Regulations for the Manufacture and Control of Live Poliovirus Vaccine: International Experience and China’s Path

The eradication of poliomyelitis is a landmark achievement in the history of public health, providing strong protection for children’s health. The introduction of the Chinese Regulations for the Manufacture and Control of Live Poliovirus Vaccine is a prerequisite and safeguard for the large-scale production and use of domestically produced live poliovirus vaccines, serving as an indispensable component of vaccine safety. This article, based on archival documents, letters, collections of essays, and oral interviews, examines the historical experience of the development of Chinese Regulations for the Manufacture and Control of Live Poliovirus Vaccine. It contends that the emphasis on localization and the active engagement in international cooperation are critical factors in the swift introduction of Chinese Regulations for the Manufacture and Control of Live Poliovirus Vaccine.

OBSERVATION ON VACCINATING NEWCASTLE DISEASE VIRUS VACCINE WITH INHALATION AND PREVENTING RECURRENCE OF NASOPHARYNGEAL CANCER AFTER RADIOTHERAPY

Objective: To understand whether the Newcastle disease virus (NDV) vaccine can successfully vaccinate the rabbits and volunteers of cancer patients by inhalation and to observe the effects of NDV vaccine on nasopharyngeal carcinoma (NRC) patients after radiotherapy. Methods: The live NDV vaccine was vaccinated through nasal cavities of rabbits, NPC patients and other cancer patients who were treated by surgery or chemotherapy with larynx spray. The blood specimens of vein from the tested rabbits and volunteers of patients with cancer were collected before and after vaccination. The anti-NDV-antibody in serum was detected by conventional blood coagulation inhibiting method. The white blood cell (WBC) amount in blood samples was counted. In addition, the NPC patients after radiotherapy were divided into both test group and control group with random match. The both were followed-up by multiple kinds of way in order to understand effects of NDV immunotherapy for NPC. Results: The anti-NDV-antibody level of the rabbits and the patients with NPC rose significantly after vaccination. The WBC amount of cancer patients treated by surgery or chemotherapy also rose significantly after vaccination. The recurrence rate (3.23%) of NRC patients in test group who received immunotherapy of NDV vaccine for 4 to 10 treatment courses within 3 years after end of radiotherapy were significantly lower than that (25.81%) of the control group (P<0.025). Conclusion: The NDV vaccine La Sota strain can vaccinate the rabbits and the cancer patients in success by inhalation. And it has remarkable effect to decrease 3 year recurrence rate of NRC patients after radiotherapy.

Construction of Live Attenuated Vaccine against Vib- rio alginolyticus and the Evaluation on Its Immuno- protection Effect

Vibrio alginolyticus, a gram-negative bacterium has been described to be one of the most common and economically important aquatic pathogens of fish and shellfish. Vaccine immunization is an effective approach preventing V. alginolyticus infection. Attenuated vaccine stimulates systemic immune response in the host, but few attenuated vaccine against V. alginolyticus is available. The type III secretion system （T3SS）, an important pathogenic factor of V. alginolyticus, is used by bacterial pathogens to inject effector proteins into the cytoplasm of their host cells. The T3SS forms a structure called needle complex with a multi-ring base that spans the bacteria and a needle-like extension that protrudes several nanometers from the bacterial surface, vscO locates at the ＂needle＂ site of T3SS, playing the role of escorting the molecular chaperone and effector proteins into host cells and further inducing the death of host cells. In this paper, an in-frame deletion mu- tant of vscO was constructed using overlap PCR and homologous recombination technology combining with chloramphenicol （Cm） and sucrose screening. The LDs0 changes of ZJO3AvscO mutant strains compared with the strain ZJ03 were e^amined in grouper （ Epinephelus coioides）. The ZJO3AvscO mutant showed about 150 times decrease in virulence in E. coioides compared with wide type ZJ03. After vaccination with ZJO3AvscO in E. coioides through injection and immersion, the spe- cific antibody titers were markedly higher than that in the saline control group （P 〈 0.05 ）. The titers of injection and immersion group on the forth week reached the maximums at 1：2 048 and 1： 128, respectively. The relative percentage survival （RPS） of injection group was 84%, while that in immersion group was 68%. These results indicate that the ZJO3AvscO of V. alginolyticus has a high immunogenicity, and can be used as live attenuated vaccine. In addition, RPS may be affected by vaccination and infection methods. This study can provide technical support for controlling fish diseases caused by V. alginolyticus.

Titration Method Validation of Live Vaccines against Infectious Bursal Disease

Validating a method of analysis goes through different steps, which aims at testing the normality of measurements distribution, estimating the uncertainty of the components of a measurement （i.e., accuracy and correctness）, and finally, define the control tests of non degradation of the method performances. This paper outlines the steps for validating a biological method of analysis. It involves the construction of an experimental design, a statistical model, and the preparation of an interne laboratory reference material （pilot vaccine）. The latter is used to study the impact of deviation and variation factors, in order to, optimize the analytical method, to evaluate the bias （random error）, and to calculate the uncertainty of measurement, and make the control charts. This method is applied in the titration of live viral vaccines of Gumboro disease on chicken＇s embryos fibroblasts. The experimental results show that potential influence factors related to the titration method had no significant influence on the obtained results. Taking into account these results, an operating mode has been elaborated. The finalized method proved to be faithful to standard deviation of repeatability and reproducibility of 0.21 and 0.22, respectively, with a confidence level of 95%. The calculated uncertainty of measurement is equal to 0.2, which represents the average error level of a titer. A homogeneous stock of interne laboratory reference vaccine （MRIL）, with an average titer of 5.9 log DIT 50, was produced and the control chart set in away to provide the laboratory with an important tool of control and monitoring of the viral titers evolution in time, as well as, the mastery of the validated titration method performances.

Live and Inactivated Salmonella Enteritidis Vaccines:Immune Mechanisms in Broiler Breeders

Salmonella is a ubiquitous pathogen which, in addition to causing poultry diseases, has a growing zoonotic impact. It has demanded the implementation of diverse control strategies, in which vaccines play a major role. The understanding of the immune pathways elicited by the different vaccines is important, contributing for the establishment of strong immune correlates of protection, for instance. With the purpose of determining the dynamics of the humoral and cellular immune responses to vaccination, broiler breeders (Cobb Slow) were immunized with live or inactivated vaccines against Salmonella Enteritidis. Lymphocyte and macrophage subsets were analyzed in the peripheral blood by flow cytometry and antigen-specific circulating IgY and mucosal IgA were quantified. The markers analyzed by flow cytometry were CD8/CD28, CD4/TCRVβ1, Kul/ MHC II and Bu-1. Both live and inactivated vaccines induced an increase in the proportion of circulating monocytes (Kul+MHCII+) in some time points compared to non-vaccinated controls. However, whereas the live vaccine leads to an increase in CD8-CD28+ and Bu-1+ lymphocytescompared to the control group, the inactivated vaccine prompteda reduction in the percentage of severalleucocyte subsets (Kul-MHCII+, Bu-1+, CD8+CD28+, CD8-CD28+, CD4+TCRVβ1-, CD4+TCRVβ1+, CD4-TCRVβ1+) after the boost dose. Both vaccines induced specific serum IgY and mucosal IgA production;however, the inactivated vaccine stimulated higher titers in a shorter period. These results contribute to the understanding of mechanisms of action of live and inactivated Salmonella vaccines in chickens.

Genomic similarity and antibody-dependent enhancement of immune serum potentially affect the protective efficacy of commercial MLV vaccines against NADC30-like PRRSV

Porcine reproductive and respiratory syndrome(PRRS)is one of the most significant diseases affecting the pig industry worldwide.The PRRSV mutation rate is the highest among the RNA viruses.To date,NADC30-like PRRSV and highly pathogenic PRRSV(HP-PRRSV)are the dominant epidemic strains in China;however,commercial vaccines do not always provide sufficient cross-protection,and the reasons for insufficient protection are unclear.This study isolated a wild-type NADC30-like PRRSV,SX-YL1806,from Shaanxi Province.Vaccination challenge experiments in piglets showed that commercial modified live virus(MLV)vaccines provided good protection against HP-PRRSV.However,it could not provide sufficient protection against the novel strain SXYL1806.To explore the reasons for this phenomenon,we compared the genomic homology between the MLV strain and HP-PRRSV or NADC30-like PRRSV and found that the MLV strain had a lower genome similarity with NADC30-like PRRSV.Serum neutralization assay showed that MLV-immune serum slightly promoted the homologous HP-PRRSV replication and significantly promoted the heterologous NADC30-like PRRSV strain replication in vitro,suggesting that antibody-dependent enhancement(ADE)might also play a role in decreasing MLV protective efficacy.These findings expand our understanding of the potential factors affecting the protective effect of PRRSV MLV vaccines against the NADC30-like strains.

Analysis for Pathogenicity and Development of Transgenic Eimeria tenella Expressing both Yellow Fluorescent Protein and TgDHFR-TS Genes

In order to determine the effect of foreign genes on a transgenic parasite,the pathogenicity and development in chickens of transgenic E.tenella strain TE1,which expresses yellow fluorescent protein (YFP) and dihydrofolate reductase thymidylate synthase derived from Toxoplasma gondii(TgDHFR-TS), were compared with that of the parental strain BJ.Results indicated that the fecundity of the transgenic parasite(TE1) was reduced at least 4 times relative to that of the BJ strain.Low dosage of the TE1 strain induced less pathogenesis in chickens than did the BJ strain,but chickens inoculated with a higher dosage of TE1 oocysts displayed severe pathogenicity and mortality as did the BJ strain.In addition,trophozoites, the first generation and the second generation meronts and merozoites,microgamonts and macrogamonts of the transgenic parasite TE1 were seen by fluorescence microscopy.More interestingly,not all four sporonts in the sporulating transgenic oocysts express YFP.These findings suggest that the expression of YFP and TgDHFR-TS genes to some extent reduced pathogenicity and reproductive potential of transgenic E.tenella.Recombination between homologous or non-homologous chromosomes occurred during zygotic meiosis in E.tenella strain TE1.

HUMAN PAPILLOMAVIRUS TYPE 16 L1 PROTEIN CAN BE EXPRESSED IN LIVE ATTENUATED SHIGELLA FLEXNERI 5A STRAIN SH42

Objective Attenuated strains of Shigella are attractive live vaccine candidates for eliciting mucosal immune responses which is a suitable carrier for the prophylactic human papillomaviruses (HPV) vaccine development, To examine the potential of a live Shigella based prophylactic HPV vaccine, HPV16L1should be expressed in attenuated shigella strain. Methods A Shigella large invasive plasmid (icsA/virG) based prokaryotic expression plasmid pHS3199 was constructed. HPV16L1 gene was inserted into plasmid pHS3199 to form pHS3199-HPV16 L1 construct, and pHS3199-hpv16L1 was electroporated into a live attenuated shigella strain sh42. The expression of HPV16L1 protein was demonstrated by Western blotting with monoclonal antibody to HPV16L1, The genetic stability of recombinant strain sh42-HPV16 L1 was monitored by consecutive passage culture. Invasive ability of sh42-HPV16L1 was evaluated by Hela cell infection assay. Results HPV16 L1 protein can be expressed in recombinant strain sh42-HPV16 L1, and the protein stably expressed over 140 generations. The invasive ability of sh42-HPV16L1 was diminished dramatically compared to its parent strain, but not abolished completely. Conclusion HPV16L1 protein was constitutively expressed in the attenuated strain of shigella flexneri sh42, and maintained partial invasive ability. Our strategy may represent a promising vaccine candidate against genital HPV16 infection.

Intranasal administration of a single dose of a candidate live attenuated vaccine derived from an NSP16-deficient SARS-CoV-2 strain confers sterilizing immunity in animals

Live attenuated vaccines might elicit mucosal and sterilizing immunity against SARS-CoV-2 that the existing mRNA,adenoviral vector and inactivated vaccines fail to induce.Here,we describe a candidate live attenuated vaccine strain of SARS-CoV-2 in which the NSP16 gene,which encodes 2′-O-methyltransferase,is catalytically disrupted by a point mutation.This virus,designated d16,was severely attenuated in hamsters and transgenic mice,causing only asymptomatic and nonpathogenic infection.A single dose of d16 administered intranasally resulted in sterilizing immunity in both the upper and lower respiratory tracts of hamsters,thus preventing viral spread in a contact-based transmission model.It also robustly stimulated humoral and cell-mediated immune responses,thus conferring full protection against lethal challenge with SARS-CoV-2 in a transgenic mouse model.The neutralizing antibodies elicited by d16 effectively cross-reacted with several SARS-CoV-2 variants.Secretory immunoglobulin A was detected in the blood and nasal wash of vaccinated mice.Our work provides proof-of-principle evidence for harnessing NSP16-deficient SARS-CoV-2 for the development of live attenuated vaccines and paves the way for further preclinical studies of d16 as a prototypic vaccine strain,to which new features might be introduced to improve safety,transmissibility,immunogenicity and efficacy.

Evaluation of the Safety and Immune Efficacy of Recombinant Human Respiratory Syncytial Virus Strain Long Live Attenuated Vaccine Candidates

Human respiratory syncytial virus(RSV)infection is the leading cause of lower respiratory tract illness(LRTI),and no vaccine against LRTI has proven to be safe and effective in infants.Our study assessed attenuated recombinant RSVs as vaccine candidates to prevent RSV infection in mice.The constructed recombinant plasmids harbored(5′to 3′)a T7 promoter,hammerhead ribozyme,RSV Long strain antigenomic cDNA with cold-passaged(cp)mutations or cp combined with temperature-sensitive attenuated mutations from the A2 strain(A2cpts)or further combined with SH gene deletion(A2cptsΔSH),HDV ribozyme(δ),and a T7 terminator.These vectors were subsequently co-transfected with four helper plasmids encoding N,P,L,and M2-1 viral proteins into BHK/T7-9 cells,and the recovered viruses were then passaged in Vero cells.The rescued recombinant RSVs(rRSVs)were named rRSV-Long/A2cp,rRSV-Long/A2cpts,and rRSV-Long/A2cptsΔSH,respectively,and stably passaged in vitro,without reversion to wild type(wt)at sites containing introduced mutations or deletion.Although rRSV-Long/A2cpts and rRSV-Long/A2cptsΔSH displayed temperature-sensitive(ts)phenotype in vitro and in vivo,all rRSVs were significantly attenuated in vivo.Furthermore,BALB/c mice immunized with rRSVs produced Th1-biased immune response,resisted wtRSV infection,and were free from enhanced respiratory disease.We showed that the combination ofΔSH with attenuation(att)mutations of cpts contributed to improving att phenotype,efficacy,and gene stability of rRSV.By successfully introducing att mutations and SH gene deletion into the RSV Long parent and producing three rRSV strains,we have laid an important foundation for the development of RSV live attenuated vaccines.

Current efforts towards safe and effective live attenuated vaccines against African swine fever: challenges and prospects

Background: African swine fever (ASF) is a fatal hemorrhagic disease in domestic pigs and wild boar caused by African swine fever virus (ASFV). Since ASF has been introduced into Europe and Asia, the major pig-raising areas, posing a huge threat to the pork industry worldwide. Currently, prevention and control of ASF are basically dependent on strict biosecurity measures and stamping-out policy once ASF occurs.Main text: The major risks of ASF spread are insufficient biosecurity measures and human behaviors. Therefore, a safe and effective vaccine seems to be a reasonable demand for the prevention and control of ASF. Due to the efficacy advantage over other types of vaccines, live attenuated vaccines (LAVs), especially virulence-associated genes deleted vaccines, are likely to be put into emergency and conditional use in restricted areas if ASF is out of control in a country with a huge pig population and pork consumption, like China. However, the safety, efficacy, and genetic stability of current candidate ASF LAVs require comprehensive clinical evaluations prior to country-wide field application. Several critical issues need to be addressed to commercialize an ideal ASF LAV, including a stable cell line for manufacturing vaccines, differentiation of infected from vaccinated animals (DIVA), and cross-protection from different genotypes.Conclusion: A safe and effective DIVA vaccine and an accompanying diagnostic assay will facilitate the prevention, control, and eradication of ASF, which is quite challenging in the near future.

Safety and immunogenicity of lyophilized, live attenuated hepatitis A vaccine in non-human primate model

Objective To evaluate the safety and immunogenicity of lyophilized, live attenuated hepatitis A vaccine (H2 strain) in rhesus monkeys Methods Nine adult rhesus monkeys were used as experimental animals The rhesus monkeys without anti HAV were divided randomly into the aqueous vaccination group (4 rhesus monkeys), the lyophilized vaccination group (3 rhesus monkeys), and the control group (2 rhesus monkeys) Monkeys were inoculated by intramuscular injection, with control monkeys being inoculated with Minimum Essential Medium Eagle (MEM) Following vaccination, the monkeys were observed for the development of diarrhoea and other adverse side effects, such as changes in appetite, frequency of defaecation and stool consistency for seven days At the weeks 2, 3, 4, 6, 8, 10 and 12 positnoculation, the peripheral blood was collected from all animals and assayed for anti HAV and alanine aminotransferase (ALT) and aspartate aminotransferase (AST), at weeks 0, 4 and 8 postinocuation, needle biopsy specimens were taken at weeks 0, 4, 8 and 12, all monkeys were sacrificed and tissue samples were taken from liver, lung, heart, kidney and brain for pathological examination at week 12 Results Animals were immunized with a dose of 7 0 logTCID 50 /ml which is stable after freeze drying During the 12 week observation, no animals showed abnormal elevations of liver enzymes (ALT and AST) and no change in appetite or activity Two monkeys (one in the aqueous group and the other in lyophilized group) showed possible lesions at week 8 The lyophilized vaccine, in addition to eliciting an anti HAV IgG response similar to aqueous vaccine ( P >0 05), also showed IgM anti HAV response at week 2 which was not observed with aqueous vaccine Conclusions These results demonstrate that lyophilized, live hepatitis A vaccine is safe and highly immunogenic in primates, supporting its further evaluation in human clinical studies

Attenuation mechanism of Brucella melitensis M5-10,implications for vaccine development and differential diagnosis

Brucellosis is a worldwide zoonosis.Vaccination is the most efficient means to prevent and control brucellosis.The current licensed attenuated vaccines for animal use were developed by sequential passage in nonnatural hosts that decreased virulence in its original hosts.The attenuation mechanism of these strains remains largely unknown.In the present study,we sequenced the genome of Brucella melitensis vaccine strain M5-10.Sequence analysis showed that a large number of genetic changes occurred in the vaccine strains.A total of 2854 genetic polymorphic sites,including 2548 SNP,241 INDEL and 65 MNV were identified.Of the 2074 SNPs in coding regions,1310(63.2%)were non-synonymous SNPs.Gene number,percent and N/S ratios were disproportionally distributed among the cog categories.Genetic polymorphic sites were identified in genes of the virB operon,flagella synthesis,and virulence regulating systems.These data indicate that changes in some cog categories and virulence genes might result in the attenuation.These attenuation mechanisms also have implications for screening and development of new vaccine strains.The genetic changes in the genome represent candidate sites for differential diagnosis between these vaccine strains and other virulence ones.Transcription analysis of virulence genes showed that expression of dnaK,vjbR were reduced in M5-10 strain when compared with that in 16M.A duplex PCR targeting virB6 and dnaK was successfully used to differentiate between M5-10 and the virulent 16M strain.The genome re-sequencing technique represents a strong strategy not only for evaluation of vaccines,but also for development of new vaccines.

Reverse genetics systems for SARS-CoV-2:Development and applications

The recent emergence of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)caused serious harm to human health and struck a blow to global economic development.Research on SARS-CoV-2 has greatly benefited from the use of reverse genetics systems,which have been established to artificially manipulate the viral genome,generating recombinant and reporter infectious viruses or biosafety level 2(BSL-2)-adapted non-infectious replicons with desired modifications.These tools have been instrumental in studying the molecular biological characteristics of the virus,investigating antiviral therapeutics,and facilitating the development of attenuated vaccine candidates.Here,we review the construction strategies,development,and applications of reverse genetics systems for SARS-CoV-2,which may be applied to other CoVs as well.

A live attenuated RHΔompdcΔuprt mutant of Toxoplasma gondii induces strong protective immunity against toxoplasmosis in mice and cats

Background Toxoplasma gondii is an obligate intracellular apicomplexan parasite and is responsible for zoonotic toxoplasmosis.It is essential to develop an effective anti-T.gondii vaccine for the control of toxoplasmosis,and this study is to explore the immunoprotective effects of a live attenuated vaccine in mice and cats.Methods First,theompdc anduprt genes of T.gondii were deleted through the CRISPR-Cas9 system.Then,the intracellular proliferation and virulence of this mutant strain were evaluated.Subsequently,the immune responses induced by this mutant in mice and cats were detected,including antibody titers,cytokine levels,and subsets of T lymphocytes.Finally,the immunoprotective effects were evaluated by challenge with tachyzoites of different strains in mice or cysts of the ME49 strain in cats.Furthermore,to discover the effective immune element against toxoplasmosis,passive immunizations were carried out.GraphPad Prism software was used to conduct the log-rank(Mantel–Cox)test,Student’st test and one-way ANOVA.Results The RHΔompdcΔuprt were constructed by the CRISPR-Cas9 system.Compared with the wild-type strain,the mutant notably reduced proliferation(P<0.05).In addition,the mutant exhibited virulence attenuation in both murine(BALB/c and BALB/c-nu)and cat models.Notably,limited pathological changes were found in tissues from RHΔompdcΔuprt-injected mice.Furthermore,compared with nonimmunized group,high levels of IgG(IgG1 and IgG2a)antibodies and cytokines(IFN-γ,IL-4,IL-10,IL-2 and IL-12)in mice were detected by the mutant(P<0.05).Remarkably,all RHΔompdcΔuprt-vaccinated mice survived a lethal challenge with RHΔku80 and ME49 and WH6 strains.The immunized sera and splenocytes,especially CD8^(+)T cells,could significantly extend(P<0.05)the survival time of mice challenged with the RHΔku80 strain compared with naïve mice.In addition,compared with nonimmunized cats,cats immunized with the mutant produced high levels of antibodies and cytokines(P<0.05),and notably decreased the shedding numbers of oocysts in feces(95.3%).Conclusions The avirulent RHΔompdcΔuprt strain can provide strong anti-T.gondii immune responses,and is a promising candidate for developing a safe and effective live attenuated vaccine.

Immure response induced by oral DNA vaccination against FMDV delivered by attenuated Salm onella choleraesuis C500

A recombinant strain of Salmonella choleraesuis C500,containing a eukaryotic expression plasmid pBO1 with the immune-dominant epitope of foot-and-mouth disease virus,was constructed.Specific immune response to this recombinant strain was evaluated by oral administration of the recombinant live bacteria pBO1/S.cho in rabbits.Results showed that T cell response and specific antibody production were elicited.This approach may present a general strategy for eliciting immune responses with DNA vaccine delivered by live bacterial vectors.The stimulated indexes of T lymphoproliferation by specific antigens of FMDV in rabbits,can reach up to 11.0 and an antibody titer of 1/32 as detected in the erum with liquid block ELISA.