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.

Evaluation of an attenuated vaccine candidate based on the genotype C of bovine parainfluenza virus type 3 in albino guinea pigs

Bovine parainfluenza virus type 3（BPIV3） is considered as one of the most important respiratory tract pathogens of both young and adult cattle, and widespread among cattle in the world. BPIV3 was first reported in China in 2008 and four strains of BPIV3 were isolated from Shandong Province, known as genotype C（BPIV3c）. Pathogen investigations had shown that BPIV3 c infection was very common among cattle in China. To date, BPIV3 can be classified into genotypes A, B and C based on genetic and phylogenetic analysis. Serological survey also demonstrates that BPIV3 infection is widespread in China, however, there is still no available vaccine for BPIV3 prevention in China nowadays. In the present study, the BPIV3 c strain SD0835 was continuously passaged on Madin-Darby bovine kidney（MDBK） cells for hundreds of times, and the pathogenicity of passage 209 was reduced in guinea pigs. The passage 209 of BPIV3 c strain SD0835 was used as a live vaccine candidate to immunize the guinea pigs. The vaccination results revealed that two vaccinations could induce excellent serum neutralizing antibody responses as well as proliferation of T lymphocytes. The vaccinated guinea pigs were well protected against challenge with a low passage of BPIV3 c strain SD0835. Additionally, the percentages of CD4~＋ and CD8~＋ T cell subsets of animals in vaccinated group increased after immunization; T cell subsets on day 2 after challenge in both groups decreased, and the decline of CD4~＋ and CD8~＋ T cell subsets levels of four guinea pigs in vaccinated group was relatively moderate, comparing with that of the control group. These data support further testing of the attenuated virus as an effective candidate vaccine.

Regulation of protein thermal stability and its potential application in the development of thermo-attenuated vaccines

The coronavirus disease 2019(COVID-19)pandemic has highlighted the importance of developing novel vaccines.An ideal vaccine should trigger an intense immune reaction without causing significant side effects.In this study we found that substitution of tryptophan located in the cores of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)protein structures with certain smaller amino acids resulted in variants with melting temperatures of 33-37℃.An enzyme activity assay indicated that the proteolytic activity of the main proteinase(3CLpro)decreased sharply when the environmental temperature exceeded the melting temperature,implying that other protein variants may lose most of their functions under the same conditions.This finding suggests that a virus variant containing engineered proteins with melting temperatures of 33-37℃ may only be functional in the upper respiratory tract where the temperature is about 33℃,but will be unable to invade internal organs,which maintain temperatures above 37℃,thus making it possible to construct temperature-sensitive attenuated vaccines.

Immune Responses to the Attenuated Mycoplasma hyopneumoniae 168 Strain Vaccine by Intrapulmonic Immunization in Piglets

To investigate the immune responses to the attenuated Mycoplasma hyopneumoniae 168 strain vaccine, 8-15 d old piglets were immunized with M. hyopneurnoniae 168 strain vaccine by intrapulmonic route. And the specific IgG antibody in serum, lymphoproliferation, IFNT, and specific secretory IgA （SIgA） antibody in bronchoalveolar lavage fluid were detected on 30 and 60 d post-immunization （DPI）, respectively. On 60 DPI, all the pigs except for those in health control group were challenged with a field M. hyopneumoniae strain JS. Necropsy was performed on 30 d post-challenge （DPC）. The results showed that IFN7 and specific SIgA were stimulated on surface of respiratory tract after immunization. And peripheral blood mononuclear cells could also be proliferated about 1.81 and 2.12 fold on 30 and 60 DPI when stimulated by M. hyopneumoniae protein in vitro. However, no serum IgG antibody against M. hyopneumoniae was detected during the whole immune phage. After challenge, vaccinated pigs were observed with only very slight histological lesion in individual lobes. None of vaccinated pigs showed any clinical signs. While the unvaccinated pigs from challenge control group showed varying degrees of clinical sign and severe macroscopical lesion of mycoplasmal pneumonia of swine （MPS）. The result suggested that the attenuated M. hyopneumoniae 168 strain vaccine inoculated by intrapulmonic route could activate the systemic cellular immunity, the local mucosal immunity and IFNγ secretion in respiratory tract to against M. hyopneumoniae infection in piglets.

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.

H2 strain attenuated live hepatitis A vaccines:protective efficacy in a hepatitis A outbreak

AIM:To investigate the protective efficacy of H2 strain attenuated live hepatitis A vaccines (H2-strain vaccines) in hepatitis A (HA) outbreaks.METHODS:With the permission of their parents, 5551 pre-school and grade 1-3 primary school children were inoculated with 1 dose (10(6.5) TCID(50)) of H2 strain vaccines in a nonrandomized, controlled trial conducted in Fucheng County, Hebei Province in May 1997.Another 6485 children in the same grades and compatible in gender and age were enrolled as controls. Epidemiological and serological survey was conducted to evaluate the protective efficacy of the vaccines. ELISA was used to detect serum IgM anti-HAV.RESULTS:HA outbreak started in early May 1998, peaked in the middle of the same month, and lasted about 80 days. Overall 302 HA cases were found, 192(63.58%) were 5-9 years old. One vaccinee and 25 control cases were found to have hepatitis A, which account for 0.28% (1/356) and 5.92% (25/422) of all vaccinees and controls in the 14 villages, respectively. The protective efficacy of vaccines was 95.27% (95% CI: 85.83%-104.72%). In subjects tested for anti-HAV IgM from 13 villages, 1(0.40%) overt and 11(4.06%) asymptomatic HAV cases were found in 271 vaccinees but 21(6.69%) of overt and asymptomatic ones were found in 314 controls.CONCLUSION:H2 strain vaccines were excellent in preventing overt hepatitis A,but not so effective in preventing asymptomatic hepatitis A virus infection.A booster dose might be needed to get permanent reliable immunity.

Inhibitory Effect of Live-Attenuated Listeria Monocytogenes-based Vaccines Expressing MIA Gene on Malignant Melanoma

Listeria monocytogenes(LM),a Gram-positive facultative intracellular bacterium,can be used as an effective exogenous antigen expression vector in tumor-target therapy.But for successful clinical application,it is necessary to construct attenuated LM stain that is safe yet retains the potency of LM based on the full virulent pathogen.In this study,attenuated LM and recombinants of LM expressing melanoma inhibitory activity(MIA) were constructed successfully.The median lethal dose(LD 50) and invasion efficiency of attenuated LM strains were detected.The recombinants were utilized for immunotherapy of animal model of B16F10 melanoma.The level of MIA mRNA expression in tumor tissue was detected by using real-time polymerase chain reaction(PCR) with specific sequence,meanwhile the anti-tumor immune response was assayed by flow cytometric analysis and enzyme-linked immunosorbent spot(ELISPOT) assay.The results showed the toxicity and invasiveness of attenuated LM were decreased as compared with LM,and attenuated LM expressing MIA,especially the double-genes attenuated LM recombinant,could significantly induce anti-tumor immune response and inhibit tumor growth.This study implicates attenuated LM may be a safer and more effective vector for immunotherapy of melanoma.

Effect of Attenuated Highly Pathogenic Pig Reproductive and Respiratory Syndrome(HP-PRRS) TJM-F92 Strain Vaccine on Immune Antibody Levels against Classical Swine Fever(CSF) and Foot-and-Mouth Disease(FMD)

Effects of attenuated highly pathogenic pig reproductive and respiratory syndrome（HP-PRRS）TJM-F92 strain vaccine on immune antibody level against classical swine fever（CSF）and foot-and-mouth disease（FMD）were studied from October 8 to November 12 in 2014,in order to optimize vaccination program of CSF,HP-PRRS and FMD and to provide scientific guidance for animal disease control and prevention work.The results showed that attenuated HP-PRRS（TJMF92 strain）vaccine had no significant effect on immune antibody level of hog cholera lapinized virus（HCLV,ST passage cell vaccine）attenuated vaccine and FMD-O inactivated vaccines（OZK/93 strain）,and single or combined use of three vaccines received good immunization effects.

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.

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.

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.

Immunogenicity and safety of a new inactivated hepatitis A vac cine in young adults: a comparative study

OBJECTIVE: To evaluate the immunogenicity, safety, and dosage of a new inactivated hepatitis A vaccine administered to young adults. METHODS: One hundred and four normal adult volunteers, seronegative for hepatitis A virus and hepatitis B surface antigen, were randomly assigned to one of three groups. The high-dose group received a primary dose of 1000 units of the new vaccine, the low-dose group received a primary dose of 500 units of the same vaccine, and the Havrix group received a primary dose of 1440 enzyme-linked immunosorbent assay units of Havrix, a licensed inactivated hepatitis A vaccine. All groups received a booster dose of the same vaccine 6 months after the primary dose. Local and systemic adverse reactions, seroconversion rates, and geometric mean titers of hepatitis A virus antibodies were measured in all three groups. RESULTS: Local and systemic reaction types and rates were similar in all three groups after primary and booster doses, although local reactions were more frequent in the Havrix group following the primary dose. No serious adverse reactions occurred. One month after the primary dose, the seroconversion rate was 87.5% in the high-dose group, 70.0% in the low-dose group, and 50.0% in the Havrix group (P = 0.001, versus the high-dose group). At month 6 (before administration of the booster dose), seroconversion rates were 96.9% in the high-dose group, 65.0% in the low-dose group (P = 0.0029), and 68.8% in the Havrix group (P = 0.007). All subjects in all groups seroconverted by one month after receipt of the booster dose. Geometric mean titers were similar in all three groups at month 1, but were higher in the high-dose group (264 mIU/ml) than those in the Havrix group (135 mIU/ml) at month 6 (P = 0.0013). One month after the booster dose, geometric mean titers in the high-dose group (2747 mIU/ml) were higher than those in the low-dose group (1657 mIU/ml) (P = 0.0223) or in the Havrix group (1316 mIU/ml) (P = 0.01). CONCLUSIONS: This new inactivated hepatitis A vaccine is immunogenic and safe; two doses of either 500 or 1000 units can induce hepatitis A virus antibodies well above the protection level.