-protection against swine and human viruses and may serve as a promising approach for influenza vaccine development.