Evaluation of the neurovirulence test for mumps vaccines.

Neurovirulence tests in Macaca fascicularis using commercial preparations of different vaccine bulks and a wild-type strain revealed that the test was unable to distinguish mixed from pure populations or a suitable vaccine from a related strain which has been shown to be associated with clinical meningitis. However, the test was able to distinguish a wild-type strain from the vaccine strains successfully. The ability of the test to discriminate between acceptable and unacceptable seeds requires further examination.     

The thermal stability of yellow fever vaccines

The assessment of yellow fever vaccine thermostability both in lyophilized form and after reconstitution were analyzed. Two commercial yellow fever vaccines were assayed for their thermal stability. Vaccines were exposed to test temperatures in the range of 8 degrees C to 45 degrees C. Residual infectivity was measured by a plaque assay using Vero cells. The titre values were used in an accelerated degradation test that follows the Arrhenius equation and the minimum immunizing dose was assumed to be 10(3) particles forming unit (pfu)/dose. Some of the most relevant results include that (i) regular culture medium show the same degradation pattern of a reconstituted 17D-204 vaccine; (ii) reconstituted YF-17D-204 showed a predictable half life of more than six days if kept at 0 degrees C; (iii) there are differences in thermostability between different products that are probably due to both presence of stabilizers in the preparation and the modernization in the vaccine production; (iv) it is important to establish a proper correlation between the mouse infectivity test and the plaque assay since the last appears to be more simple, economical, and practical for small laboratories to assess the potency of the vaccine, and (v) the accelerated degradation test appears to be the best procedure to quantify the thermostability of biological products.     

Pre-clinical efficacy and safety of experimental vaccines based on non-replicating vaccinia vectors against yellow fever

Background

Currently existing yellow fever (YF) vaccines are based on the live attenuated yellow fever virus 17D strain (YFV-17D). Although, a good safety profile was historically attributed to the 17D vaccine, serious adverse events have been reported, making the development of a safer, more modern vaccine desirable.

Methodology/Principal Findings

A gene encoding the precursor of the membrane and envelope (prME) protein of the YFV-17D strain was inserted into the non-replicating modified vaccinia virus Ankara and into the D4R-defective vaccinia virus. Candidate vaccines based on the recombinant vaccinia viruses were assessed for immunogenicity and protection in a mouse model and compared to the commercial YFV-17D vaccine. The recombinant live vaccines induced γ-interferon-secreting CD4- and functionally active CD8-T cells, and conferred full protection against lethal challenge already after a single low immunization dose of 10⁵ TCID₅₀. Surprisingly, pre-existing immunity against wild-type vaccinia virus did not negatively influence protection. Unlike the classical 17D vaccine, the vaccinia virus-based vaccines did not cause mortality following intracerebral administration in mice, demonstrating better safety profiles.

Conclusions/Significance

The non-replicating recombinant YF candidate live vaccines induced a broad immune response after single dose administration, were effective even in the presence of a pre-existing immunity against vaccinia virus and demonstrated an excellent safety profile in mice.     

Experience in Canada with the new revised monkey neurovirulence test for oral poliovirus vaccine

Nine years of experience in our laboratory, using more than 1500 cynomolgus monkeys in 138 tests, has shown that the new neurovirulence test (NVT) adopted by the World Health Organization (WHO) for live, oral monovalent vaccine of each poliovirus type, was a reproducible and sensitive assay likely to ensure the safety of this vaccine in humans. Our findings were the following: (1) when the test vaccine and the appropriate homotypic reference vaccine were tested in a single group of monkeys, the concurrent use of the reference vaccine considerably increased the reproducibility of the NVT; (2) in the assessment of the degree of attenuation of each lot of vaccine, the use of 12 monkeys for types 1 and 2 vaccines and 20 monkeys for type 3 vaccine (inoculated intraspinally each for reference and test vaccine) was satisfactory; (3) the virus dose used per monkey (10(5.6) to 10(6.6) pfu per monkey) was found not to be critical, i.e. the lower virus dose yielded mean lesion scores in the central nervous system of monkeys at least as high or higher than the tenfold higher virus dose; (4) the statistical analysis of our data showed that the old intrathalamic (IT) assay was considerably less sensitive than the new intraspinal (IS) assay, i.e., a test vaccine with a twofold increase in monkey neurovirulence would have a 41% chance of failing in the IT test (using 30 monkeys per vaccine), while this chance increased to 99% in the WHO IS assay (using 12 or 20 monkeys per vaccine). Since the introduction of the WHO NVT in Canada, the laboratory findings in monkeys were confirmed by vaccine experience in humans; the number of vaccine-associated paralytic poliomyelitis cases in the population showed a further decline.     

In vitro potency assay for yellow fever vaccines: comparison of three vero cell lines sources.

Quality control of Yellow Fever vaccines performed by Control Authorities prior to marketing vaccines batches requires in vitro potency assays. The two currently available methods are the plaque formation assay and the cytopathic effect assay based on the use of porcine kidney PS cells or monkey kidney Vero cells. Among several sources of variation in virus titration, the cell systems are considered as important issues and Quality Assurance strongly recommends working with cell banks from certified suppliers. The aim of our study was to compare the behaviour and the sensitivity of three Vero cell sources obtained from ATCC, WHO and EP used at different passage levels in a plaque formation test. The conclusion of this work was that the yellow fever live attenuated virus titration, adapted in Vero cell lines appeared as a reliable method applicable for routine in vitro potency assay. The comparison of Vero cell lines, originated from three different sources, showed that they could be equally used as substrates by laboratories having the basic facility of cell culture, without influence on the final viral titre.     

口服脊髓灰质炎减毒活疫苗猴体神经毒力试验参考品合格标准的统计分析

验证口服脊髓灰质炎减毒活疫苗的猴体神经毒力试验参考品合格标准是否发生改变及其对疫苗猴体神经毒力试验检测结果的影响。按照WHO推荐的猴体神经毒力中枢神经系统病理学检定方法,统计分析了1996~2002年中猴体神经毒力试验三个型参考品各五批以上的实验数据。并对Ⅰ、Ⅱ、Ⅲ型参考品的病变好发部位腰、颈、脑的病变分数进行了比较。结果表明,Ⅰ、Ⅱ型参考品合格标准上、下限及C值有所下降;Ⅲ型参考品合格标准上、下限及C值略有升高。从病理学记分结果显示,三型参考品在腰、颈、脑部的致病力存在着差异;Ⅲ型参考品的致病变力向颈、脑部位扩散的指数明显增大。Ⅰ、Ⅱ型参考品合格标准对疫苗猴体神经毒力试验合格界线趋于严格,而Ⅲ型参考品猴体神经毒力病变指数由腰部向颈、脑部蔓延趋势的增大导致病变扩散指数和强度指数增大,从而使Ⅲ型参考品合格标准对疫苗的猴体神经毒力试验合格界线范围趋大,有可能对Ⅲ型疫苗制品合格率增大。     

Molecular basis for attenuation of neurovirulence of a yellow fever Virus/Japanese encephalitis virus chimera vaccine (ChimeriVax-JE)

A yellow fever virus (YFV)/Japanese encephalitis virus (JEV) chimera in which the structural proteins prM and E of YFV 17D are replaced with those of the JEV SA14-14-2 vaccine strain is under evaluation as a candidate vaccine against Japanese encephalitis. The chimera (YFV/JEV SA14-14-2, or ChimeriVax-JE) is less neurovirulent than is YFV 17D vaccine in mouse and nonhuman primate models (F. Guirakhoo et al., Virology 257:363-372, 1999; T. P. Monath et al., Vaccine 17:1869-1882, 1999). Attenuation depends on the presence of the JEV SA14-14-2 E protein, as shown by the high neurovirulence of an analogous YFV/JEV Nakayama chimera derived from the wild JEV Nakayama strain (T. J. Chambers, A. Nestorowicz, P. W. Mason, and C. M. Rice, J. Virol. 73:3095-3101, 1999). Ten amino acid differences exist between the E proteins of ChimeriVax-JE and the YFV/JEV Nakayama virus, four of which are predicted to be neurovirulence determinants based on various sequence comparisons. To identify residues that are involved in attenuation, a series of intratypic YFV/JEV chimeras containing either single or multiple amino acid substitutions were engineered and tested for mouse neurovirulence. Reversions in at least three distinct clusters were required to restore the neurovirulence typical of the YFV/JEV Nakayama virus. Different combinations of cluster-specific reversions could confer neurovirulence; however, residue 138 of the E protein (E(138)) exhibited a dominant effect. No single amino acid reversion produced a phenotype significantly different from that of the ChimeriVax-JE parent. Together with the known genetic stability of the virus during prolonged cell culture and mouse brain passage, these findings support the candidacy of this experimental vaccine as a novel live-attenuated viral vaccine against Japanese encephalitis.     

Insights into human CD8(+) T-cell memory using the yellow fever and smallpox vaccines

Live virus vaccines provide a unique opportunity to study human CD8(+) T-cell memory in the context of a controlled, primary acute viral infection. Yellow fever virus-17D and Dryvax are two such live-virus vaccines that are highly efficacious, used worldwide and provide long-term immunity against yellow fever and smallpox respectively. In this review, we describe the properties of virus-specific memory CD8(+) T cells generated in smallpox and yellow fever vaccinees. We address fundamental questions regarding magnitude, functional quality and longevity of the CD8(+) T-cell response, which are otherwise challenging to address in humans. These findings provide insights into the attributes of the human immune system as well as provide a benchmark for the optimal quality of a CD8(+) T-cell response that can be used to evaluate novel candidate vaccines.     

The effect of serial passage on Sabin oral poliomyelitis vaccine virus in secondary monkey kidney versus Vero cells as measured by the monkey neurovirulence test

The three Sabin strains of poliomyelitis seed virus were serially passaged in either secondary monkey kidney or Vero cell cultures and the tenth passage of each virus harvest compared to non-passaged Sabin reference virus of the same type using the monkey neurovirulence test. All three types were further attenuated by passage in Vero cells, whereas only type 2 became further attenuated after passage in secondary monkey kidney cells. After passage in Vero cells, type 3 poliomyelitis virus became more heat stable, as measured by its replicative capacity at 40°C.     

Safety testing for neurovirulence of novel live, attenuated flavivirus vaccines: infant mice provide an accurate surrogate for the test in monkeys.

Current requirements for control of live viral vaccines, including yellow fever 17D, produced from potentially neurotropic wild-type viruses include tests for neurovirulence in nonhuman primates. We have used yellow fever 17D virus as a live vector for novel flavivirus vaccines (designated ChimeriVax) against dengue, Japanese encephalitis (JE), and West Nile (WN) viruses. For control of these vaccines, it would be preferable to substitute a test in mice for the test in a higher species (monkeys). In this study, we compare the neurovirulence of ChimeriVax vaccine candidates in suckling mice inoculated by the intracerebral (IC) route with graded doses of the test article or yellow fever 17D vaccine as a reference control. Mortality ratio and survival distribution are the outcome measures. The monkey safety test is performed as described for control of yellow fever vaccines. In both mice and monkeys, all chimeric vaccines were significantly less neurovirulent than yellow fever 17D vaccine. The test in suckling mice discriminated between strains of two different vaccines (ChimeriVax-JE and ChimeriVax-DEN1) differing by a single amino acid change, and was more sensitive for detecting virulence differences than the test in monkeys. The results indicate that the suckling mouse test is simple to perform, highly sensitive and, with appropriate validation, could complement or possibly even replace the neurovirulence component of the monkey safety test. The test in infant mice is particularly useful as a means of demonstrating biological consistency across seed virus and vaccine lots.     

Interaction of yellow fever virus French neurotropic vaccine strain with monkey brain: characterization of monkey brain membrane receptor escape variants

Binding of yellow fever virus wild-type strains Asibi and French viscerotropic virus and vaccine strains 17D and FNV to monkey brain and monkey liver cell membrane receptor preparations (MRPs) was investigated. Only FNV bound to monkey brain MRPs, while French viscerotropic virus, Asibi, and FNV all bound to monkey liver MRPs. Four monkey brain and two mouse brain MRP escape (MRP(R)) variants of FNV were selected at pH 7.6 and 6.0. Three monkey brain MRP(R) variants selected at pH 7.6 each had only one amino acid substitution in the envelope (E) protein in domain II (E-237, E-260, or E274) and were significantly attenuated in mice following intracerebral inoculation. Two of the variants were tested in monkeys and retained parental neurotropism following intracerebral inoculation at the dose tested. We speculate that this region of domain II is involved in binding of FNV E protein to monkey brain and is, in part, responsible for the enhanced neurotropism of FNV for monkeys. A monkey brain MRP(R) variant selected at pH 6.0 and two mouse brain MRP(R) variants selected at pH 7.6 were less attenuated in mice, and each had an amino acid substitution in the transmembrane region of the E protein (E-457 or E-458).     

Immunogenicity and safety of primary fractional-dose yellow fever vaccine in autoimmune rheumatic diseases

BackgroundBrazil faced a yellow fever(YF) outbreak in 2016–2018 and vaccination was considered for autoimmune rheumatic disease patients(ARD) with low immunosuppression due to YF high mortality.ObjectiveThis study aimed to evaluate, prospectively for the first time, the short-term immunogenicity of the fractional YF vaccine(YFV) immunization in ARD patients with low immunossupression.Methods and ResultsA total of 318 participants(159 ARD and 159 age- and sex-matched healthy controls) were vaccinated with the fractional-dose(one fifth) of 17DD-YFV. All subjects were evaluated at entry(D0), D5, D10, and D30 post-vaccination for clinical/laboratory and disease activity parameters for ARD patients. Post-vaccination seroconversion rate(83.7%vs.96.6%, p = 0.0006) and geometric mean titers(GMT) of neutralizing antibodies[1143.7 (95%CI 1012.3–1292.2) vs.731 (95%CI 593.6–900.2), p<0.001] were significantly lower in ARD compared to controls. A lower positivity rate of viremia was also identified for ARD patients compared to controls at D5 (53%vs.70%, p = 0.005) and the levels persisted in D10 for patients and reduced for controls(51%vs.19%, p = 0.0001). The viremia was the only variable associated with seroconvertion. No serious adverse events were reported. ARD disease activity parameters remained stable at D30(p>0.05).ConclusionFractional-dose 17DD-YF vaccine in ARD patients resulted in a high rate of seroconversion rate(>80%) but lower than controls, with a longer but less intense viremia. This vaccine was immunogenic, safe and did not induce flares in ARD under low immunosuppression and may be indicated in YF outbreak situations and for patients who live or travel to endemic areas.Trial registrationThis clinical trial was registered with Clinicaltrials.gov (#{"type":"clinical-trial","attrs":{"text":"NCT03430388","term_id":"NCT03430388"}}NCT03430388).     

A mouse-based assay for the pre-clinical neurovirulence assessment of vaccinia virus-based smallpox vaccines

Post-vaccinal encephalitis, although relatively uncommon, is a known adverse event associated with many live, attenuated smallpox vaccines. Although smallpox vaccination ceased globally in 1980, vaccine manufacture has resumed in response to concerns over the possible use of smallpox virus as an agent of bioterrorism. To better support the production of safer smallpox vaccines, we previously reported the development of a mouse model in which a relatively attenuated vaccine strain (Dryvax^®) could be discerned from a more virulent laboratory strain (WR). Here we have further tested the performance of this assay by evaluating the neurovirulence of several vaccinia virus-based smallpox vaccines spanning a known range in neurovirulence for humans. Our data indicate that testing of 10–100 pfu of virus in mice following intracranial inoculation reliably assesses the virus's neurovirulence potential for humans.     

Recombinant yellow fever viruses are effective therapeutic vaccines for treatment of murine experimental solid tumors and pulmonary metastases

We have genetically engineered an attenuated yellow fever (YF) virus to carry and express foreign antigenic sequences and evaluated the potential of this type of recombinant virus to serve as a safe and effective tumor vaccine. Live-attenuated YF vaccine is one of the most effective viral vaccines available today. Important advantages include its ability to induce long-lasting immunity, its safety, its affordability, and its documented efficacy. In this study, recombinant live-attenuated (strain 17D) YF viruses were constructed to express a cytotoxic T-lymphocyte epitope derived from chicken ovalbumin (SIINFEKL). These recombinant viruses replicated comparably to the 17D vaccine strain in cell culture and stably expressed the ovalbumin antigen, and infected cells presented the antigen in the context of major histocompatibility complex class I. Inoculation of mice with recombinant YF virus elicited SIINFEKL-specific CD8(+) lymphocytes and induced protective immunity against challenge with lethal doses of malignant melanoma cells expressing ovalbumin. Furthermore, active immunotherapy with recombinant YF viruses induced regression of established solid tumors and pulmonary metastases. Thus, recombinant YF viruses are attractive viral vaccine vector candidates for the development of therapeutic anticancer vaccines.     

The clinical evaluation of diagnostic prostaglandin test

An oral prostaglandin test with PGE2 in a dose 0.5 mg was performed on 44 women in the last week of pregnancy. During 30 minutes of cardiotocographic recording, two types of contractile activity of the uterus were observed: regular and irregular. Women with regular uterine activity have statistically significantly higher serum oxytocinase level and also a shorter duration of labour.Thus, these two types of uterine activity may also be distinguished by enzyme as well as labour data.