System Vaccinology for the Evaluation of Influenza Vaccine Safety by Multiplex Gene Detection of Novel Biomarkers in a Preclinical Study and Batch Release Test

Vaccines are beneficial and universal tools to prevent infectious disease. Thus, safety of vaccines is strictly evaluated in the preclinical phase of trials and every vaccine batch must be tested by the National Control Laboratories according to the guidelines published by each country. Despite many vaccine production platforms and methods, animal testing for safety evaluation is unchanged thus far. We recently developed a systems biological approach to vaccine safety evaluation where identification of specific biomarkers in a rat pre-clinical study evaluated the safety of vaccines for pandemic H5N1 influenza including Irf7, Lgals9, Lgalsbp3, Cxcl11, Timp1, Tap2, Psmb9, Psme1, Tapbp, C2, Csf1, Mx2, Zbp1, Ifrd1, Trafd1, Cxcl9, β2m, Npc1, Ngfr and Ifi47. The current study evaluated whether these 20 biomarkers could evaluate the safety, batch-to-batch and manufacturer-to-manufacturer consistency of seasonal trivalent influenza vaccine using a multiplex gene detection system. When we evaluated the influenza HA vaccine (HAv) from four different manufactures, the biomarker analysis correlated to findings from conventional animal use tests, such as abnormal toxicity test. In addition, sensitivity of toxicity detection and differences in HAvs were higher and more accurate than with conventional methods. Despite a slight decrease in body weight caused by HAv from manufacturer B that was not statistically significant, our results suggest that HAv from manufacturer B is significantly different than the other HAvs tested with regard to Lgals3bp, Tapbp, Lgals9, Irf7 and C2 gene expression in rat lungs. Using the biomarkers confirmed in this study, we predicted batch-to-batch consistency and safety of influenza vaccines within 2 days compared with the conventional safety test, which takes longer. These biomarkers will facilitate the future development of new influenza vaccines and provide an opportunity to develop in vitro methods of evaluating batch-to-batch consistency and vaccine safety as an alternative to animal testing.     

The Target Animal Safety Test—Is it Still Relevant?

In Europe, the target animal safety test (TAST) is stipulated by 52 European Pharmacopoeia monographs, by three European Union (EU) Directives and a number of EU guidelines as a routine test for veterinary immunologicals, to be carried out on the finished product. TAST data from seven European Official Member States Control Laboratories (OMCLs) and 14 manufacturers were retrospectively analysed. During 1994–1997, 11 185 vaccine batches had been submitted for batch release, and the OMCLs had tested 670 batches in the TAST (665 passed, 4 passed after retesting, 1 failed). In total, 82 of these batches were not released; however, in only one case this was due to failure in the TAST. The data received from the 14 manufacturers covered the years from 1997 to 1999. 11 386 batches were tested in the TAST, of which 215 passed after retesting and 7 failed. Although only 30% of the OMCLs provided data and the data of the manufacturers are not complete they clearly indicate that the TAST does not contribute to the safety of veterinary vaccines and should therefore not be required as a routine batch test. In cases, where it appears to be necessary, detailed guidance on the test design and evaluation should be given. Copyright 2002 Published by Elsevier Science Ltd on behalf of The International Association for Biologicals.     

Reduction of numbers of animals used in the quality control of biologicals

Laboratory animals are used for the quality control of vaccines. In particular, the potency testing of batches of inactivated vaccine requires large numbers of animals. The possibilities for reduction have been evaluated, and the results are summarised in this paper. Several approaches were studied, including the retrospective analysis of test data, with the objectives of determining the minimum number of animals required per vaccine dilution group, and evaluating the feasibility of a single-dose potency test. Other studies focused on the development of serology-based models and the use of genetically uniform animals. Based on the outcome of these studies, a substantial reduction in the number of animals used for the potency testing of toxoid vaccines has been achieved or will be achieved in the near future. As reduction alternatives can generally be explored in a relatively simpler and less time-consuming way than replacement alternatives, more emphasis should be placed on reduction strategies than at present.     

Rapid quality control of a live attenuated Peste des petits ruminants (PPR) vaccine by monoclonal antibody based sandwich ELISA.

Peste des petits ruminants (PPR) is a highly contagious and economically important viral disease of goats and sheep. A homologous Vero cell-based attenuated PPR vaccine developed in our laboratory and used extensively throughout the country, is available for control of PPR. The presently used quality control test, titration in Vero cells for PPR virus titre in vaccine batches, takes at least 6-8days to determine the quality and dose of vaccine. In this study, 74 freeze-dried PPR vaccine batches were tested simultaneously by both virus titration and PPR sandwich ELISA (S-ELISA) to correlate the titre of the vaccine virus with reactivity in S-ELISA. It was found that the vaccine batches with titre more than 10(3)TCID(50)/ml gave positive results in S-ELISA and correlated well with the virus titre of the freeze-dried vaccines. The correlation coefficient between the virus titration and S-ELISA reactivity was estimated as 0.96, indicating a high correlation between the two parameters based on 74 batches of freeze-dried PPR vaccine. The vaccine batches with titres of 3.0, 4.3, 4.5, 5.0, 6.5 and 7.0 had shown a positive reaction when tested in two-fold dilutions in S-ELISA at 1, 5, 6, 7, 8 and 9log2 titres, respectively. The test vaccine batches were found to be negative in S-ELISA when the titre of the vaccine was less than 10(3)TCID50/ml, suggesting that the vaccine could not be passed for field use. It is concluded that S-ELISA could be a preliminary tool useful for the quality control of PPR vaccine as it is rapid and easy to perform when compared to virus titration.     

流行性感冒裂解疫苗免疫原性试验及电镜观察

实验报告了以三硝基甲苯X-100(Triton X-100)为裂解剂对流行性感冒病毒裂解效果的电镜观察结果及裂解疫苗的免疫原性。通过对病毒纯化、裂解、再纯化制备的3批流行性感冒裂解疫苗样品的电镜观察,表明使用此裂解剂能使疫苗中的病毒裂解完全,无完整病毒颗粒,裂解效果好。安全试验和免疫试验结果表明疫苗的安全性好,免疫效果好。     

流感病毒裂解疫苗裂解剂去除工艺改进及稳定性研究

目的改进流感病毒裂解疫苗裂解剂去除工艺,降低残余卵清蛋白和裂解剂含量,提高疫苗质量,降低成本。方法分别将A1、A3和B型流感病毒纯化液用磷酸缓冲液（PB）沉淀法去除裂解剂,经超滤、除菌制备原液,配制6批半成品,其中3批不含硫柳汞,3批含硫柳汞,经全面检定,并观察放置37℃、25℃和2～8℃不同时间的稳定性。结果该疫苗各项指标均符合《中国药典》（2010年版）三部要求,其中卵清蛋白平均为3．83ng／mL,裂解剂平均为57μg／mL,比改进前分别降低97．9％和69％。37℃放置4周、25℃3个月及2-8℃12个月后检定全部合格。结论该工艺步骤简单,去除卵清蛋白和裂解剂效果明显,是进一步提高疫苗质量和降低成本的有效工艺。     

伤寒Vi多糖菌苗多糖含量及分子大小测定试验

伤寒Ｖｉ多糖菌苗是我国新近研制成功的一种多糖菌苗。为了严格控制该制品的质量,经反复试验,建立了多糖含量和分子大小的测定方法。本文报导了（１）用火箭电泳法测定伤寒Ｖｉ多糖菌苗多糖含量。经对不同实验条件进行比较,选择出较为理想的条件。用该法测定８批样品。结果均符合规程要求。对其中５批样品进行６次重复试验表明,该法的重复性好,操作简单,是测定多糖含量较为理想的方法。（２）用琼脂糖柱层析法对２８批伤寒Ｖｉ多糖菌苗的分子大小进行测定。对用该法所得柱层析收集液分别用Ｈｅｓｔｒｉｎ法和２０６ｎｍ扫描法测定其多糖回收率,对测定结果进行比较。结果表明,两种方法的测定结果无显著性差异（Ｐ＞０．０１）,而且重复性均好。可根据实验室条件选择测定方法。     

Risks and Benefits of Early Antithrombotic Therapy after Thrombolytic Treatment in Patients with Acute Stroke

Background

Current guidelines recommend withholding antithrombotic therapy (ATT) for at least 24 h in patients with acute ischemic stroke treated with thrombolytic therapy. Herein, we report a retrospective analysis of a single-centre experience on the safety and efficacy of antithrombotic therapy (ATT) started before or after 24 h of intravenous thrombolysis in a cohort of acute ischemic stroke patients.

Methods

A total of 139 patients (Rapid ATT group) received antithrombotic therapy before 24 h of thrombolysis, and 33 patients (Standard ATT group) after 24 h. The brain parenchyma and vessel status were assessed using simple CT scan on admission, multimodal CT scan at the end of thrombolysis, and angio-CT/MRI scan at day 3. Functional outcome was scored using the modified Rankin Scale (mRS) at day 90.

Results

The two ATT groups had similar demographics, stroke subtypes, baseline NIHSS, thrombolytic strategies, vessel-patency rates at the end of thrombolysis, and incidence of bleeding complications at follow up. At day 3, the Rapid ATT group had a non-significant improved vessel-patency rate than the Standard ATT group. At day 90, a greater proportion of patients in the rapid ATT group had shifted down the mRS, and had improved in the NIHSS score.

Conclusions

ATT initiated before 24 h of intravenous thrombolytic therapy in acute stroke patients disclosed no safety concerns compared with a conventional antithrombotic therapy delay of 24 h and showed better functional outcome at follow up. The value of early initiation of ATT after thrombolysis deserves further assessment in randomized controlled trials.     

Development and validation of a detection system for wild-type Vibrio cholerae in genetically modified cholera vaccine.

Orochol, a live oral cholera vaccine licensed in Switzerland and in other countries, is based on the genetically modified Vibrio cholerae strain CVD103-HgR. This strain is derived from the wild-type O1 strain Inaba 569B by deletion of a fragment internal to the ctxA gene encoding the A1 subunit of cholera toxin and by replacement of an internal fragment of the hlyA gene with a fragment carrying the mer operon mediating mercury resistance. In this study we describe a polymerase chain reaction (PCR) system for the detection of wild-type Vibrio cholerae and the identification of the vaccine strain for the quality control of production batches. A multiplex PCR system that targets the intact ctxA gene of the wild-type strain and simultaneously the integration site of the mer operon in the hlyA gene (hlyA::mer) of the vaccine strain CVD103-HgR was developed. To evaluate the detection limit of the system, vaccine suspensions were artificially contaminated with wild-type V. cholerae 569B cells and tested by PCR. The detection limit of the system was statistically evaluated and found to be at 11625 wild-type cells per vaccine sachet (95% confidence limit). This number is below the infective dose of wild-type Vibrio cholerae. In Switzerland this test is used in combination with other tests in the official batch-release procedure to assure the safety of each batch of the cholera vaccine Orochol.     

A need for careful evaluation of endotoxin contents in acellular pertussis-based combination vaccines

Two batches each of diphtheria-tetanus-acellular pertussis vaccine (DTaP) and that combined with inactivated polio vaccine purchased from foreign markets were tested by mouse body weight decreasing (BWD) toxicity test and Limulus amaebocyte lysate (LAL) test. Three out of the four imported vaccine batches showed the levels of BWD toxicity even comparable to that of DT-whole cell pertussis vaccine. BWD toxicity test is based on endotoxin dose-dependent weight loss of mice and has been used for controlling endotoxin in DTaP. Although of the strong BWD toxicity of the imported vaccines, there was no marked difference in LAL test results between the imported vaccines and Japanese DTaP. However, one imported DTaP batch showed very strong interference with LAL activity of spiked lipopolysaccharide (LPS). The batch interfered not only with LAL activity but also with pyrogenicity and prostaglandin E₂ induction activity. However, the pyrogenicity of the spiked LPS could be recovered from the precipitated fraction of the batch by treating with phosphate buffer to suggest the possibility of recovering in vivo toxicity. As an adequate in vitro test method could not be identified for controlling the safety of the interfering batch, an appropriate in vivo test would be required for testing such vaccines.     

Neurovirulence of mumps virus: intraspinal inoculation test in marmosets.

An intraspinal inoculation test of mumps virus using marmosets was performed in order to develop a neurovirulence test of mumps vaccines. In the group inoculated with non-neurovirulent Jeryl Lynn vaccine strain at 10(2.0) pfu/dose, there were only minimal histopathological changes in 3 of the 5 marmosets. In contrast, all marmosets inoculated with neurovirulent Urabe and NK-M46 vaccine strains developed extensive encephalitis and meningitis. Thus, this marmoset model, which can distinguish between non-neurovirulent and neurovirulent vaccine strains, is useful for evaluating neurovirulence of vaccine strains and elucidating the molecular pathogenesis of mumps.     

HPV16/18假病毒中和抗体检测方法的验证

目的应用假病毒中和法建立检测血清HPV16/18中和抗体滴度检测方法并进行验证。方法分别采用不同批次假病毒以及不同代次细胞对不同滴度的HPV16/18阳性血清进行多次平行检测,考察这些因素对检验结果的影响;同时通过对抗HPV16/18双价阳性血清、抗HPV16单价阳性血清和抗HPV18单价阳性血清的检测进一步评估中和抗体检测法的准确性、特异性及重复性。结果不同批次假病毒和不同代次细胞对检验结果的影响均在4倍范围内,此外该检测法的准确性、特异性、重复性均在可接受标准范围之内。结论建立的假病毒法可满足中和抗体效价检测的要求,可用于评价疫苗的免疫效果。     

Effect of the contents and form of rabies glycoprotein on the potency of rabies vaccination in cattle

One of the methods used for controlling cattle rabies in Brazil consists of vaccination. Sometimes, however, rabies occurs in cattle supposedly protected. Since rabies vaccine batches are officially controlled by tests performed on laboratory animals, it is questionable whether the minimal mandatory requirements really correspond to immunogenicity in the target species. We have analyzed the association among potencies of rabies vaccines tested by the NIH test, the contents and form (free-soluble or virus-attached) of rabies glycoprotein (G) in the vaccine batches, and the virus-neutralizing antibodies (VNA) titers elicited in cattle. No correlation was found between G contents in the vaccine batches and the NIH values, whatever the presentation of G. There was no correlation either between NIH values and VNA titers elicited in cattle. There was, however, a positive correlation (r = 0.8681; p = 0.0001) between the amounts of virion-attached G present in the vaccine batches and VNA elicited in cattle. This was not observed when the same analysis was performed with total-glycoprotein or free-soluble glycoprotein. The study demonstrated that NIH values can not predict the effect of the immunogen in cattle. On the other hand, the quantification of virus-attached rabies glycoprotein has a strong correlation with VNA elicited in cattle.     

A test for the relative potency of herpes simplex virus vaccines based upon the female guinea-pig model of HSV 2 genital infection

An ELISA for total herpes simplex virus (HSV) 1 antigen content and a test of immunogenicity based upon the female guinea-pig model of HSV 2 genital infection were applied to two samples from batches of HSV 1 subunit ('Skinner') vaccine. The ELISA was reproducible within an approximately threefold limit of error and batches 1 and 2 were indistinguishable in antigen content. The effects of vaccination in the guinea-pig model were assessed by a statistical analysis of scores derived from the principal clinical signs, vaginal oedema and lesions on the external genitalia. The statistical power of the guinea-pig assay was such that reductions in the severity of illness approaching 40% would be significant (P less than 0.05) on 90% of occasions. The ability to make quantitative estimates of immunogenicity will prove useful in the quality control of HSV vaccine batches which are destined for clinical trials in man.     

Experimental screening of BCG preparations produced for cancer immunotherapy: Safety and immunostimulating and antitumor activity of four consecutively produced batches

Summary Four consecutively produced batches of Bacillus Calmette-Guérin (BCG) especially intended to be used for cancer immunotherapy were investigated for consistency of the vaccine. Each batch was investigated directly after production of the vaccine, so that the four batches were not tested simultaneously. The activity of the four batches was investigated in general safety assays, immunostimulation assays, and two different tumor models.General safety assays showed dose-dependent growth retardation and increased serum glutamic pyruvic transaminase activity in mice, and a long-lasting temperature rise in rabbits after IV administration of the BCG preparations. In a skin reactivity assay, reactions were found acceptable for all preparations when compared with a reference batch.The results of the immunostimulation and antitumor studies can be summarized as follows. All four batches induced a specific delayed-type hypersensitivity reaction to PPD, indicating the induction of cell-mediated immunity. A stimulating effect on lymphoreticular organs was concluded from increased spleen weight and enhanced cell proliferation in draining lymph nodes. Enhanced macrophage function (phagocytosis and killing of bacteria) was demonstrated by an increased resistance to Listeria monocytogenes. YAC lymphoma target cells were killed nonspecifically by BCG-activated peritoneal exudate cells (PEC), indicating the induction of natural killing activity by BCG.Intralesional injection of BCG induced tumor regression in the guinea pig line 10 hepatocellular carcinoma, followed by immunity to the line 10 tumor. In the murine 5D04 squamous cell carcinoma, BCG had no effect on the primary tumor. However, IV-injected BCG resulted in a decreased number of lung metastases.In general, the four consecutively produced batches showed similar safety and activity in the immunostimulation assays and antitumor activity. Since only minor differences between the batches were found, which can also be attributed to the variation in experimental conditions common to biological assays, it is concluded that the vaccine batches produced show an acceptable consistency.Abbreviations used BCG, Bacillus Calmette-Guérin; C. parvum, Corynebacterium parvum; c. p., culturable particles; IU, international unit; PPD, purified protein derivative; PEC, peritoneal exudate cells     

Summary of the diverse situation of similar biotherapeutic products in the selected countries (August 2010)

The WHO guidelines on evaluating similar biotherapeutic products (SBPs) were adopted by the Expert Committee on Biological Standardization in 2009. The fundamental messages of the guidelines are that a) generic approach is not suitable for licensing SBPs, b) only products that have been subjected to a comparability exercise and show similarity to the reference biotherapeutic product (RBP) in terms of their quality, safety and efficacy are defined as SBPs, and c) the products that are not shown to be similar to the originator products as indicated in the guidelines should neither be described as "similar" nor called SBPs. In view of these, the products which have not been subjected to a head to head comparison with the RBP should be referred to as another term, e.g. 'non-innovator' therapeutic products. In order to review the current situation in each country, a survey was planned in line with the implementation workshop of the guidelines in August 2010. The results show that the diversity of regulatory framework for licensing SBPs and the ambiguous use of the terms, 'similar' or 'generic', present considerable challenges for the future use of SBPs.     

Overview of marker vaccine and differential diagnostic test technology.

Recent advances in molecular biology, immunology, microbiology, genetics and microbial pathogenesis have lead to the development of a wide variety of new approaches for developing safer and more effective vaccines based on designs such as subunit vaccines, gene deleted vaccines, live vectored vaccines, and DNA mediated vaccines. Technology tools can be as basic as identifying naturally occurring strains with deletions that support differentiating infected from vaccinated animal (DIVA) needs or be based on higher technology developments such as improved protein expression and purification methods, transgenic plant- and plant virus-based antigen production, and novel adjuvants that target specific immune responses. These new approaches, when applied to the development of marker vaccines and companion diagnostic test kits hold tremendous potential for developing improved tools for eradication and control programs. Marker vaccines and companion diagnostic test kits must meet the established licensing requirements for purity, potency, safety and efficacy. Efficacy claims are based on evaluation of the level of protection demonstrated in host animal trials and may range from "prevents infection with (a specific agent)", to "for use as an aid in the reduction of disease due to (a specific agent)." The differences in claims and recommendations are a function of the variation in protection elicited by various vaccines. For designing effective eradication programs, vaccine efficacy characteristics such as for reducing susceptibility to infections and spread of infections must be well defined; similarly, diagnostic test performance characteristics (efficacy) must be determined. In addition to data to support efficacy claims, it is imperative that safety of production and use of vaccines be evaluated. During the design of marker vaccines and diagnostic tests, it is important to consider the application of appropriate technologies to improve the safety of these products. Use of recombinant technologies for production of vaccines and/or diagnostic test antigens can reduce the biosafety concerns during production and during use, including human exposure to zoonotic pathogens during production and use, and potential spread of foreign animal disease agents due to loss of biocontainment. In addition, vaccines may induce adverse reactions. It is important to determine the frequency of adverse events and to reduce the likelihood of induction of adverse reactions through proper design.     

The use of the Toxin Binding Inhibition (ToBI) test for the estimation of the potency of the diphtheria component of vaccines

The Toxin Binding Inhibition (ToBI) test, previously developed for the estimation of diphtheria and tetanus antitoxin in human sera, was adapted for the estimation of the potency of diphtheria components in vaccines. Data are presented to show that antitoxin titres of individual sera of mice obtained by the ToBI test are in good agreement with those obtained in the Vero cell test. In addition, diphtheria potency and 95% confidence interval of twelve batches of vaccine in different compositions were estimated by the ToBI test and the results were compared with those obtained in Vero cells. A significant correlation could be demonstrated. It is concluded from this study that the ToBI test is a valuable model in the potency assay of diphtheria toxoids, based on antitoxin induction in mice.     

火箭电泳法测定伤寒Vi多糖菌苗多糖含量

伤寒沙门氏菌Ｖｉ抗原系ａ─１,４─Ｎ─乙酰─半乳糖醛酸高度聚合体,其Ｃ２和Ｃ３位分别被Ｎ─乙酰基和０─乙酰基取代,它不能用分光光度法测定其含量,实验表明火箭电泳的火箭峰高与其样品中多糖含量的对数成直线相关关系,作者采用火箭电泳方法对全国六个生物制品研究所的１８批伤寒Ｖｉ多糖菌苗中Ｖｉ多糖含量进行了测定,结果表明多数制品符合规程要求,但部分制品多糖含量偏高,值得引起注意,各所三批制品差别不大,表明各所的生产工艺相对稳定,利用火箭电泳测定伤寒Ｖｉ多糖,操作简单,用时较短,所需样品极少,是目前较为合适的测定伤寒Ｖｉ多糖菌苗多糖含量的方法。     

DNA shuffling and screening strategies for improving vaccine efficacy

The efficacy of vaccines can be improved by increasing their immunogenicity, broadening their crossprotective range, as well as by developing immunomodulators that can be coadministered with the vaccine antigen. One technology that can be applied to each of these aspects of vaccine development is MolecularBreeding directed molecular evolution. Essentially, this technology is used to evolve genes in vitro through an iterative process consisting of recombinant generation followed by selection of the desired recombinants. We have used DNA shuffling and screening strategies to develop and improve vaccine candidates against several infectious pathogens including Plasmodium falciparum (a common cause of severe and fatal human malaria), dengue virus, encephalitic alphaviruses such as Venezuelan, western and eastern equine encephalitis viruses (VEEV, WEEV, and EEEV, respectively), human immunodeficiency virus-1 (HIV-1), and hepatitis B virus (HBV). By recombining antigen-encoding genes from different serovar isolates, new chimeras are selected for crossreactivity; these vaccine candidates are expected to provide broader crossprotection than vaccines based on a single serovar. Furthermore, the vaccine candidates can be selected for improved immunogenicity, which would also improve their efficacy. In addition to vaccine candidates, we have applied the technology to evolve several immunomodulators that when coadministered with vaccines can improve vaccine efficacy by fine-tuning the T cell response. Thus, DNA shuffling and screening technology is a promising strategy to facilitate vaccine efficacy.