猪瘟活疫苗佐剂稀释剂的制备及免疫效果评价

本试验旨在针对性的研发一种安全性好、能有效增强免疫效果、使用方便的猪瘟活疫苗新型佐剂稀释剂(AD),并对AD的免疫增强效果进行评价。采用高压均质技术制备了AD,采用响应面试验设计优化了AD的处方和制备工艺,并对其稳定性、粒径、多分散指数(PDI)及Zeta电位进行表征。将AD与猪瘟活疫苗混合后,肌肉注射免疫BALB/c小鼠,进行佐剂增强免疫效果评价。优化处方和工艺制备的AD平均粒径为100.4 nm,PDI为0.147,Zeta电位为-28.7 mV,试验结果表明其稳定性良好。免疫效果评价试验结果显示,佐剂组小鼠血清中抗猪瘟病毒(CSFV)抗体水平与对照组相比有显著差异(P<0.05),抗体水平能持续较长时间,表明AD能有效诱导机体产生体液免疫应答;佐剂组小鼠血清中IL-4、IL-6和IFN-γ的水平均有明显提高,表明该疫苗佐剂能诱导机体产生细胞免疫应答。本试验制备的佐剂稀释剂与疫苗混合使用,能显著提高体液免疫和细胞免疫水平,从而增强疫苗的免疫刺激能力。     

Toxoplasma gondii rhomboid protein 1 (TgROM1) is a potential vaccine candidate against toxoplasmosis

Infection with the intracellular protozoan parasite Toxoplasma gondii causes serious public health problems and is of great economic importance worldwide. The rhomboid proteins which are responsible for adhesion and invasion of host cells have been suggested as vaccine candidates against toxoplasmosis. A DNA vaccine (pVAX-ROM1) encoding T. gondii rhomboid protein 1 (TgROM1) gene was constructed and the immune response and protective efficacy of this vaccine against lethal challenge in BALB/c mice were evaluated. The results indicated that specific antibody and lymphocyte proliferative responses were elicited in mice receiving pVAX-ROM1. The production levels of IFN-γ, IL-2, IL-4, and IL-10, as well as the percentage of CD4(+) cells in mice vaccinated with pVAX-ROM1 were significantly increased respectively, compared to controls receiving either pVAX1 alone or PBS. After lethal challenge, the mice immunized with pVAX-ROM1 showed an increased survival time compared with the mice in the controls. Our data suggested that a DNA vaccine pVAX-ROM1 encoding T. gondii rhomboid protein 1 triggered strong humoral and cellular responses, and prolonged survival time against T. gondii infection in BALB/c mice.     

Immune response in mice and cattle after immunization with a Boophilus microplus DNA vaccine containing bm86 gene

Plasmid pBMC2 encoding antigen Bm86 from a Colombian strain of cattle tick Boophilus microplus, was used for DNA-mediated immunization of BALB/c mice, employing doses of 10 and 50microg, delivered by intradermic and intramuscular routes. Anti-Bm86 antibody levels were significantly higher compared to control mice treated with PBS. In the evaluation of immunoglobulin isotypes, significant levels of IgG2a and IgG2b were observed in mice immunized with 50microg of pBMC2. Measurement of interleukine (IL) levels (IL-4, IL-5, IL-12(p40)) and interferon-gamma (IFN-gamma) in the sera of mice immunized with pBMC2 indicated high levels of IL-4 and IL-5, although there were also significant levels of IFN-gamma. Mice immunized with pBMC2 showed antigen-specific stimulation of splenocytes according to the incorporation of bromodeoxyuridine and IFN-gamma secretion. In all trials, mice injected intramuscularly with 50microg of pBMC2 presented the highest immune response. Moreover, cattle immunized with this DNA vaccine showed antibody production significantly different to the negative control. In conclusion, these results suggest the potential of DNA immunization with pBMC2 to induce humoral and cellular immune responses against B. microplus.     

Evaluation of immune responses induced by SAG1 and MIC3 vaccine cocktails against Toxoplasma gondii

The aim of this study was to evaluate the immune responses of a SAG1 and MIC3 vaccine cocktail in BALB/c mice. Ninety-six BALB/c mice were randomly divided into eight groups, including three plasmid DNA vaccine groups (pcDNA-MIC3, pcDNA-SAG1, pcDNA-MIC3+pcDNA-SAG1), three recombinant pseudotype baculovirus vaccine groups (BV-G-MIC3, BV-G-SAG1, BV-G-SAG1+BV-G-MIC3) and two control groups (PBS and BV-G-EGFP). All groups were immunized intramuscularly twice at three-week intervals. The production of anti-Toxoplasma gondii lysate antigen (TLA) antibodies, lymphoproliferation, levels of IFN-γ, IL-4 and IL-10 and the survival time were monitored after vaccination. The results showed that immunization of BALB/c mice with MIC3 and SAG1 vaccines stimulated both the cellular and humoral immune responses with the production of anti-T. gondii TLA antibodies. The vaccine cocktails of pcDNA-MIC3+pcDNA-SAG1 or BV-G-SAG1+BV-G-MIC3 induced significantly higher immunogenicity than a single-gene vaccine (P<0.05). Splenocytes from the immunized mice significantly proliferated in response to the TLA and released interferon (IFN)-γ (P<0.05). However, the levels of IL-4 and IL-10 in the sera of the immunized mice were not significantly different from those of the controls (P>0.05). Immunization with the vaccine cocktail (BV-G-SAG1+BV-G-MIC3) in mice significantly prolonged survival (50%; P<0.05) against a lethal challenge of T. gondii (RH tachyzoites), while all mice in the other immunized groups and control groups died within 20 and 4 days post-infection, respectively. Furthermore, the recombinant pseudotype baculovirus vaccines induced better immunogenicity than the plasmid DNA vaccines (P<0.05). These results suggest that an excellent vector-mediated vaccine cocktail strategy might be used to develop a new generation of vaccines against T. gondii infection.     

Immune response and protective efficacy against homologous challenge in BALB/c mice vaccinated with DNA vaccine encoding Toxoplasma gondii actin depolymerizing factor gene

A DNA vaccine (pVAX1-TgADF) encoding Toxoplasma gondii actin depolymerizing factor (ADF) gene was constructed and the immune response and protective efficacy of this vaccine against homologous challenge in BALB/c mice were evaluated. High titers of specific antibody and increases in the percentage of CD4(+) and CD8(+) T lymphocyte cells were observed from BALB/c mice vaccinated with pVAX1-TgADF (P<0.05), when PBS group was used as control. The survival time of BALB/c mice in pVAX1-TgADF group was longer than those in control groups. The numbers of brain cysts in the experimental BALB/c mice immunized with pVAX1-TgADF reduced significantly compared with those in PBS group (P<0.05), and the rate of reduction could reach to around 42.8%. These results suggested that the DNA vaccine pVAX1-TgADF could generate specific humoral and cellular immune responses, prolong survival times, and reduce brain cysts load against T. gondii infection in BALB/c mice.     

Assessment in mice of vapA-DNA vaccination against Rhodococcus equi infection

There is a need to produce a vaccine against Rhodococcus equi pneumonia in foals in which immunity against infection is largely based on a type 1, cell-mediated, immune response. The VapA protein of the virulence plasmid of R. equi is highly immunogenic. To assess the potential of vapA-DNA to produce immunity, C57BL/6 and BALB/c mice were immunized with a DNA vaccine constructed from vapA incorporated into pcDNA3.1. The plasmid construct expressed VapA in a COS-7 cell line. Intramuscular immunization of mice resulted in enhanced clearance of R. equi from the liver of intravenously challenged mice compared to non-immunized controls. This effect was more marked when pORF-IL-12, a plasmid expressing murine IL12, was included with the vaccine. Antibody developed to VapA, with an IgG2a response being more marked in mice immunized with pcDNA-vapA than in non-immunized or in mice immunized with the mixed vapA and IL-12 plasmid constructs. In conclusion, this study has shown for the first time that DNA immunization with vapA enhances the immune responses of mice against R. equi infection, that the IgG subisotype response is consistent with a type 1-based immune response, and that this can be enhanced by injection of the IL-12 gene.     

几种佐剂对肌肉注射猪支原体肺炎活疫苗免疫刺激能力的增强作用研究

为增强现有猪支原体肺炎活疫苗的免疫刺激能力以实现肌肉注射免疫,向现有活疫苗中添加几种不同佐剂,考察其经肌肉免疫后机体的免疫应答情况。结果表明,免疫刺激复合物基质佐剂和左旋咪唑+壳聚糖混合佐剂能有效激发细胞免疫应答和体液免疫应答;左旋咪唑+黄芪多糖混合佐剂能增强疫苗的细胞免疫刺激能力,而对体液免疫无作用;皂角素佐剂未显示明显的免疫增强效果。本试验结果为肌肉注射猪支原体肺炎活疫苗的开发奠定了数据基础。     

The adjuvant effect of a single dose of interleukin-12 on murine immune responses to live or killed Brucella abortus strain RB51.

This study was designed to determine if a single 0.5 microg administration of recombinant murine interleukin-12 (IL-12) would influence immune responses of mice vaccinated with live or killed Brucella abortus strain RB51 (SRB51). Mice were vaccinated intraperitoneally with 5 x 10(8) cfu of live or gamma-irradiated SRB51 bacteria alone, or in combination with 0.5 microg of IL-12. Control mice received saline or 0.5 microg of IL-12. Serologic responses and spleen weights after vaccination were greater in mice vaccinated with live SRB51 when compared to mice receiving killed SRB51 or control treatments. Administration of a single dose of IL-12 as a vaccine adjuvant did not influence immune responses, clearance of live SRB51, or resistance against B. abortus strain 2308 (S2308) challenge. The results of this study suggest that a single administration of 0.5 microg of IL-12 at the time of vaccination does not have significant adjuvant effects on vaccine-induced immune responses against live or killed Brucella.     

法氏囊提取液对新城疫活疫苗免疫效力的影响

本文对鸡法氏囊提取液与ND活疫苗的使用方法进行了分析。通过血凝抑制抗体滴度的测定结果表明,预先接种鸡法氏囊提取液时,经肌肉注射、口服和点眼滴鼻接种方式均能提高ND疫苗免疫鸡的抗体产生能力,且经肌肉注射法最佳。但是,在整个试验期间,法氏囊提取液只能增强鸡群对ND活疫苗的早期免疫应答,而法氏囊提取液与ND活疫苗同时使用时更能刺激免疫鸡群的早期抗体产生水平。     

Simultaneous vaccination of piglets against foot-and-mouth disease and classical swine fever

Six-week-old piglets, born of unvaccinated sows, were vaccinated against foot-and-mouth disease (FMD) with a trivalent, inactivated vaccine containing an adjuvant or vaccinated against classical swine fever (CSF) with a live attenuated vaccine or against both diseases simultaneously at two different sites. The antibody response to the FMD vaccine was not significantly influenced by the simultaneous vaccination against CSF. FMD vaccine administered simultaneously with the CSF vaccine produced a significantly higher antibody response to CSF than occurred with CSF vaccination only.     

DNA Fragment Encoding Human IL-1β 163–171 Peptide Enhances the Immune Responses Elicited in Mice by DNA Vaccine against Foot-and-Mouth Disease

DNA vaccine has been tested for protection against foot-and-mouth disease. However, the relatively low efficacy of DNA vaccine in inducing immune responses in large animals has restricted its practical use. Interleukin-1 plays an essential role in amplifying both the cellular and humoral immune responses to foreign antigens, and may therefore represent a good candidate as an adjuvant of DNA vaccines. Since the inflammatory activity of IL-I may restrict its application in DNA vaccine treatment, we explored the possibilities of augmenting immune responses without unwanted inflammatory effects using the IL-1beta fragment (amino acids (aa) 163-171), which is essential for IL-1 receptor-1 binding. The DNA fragment encoding the human IL-1beta fragment (aa 163-171) was fused to foot-and-mouth disease virus (FMDV) DNA vaccine, and injected into mice to analyse its immune response. Compared with control mice receiving FMDV DNA vaccine alone, significant increases in the FMDV-specific antibody response and also in T cell proliferation were observed in mice receiving IL-1beta (163-171)-FMDV. These results suggested that DNA fragment encoding IL-1beta 163-171 peptide might represent a good candidate for an adjuvant of FMDV DNA vaccine.     

Evaluation of the immune response induced by intradermal vaccination by using a needle-less system in comparison with the intramuscular route in conventional pigs

The immune response induced by intradermal vaccination using a needle-less device was evaluated in conventional pigs in comparison with the more conventional intramuscular vaccination; to this purpose, vaccination against Aujeszky’s Disease (AD) was used as a model of antiviral immunity. Two groups of pigs (n = 10 each) were vaccinated 4 weeks apart respectively by the intramuscular (IM group) and intradermal route (ID group; needle-less I.D.A.L.® vaccinator) with an AD modified live virus. Ten pigs injected with the vaccine adjuvant only were kept as sham-vaccinated controls (C group).On blood samples collected at 0, 2, 4, 5, 6 and 7 weeks post-vaccination (PV) ADV-specific virus neutralizing (VN) antibodies, IFN-γ secreting cells (SC), lymphocyte subsets and IFN-γ gene expression in PBMC were evaluated.VN antibodies increased after the 1st vaccination and peaked after the 2nd vaccination in both vaccinated groups. Also IFN-γ SC reached maximum levels in both groups after administration of the booster dose. Pigs in the control group remained negative for both parameters throughout the study. Flow cytometry showed persistently higher levels of CD3−CD8α+ Natural Killer cells in both vaccinated pigs. The ID group showed an earlier and regulated activation characterized by an increase of cytotoxic CD8β+ T lymphocytes and CD25+ cells after the boosting dose. No statistically significant differences between treated and control groups were detected for memory CD4+CD8α+^low T cells. Upregulation of IFN-γ gene expression in PBMC was detected in ID and IM pigs after both vaccine administrations, although at a different extent. Overall, the results showed that the intradermal vaccine delivery by a needle-less device can prime a strong humoral and cellular immune response comparable to that obtained by the intramuscular vaccination.     

马流感病毒血凝素基因甲病毒复制子重组表达质粒的构建及其免疫效力评价

为评价马流感病毒(EIV)HA基因核酸免疫效果,本研究以甲病毒复制子载体pSFV1CS分别构建了表达EIV H3N8亚型的美洲型和欧洲型HA基因的重组真核表达质粒。并将其转染293T细胞,经间接免疫荧光鉴定表明HA基因获得表达;以重组质粒免疫的BALB/c鼠能够检测到特异性抗体产生,而且HI抗体水平持续升高,同时小鼠体内IFN-γ、IL-4分泌水平也有所升高。攻毒后小鼠表现轻度临床症状,但病毒分离和RT-PCR均未检测到病毒。上述结果表明,该重组质粒pSFV1CS-EIV-HA具有良好的免疫原性并且可以诱导免疫动物产生较高免疫应答的能力。     

基因枪与肌肉注射猪繁殖与呼吸综合征病毒ORF5基因疫苗诱导小鼠体液及细胞免疫的比较研究

猪繁殖与呼吸综合征病毒ORF5基因疫苗（pcDNA—PRRSV—ORF5）以不同免疫途径（基因枪和肌肉注射）免疫BALB／c小鼠，以PBS和空载质粒pcDNA3．1（＋）为对照，采用流式细胞仪（FACS）、淋巴细胞增殖试验（MTT法）及间接ELISA试验分别对小鼠外周血中CD4^＋、CD8^＋T淋巴细胞数、T淋巴细胞的转化功能及小鼠血清中特异性PRRSV血清抗体IgG动态变化进行了检测。结果表明，pcDNA—PRRSV-ORF5基因疫苗接种小鼠后外周血对ConA有明显的反应性，试验组与对照组比较差异极显著（P〈0．01）或显著（P〈0．05），CD4^＋T淋巴细胞数在免疫后7d高于对照组（P〈0．01），CD8^＋T淋巴细胞在免疫后28d高于对照组，不同途径基因疫苗接种小鼠后均诱导小鼠产生PRRSV特异性IgG。在诱导细胞免疫方面，基因枪和肌肉注射各组间无明显差异；在诱导体液免疫方面，基因枪法优于肌肉注射。研究表明制备的pcDNA—PRRSV—ORF5基因疫苗免疫小鼠能够诱导其机体产生良好的体液和细胞免疫应答，基因枪法较肌肉注射更能诱导体液免疫应答的产生。     

Immunogenicity and protective efficacy of two recombinant pseudorabies viruses expressing Toxoplasma gondii SAG1 and MIC3 proteins

Toxoplasma gondii is one of the most common parasitic pathogens in humans and warm-blooded animals, causing toxoplasmosis. One of the efficient ways to control this disease is immunization. In this study, two recombinant pseudorabies virus (PRV) expressing TgSAG1 (rPRV-SAG1) and TgMIC3 (rPRV-MIC3) based on the PRV vaccine strain were developed by homologous recombination and used for immunizing BALB/c mice. Ninety BALB/c mice were randomly divided into five groups including four experimental groups (inoculated twice in 4 weeks interval with PRV TK-/gG-/EGFP+, rPRV-SAG1, rPRV-MIC3, rPRV-SAG1+rPRV-MIC3, respectively) and one control group (inoculated with medium). All mice vaccinated with rPRV developed a high level of specific antibody responses against T. gondii lysate antigen (TLA), a strong increase of the splenocyte proliferative response, and significant levels of IFN-γ and IL-2 production. These results demonstrated that rPRV could induce significant humoral and cellular Th1 immune responses. Moreover, rPRV immunization induced partial protection against a lethal challenge with T. gondii RH strain, and neutralizing antibodies against PRV in a BALB/c mouse model. The mice immunized with the rPRV-SAG1 and rPRV-MIC3 cocktail could develop higher T. gondii-specific IgG antibodies and lymphocyte proliferative responses and conferred more efficient protection against T. gondii challenge. These results suggested that expression of protective antigens of T. gondii in PRV is a novel approach towards the development of a vaccine against both animal pseudorabies and toxoplasmosis.     

Porcine interleukin-2 gene encapsulated in chitosan nanoparticles enhances immune response of mice to piglet paratyphoid vaccine

Interleukin-2 (IL-2) is vital to elicit and amplify the cellular and humoral immune responses to foreign antigens, which is extensively utilized in the control of infectious disease and treatment of various cancers. Porcine and murine IL-2 genes were, respectively, subcloned into VR1020, designated as VPIL-2 and VMIL-2, and then encapsulated in chitosan nanoparticles (CNP) prepared by ionic linkage. The BALB/c mice were intramuscularly co-administrated with chitosan-IL-2 nanoparticles (CNP-IL2) and paratyphoid vaccine to test the adjuvant effect of CNP-IL2. On day 35, the immunized mice were orally challenged with virulent Salmonella. The content of IgG, IgA, IgM, IL-2, IL-4, IL-6 and specific antibody titer as well as the number of immunocompetent cells were systematically analyzed in the vaccinated mice. The results revealed that the levels of immunoglobulins, cytokines, the specific antibodies, together with the numbers of lymphocytes significantly increased in vaccinated mice inoculated with CNP-VPIL2 in contrast with those with naked IL-2 plasmids and blank plasmids. The CNP-VPIL2 immunized mice exhibited higher humoral and cellular immune responses, less severe clinical signs and lesions of disease caused by the bacteria than the other groups after challenge. These findings suggest that CNP-VPIL2 has a significant enhancement effect on immune responses of mice, which results in better immunoprotection against Salmonella infection, indicating that CNP-VPIL2 could be employed as an effective immunoadjuvant to elevate immunity of animals to conventional vaccines.     

Evaluation of efficacy of a new live Salmonella Typhimurium vaccine candidate in a murine model

Efficacy of a new live Salmonella Typhimurium vaccine candidate attenuated by two virulence gene deletions was evaluated in this study. Live form of the vaccine was orally administered and formalin-inactivated form was intramuscularly (IM) administered. 105 female BALB/c mice were used and divided into 5 groups, A to E, containing 21 mice per group. Serum IgG and secretory IgA titers and levels of serum IFN-γ, IL-4, TNF-α, IL-12 of the immunized groups, especially group C (oral prime-oral booster) significantly increased. In addition, all animals in groups C showed no clinical symptoms and survived the virulent challenges, whereas all or some of mice from the other groups died of the challenge. These indicate that the vaccine candidate can be an effective tool for prevention of Salmonella infections by inducing robustly protective immune responses and leading to the production of inflammatory cytokines. Booster immunization, especially via oral administration, is necessary to optimize its protection.     

The Mycoplasma hyopneumoniae recombinant heat shock protein P42 induces an immune response in pigs under field conditions

Enzootic pneumonia (EP), resulting from Mycoplasma hyopneumoniae infection is one of the most prevalent diseases in pigs and is a major cause of economic losses to the swine industry worldwide. EP is often controlled by vaccination with inactivated, adjuvanted whole-cell bacterin. However, these bacterins provide only partial protection and do not prevent M. hyopneumoniae colonization. Attempts to develop vaccines that are more efficient have made use of the recombinant DNA technology. The objective of this study was to assess the potential of recombinant M. hyopneumoniae heat shock protein P42 in vaccine preparations against EP, using piglets housed under field conditions in a M. hyopneumoniae-positive farm. The cellular and humoral immune responses were elicited after a single intramuscular inoculation of rP42 in an oil-based adjuvant, or in conjunction with whole-cell vaccine preparation. The production of INF-γ and IL-10 cytokines was quantified in the supernatant of the cultured mononuclear cells. The rP42 emulsified in oil-based adjuvant was able to trigger a strong humoral immune response. Further, it induced a cellular immune response, accompanied by the production of antibodies that reacted with the native M. hyopneumoniae protein. The rP42 mediated induction of cellular and humoral immune response in the host suggests that rP42 emulsified in an oil-based adjuvant holds promise as an effective recombinant subunit vaccine against EP.     

A recombinant DNA vaccine encoding C. andersoni oocyst wall protein induces immunity against experimental C. parvum infection

Cryptosporidium andersoni parasited in the abomasum has been demonstrated as a cause of reduction of milk production in dairy cow. In this study, a novel chimeric DNA vaccine pVAX1-AB was constructed and the efficacy against Cryptosporidium parvum was determined. BALB/c mice were divided into 3 groups and immunized with DNA vaccine expressing the oocyst wall protein, AB protein of C. andersoni, the recombinant plasmid containing the AB gene, respectively. After inoculation of 1 × 10(6) oocysts of C. parvum, the humoral and cellular immune responses were detected. Experimental results showed that the recombinant plasmid can induce corresponding specific antibody response, simultaneously influenced cellular immune responses, and provided greater protection rate (48.6%) than the other groups. These results indicated that chimeric DNA vaccine has a potential in Cryptosporidium vaccine development.     

Comparison of intramuscular and footpad subcutaneous immunization with DNA vaccine encoding HSV-gD2 in mice

Herpes simplex virus type 2 is the most common infectious agent in humans that causes genital herpes disease and vaccination is a desirable method to prevent herpes infections. An effective therapeutic vaccine will need to elicit virus-specific immune responses. The route of immunization has important role in immune responses. In this study, DNA vaccine encoding glycoprotein D of herpes simplex virus type 2 (HSV-gD2) was prepared and injected via intramuscular and footpad routes to determine the optimal method of delivery for immune stimulation. The control manipulation of immune response by concerning route of administration is highly appreciated issue by researches. Although DNA vaccine containing HSV-gD2 is effective in both intramuscular and footpad injection routes, the latter could induce significantly higher cellular responses against HSV-2.