Development of an ELISA system for tick-borne encephalitis virus infection in rodents

Tick-borne encephalitis (TBE) virus causes severe encephalitis with serious sequelae in humans. An epizootiological survey of wild rodents is effective to detect TBE virus-endemic areas; however, limited serological diagnostic methods are available to detect anti-TBE virus antibodies in wild rodents. In this study, ELISAs for the detection of rodent antibodies against the TBE virus were developed using two recombinant proteins, domain III of the E protein (EdIII) and subviral particles (SPs), as the antigens. As compared with the neutralization test, the ELISA using EdIII had 77.1% sensitivity and 80.0% specificity, and the ELISA using SPs had 91.4% sensitivity and 100% specificity. Furthermore, when the ELISAs were applied to the epizootiological survey in the TBE virus-endemic area, both of the ELISAs was able to detect wild rodents with TBE virus-specific antibodies. This is the first study to show that ELISAs using recombinant antigens can be safe and useful in the detection of TBE virus-infected wild rodents in epizootiological research.     

Evaluation of the European tick‐borne encephalitis vaccine against Omsk hemorrhagic fever virus

This study focused on the antigenic cross‐reactivity between tick‐borne encephalitis virus (TBEV) and Omsk hemorrhagic fever virus (OHFV) to assess the efficacy of the commercial TBE vaccine against OHFV infection. Neutralization tests performed on sera from OHFV‐ and TBEV‐infected mice showed that neutralizing antibodies are cross‐protective. The geometric mean titers of antibodies against TBEV and OHFV from TBEV‐infected mice were similar. However, the titers of anti‐TBEV antibodies in OHFV‐infected mice were significantly lower than those of anti‐OHFV antibodies in the same animals. In mouse vaccination and challenge tests, the TBE vaccine provided 100% protection against OHFV infection. Eighty‐six percent of vaccinees seroconverted against OHFV following complete vaccination, and the geometric mean titers of neutralizing antibodies against OHFV were comparable to those against TBEV. These data suggest that the TBE vaccine can prevent OHFV infection.     

Characterization of monoclonal antibodies against Hokkaido strain tick-borne encephalitis virus

A tick-borne encephalitis (TBE) patient was found in Hokkaido in 1993, and TBE viruses were isolated from animals and ticks in our previous studies. To develop a diagnostic reagent to identify TBE viruses, monoclonal antibodies (Mabs) were produced against the TBE virus strain Hokkaido (Oshima 5-10). Seven Mabs were obtained which reacted with the envelope protein of the Oshima 5-10 strain. These Mabs were flavivirus genus-specific, TBE virus complex-specific or TBE virus type-specific. The Mabs are applicable for identification of TBE virus strains.     

A plasmid encoding Japanese encephalitis virus PrM and E proteins elicits protective immunity in suckling mice

A plasmid encoding Japanese encephalitis virus (JEV) prM and E proteins was constructed, and its efficacy as a candidate vaccine against JEV was evaluated in suckling mice. Groups of 10 BALB/c mice (5-7 days old) were immunized twice via muscular injection with this DNA vaccine, an empty vector or PBS at an interval of 3 weeks, and were challenged with a lethal dose of JEV 3 weeks after the second inoculation. Both cellular and humoral immune responses were examined before the challenge. Two animals from each group were sacrificed to detect the JEV-specific cytotoxic T lymphocyte activity. JEV-specific lactate dehydrogenase release in the DNA vaccine, empty vector and PBS groups was 37.5%, 18% and 8.5% respectively. JEV-specific antibody was detected in 8 of 10 animals in DNA vaccine group with a geometrical mean titer of 1: 28.3. The pooled serum from the same group also showed a neutralizing activity. Six of 8 mice in the DNA vaccine group survived the challenge, with a protection rate of 75%, but all the mice died in the two control groups. These results show that this JEV prM and E DNA vaccine is immunogenic and protective against JEV infection in the mouse model.     

Immunization with chlamydial plasmid protein pORF5 DNA vaccine induces protective immunity against genital chlamydial infection in mice

To validate the immune protective efficacy of pORF5 DNA vaccine and to analyze potential mechanisms related to this protection. In this study, pORF5 DNA vaccine was constructed and evaluated for its protective immunity in a mouse model of genital chlamydial infection. Groups of BALB/c mice were immunized intranasally with pORF5 DNA vaccine. Humoral and cell mediated immune responses were evaluated. The clearance ability of chlamydial challenge from the genital tract and the chlamy- dia-induced upper genital tract gross pathology and histopathological characterization were also de- tected. The results showed that the total and the IgG2a anti-pORF5 antibody levels in serum were sig- nificantly elevated after pcDNA3.1-pORF5 vaccination, as were the total antibody and IgA levels in vaginal fluids. pcDNA3.1-pORF5 induced a significantly high level of Th1 response as measured by robust gamma interferon (IFN-γ). Minimal IL-4 was produced by immune T cells in response to the re-stimulation with pORF5 protein or the inactive elementary body in vitro. pcDNA3.1-pORF5-vacci- nated mice displayed significantly reduced bacterial shedding upon a chlamydial challenge and an accelerated resolution of infection. 100% of pcDNA3.1-pORF5 vaccinated mice successfully resolved the infection by day 24. pcDNA3.1-pORF5-immunized mice also exhibited protection against patho- logical consequences of chlamydial infection. The stimulated index was significantly higher than that of mice immunized with pcDNA3.1 and PBS (P<0.05). Together, these results demonstrated that immu- nization with pORF5 DNA vaccine is a promising approach for eliciting a protective immunity against a genital chlamydial challenge.     

Immunogenicity of a recombinant MVA and a DNA vaccine for Japanese encephalitis virus in swine

We previously reported that mice immunized with recombinant modified vaccinia virus Ankara (MVA) encoding Japanese encephalitis virus (JEV) prM and E genes were completely protected against JEV challenge (Nam, J.H., Wyatt, L.S., Chae, S.L., Cho, H.W., Park, Y.K., Moss, B. Vaccine 1999,17: 261-268). In this study, we examined the immunogenicity in swine of this recombinant MVA (vJH9) or a DNA vaccine (pcJH-1) expressing the same JEV genes. Although the booster effect in mice with a combination of vJH9, pcJH-1 and inactivated JEV commercial vaccine was not apparent by measuring JEV antibodies, the recombinant MVA vaccine (vJH9) and the DNA vaccine (pcJH-l) efficiently produced neutralizing antibodies in swine and 2 doses of each showed a booster effect in mice and swine. Therefore, both vJH9 and pcJH-1 are good candidates for a second generation JEV vaccine.     

Sublingual immunization with Japanese encephalitis virus vaccine effectively induces immunity through both cellular and humoral immune responses in mice

The Japanese encephalitis virus (JEV) is the leading cause of viral encephalitis. Although there are four classes of vaccines against JEV, all of them are administered by s.c or i.m injection. Here, the effectiveness of sublingual (s.l.) administration of a JEV live‐attenuated vaccine or recombinant modified vaccinia virus Ankara (MVA) vaccine, including JEV prM/E, was investigated. The mice were immunized three times i.m. or s.c. One week after the final immunization by both s.l. and i.m. routes, the titers of IgG1 induced by the recombinant MVA vaccine were higher than those induced by the live‐attenuated vaccine, whereas the titers of IgG2a induced by the live‐attenuated vaccine were higher than those induced by the recombinant MVA vaccine. However, both vaccines induced neutralizing antibodies when given by either s.l. or i.m. routes, indicating that both vaccines induce appropriate Th1 and Th2 cell responses through the s.l. and i.m. routes. Moreover, both vaccines protected against induction of proinflammatory cytokines and focal spleen white pulp hyperplasia after viral challenge. Virus‐specific IFN‐γ⁺ CD4⁺ and CD8⁺ T cells appeared to increase in mice immunized via both s.l. and i.m. routes. Interestingly, virus‐specific IL‐17⁺ CD4⁺ T cells increased significantly only in the mice immunized via the s.l. route; however, the increased IL‐17 did not affect pathogenicity after viral challenge. These results suggest that s.l. immunization may be as useful as i.m. injection for induction of protective immune responses against JEV by both live‐attenuated and recombinant MVA vaccines.     

In vivo kinetics and biodistribution of a Hantaan virus DNA vaccine after intramuscular injection in mice

To study the kinetics in vivo of a Hantaan virus DNA vaccine, we constructed a fusion DNA vaccine,pEGFP/S, by cloning the S segment of Hantavirus into the vector, pEGFP-C1, which encodes Green fluorescent protein EGFP. In this report, we provide evidence that pEGFP/S was distributed and persistently expressed for more than 60 days in several organs after inoculation. Our findings suggest that the persistent immune responses induced by a Hantaan virus DNA vaccine are likely due to the plasmid pEGFP/S deposited in vivo, which acts as a booster immunization.     

Determinants of tick-borne encephalitis virus antibody presence in roe deer (Capreolus capreolus) sera

In order to identify variables associated with the presence of the tick-borne encephalitis (TBE) virus, we conducted a serological survey of roe deer [Capreolus capreolus (Artiodactyla: Cervidae, Linnaeus 1758)] in three forest districts of southern Hesse, Germany. Overall, 24 out of 105 (22.9%) of the sera were positive (≥1 : 10 plaque reduction neutralization test). Using a logistic regression approach, we found that unexplained spatial variation, indexed roe deer density (positive correlation), hind foot length of the tested roe deer (positive correlation) and infestation with female Ixodes spp. ticks (negative correlation) predicted the probability of TBE virus antibody presence in individual roe deer sera. Spring temperature increase and host sex were rejected as explanatory variables. We found considerable differences in TBE virus antibody seroprevalence (50.0% vs. 17.6%) between two forest districts located in the same county; this finding questions the current county-resolution of public health recordings. Given the high seroprevalence of roe deer and the considerable explanatory power of our model, our approach appears suitable to delineate science-based risk maps at a smaller spatial scale and to abandon the current human incidence per county criterion. Importantly, using roe deer as sentinels would eliminate the inherent bias of risk maps based on human incidence (varying levels of immunization and exposure of humans).     

A single intramuscular injection of recombinant plasmid DNA induces protective immunity and prevents Japanese encephalitis in mice

Plasmid vectors containing Japanese encephalitis virus (JEV) premembrane (prM) and envelope (E) genes were constructed that expressed prM and E proteins under the control of a cytomegalovirus immediate-early gene promoter. COS-1 cells transformed with this plasmid vector (JE-4B clone) secreted JEV-specific extracellular particles (EPs) into the culture media. Groups of outbred ICR mice were given one or two doses of recombinant plasmid DNA or two doses of the commercial vaccine JEVAX. All mice that received one or two doses of DNA vaccine maintained JEV-specific antibodies 18 months after initial immunization. JEVAX induced 100% seroconversion in 3-week-old mice; however, none of the 3-day-old mice had enzyme-linked immunosorbent assay titers higher than 1:400. Female mice immunized with this DNA vaccine developed plaque reduction neutralization antibody titers of between 1:20 and 1:160 and provided 45 to 100% passive protection to their progeny following intraperitoneal challenge with 5,000 PFU of virulent JEV strain SA14. Seven-week-old adult mice that had received a single dose of JEV DNA vaccine when 3 days of age were completely protected from a 50, 000-PFU JEV intraperitoneal challenge. These results demonstrate that a recombinant plasmid DNA which produced JEV EPs in vitro is an effective vaccine.     

Synthetic fragments of the NS1 protein of the tick-borne encephalitis virus exhibiting a protective effect

Potentially immunoactive regions of the NS1 nonstructural protein of the tick-borne encephalitis virus that can stimulate the antibody formation in vivo and protect animals from this disease were chosen on the basis of theoretical calculations. Eleven 16-to 27-aa peptides containing the chosen regions were synthesized. The ability of the free peptides (without any high-molecular-mass carrier) to stimulate the production of antipeptide antibodies in mice of three lines and ensure the formation of protective immunity was studied. Most of these peptides were shown to exhibit the immunogenic activity in a free state. Five fragments that can protect mice from the infection by a lethal dose of tick-borne encephalitis virus were found.     

DNA vaccination using the fragment C of botulinum neurotoxin type A provided protective immunity in mice

Botulinum neurotoxin (BoNT) is one of the most toxic substances known to produce severe neuromuscular paralysis. The currently used vaccine is prepared mainly from biohazardous toxins. Thus, we studied an alternative method and demonstrated that DNA immunization provided sufficient protection against botulism in a murine model. A plasmid of pBoNT/A-Hc, which encodes the fragment C gene of type A botulinum neurotoxin, was constructed and fused with an Igkappa leader sequence under the control of a human cytomegalovirus promoter. After 10 cycles of DNA inoculation with this plasmid, mice survived lethal doses of type A botulinum neurotoxin challenges. Immunized mice also elicited cross-protection to the challenges of type E botulinum neurotoxin. This is the first study demonstrating the potential use of DNA vaccination for botulinum neurotoxins.     

DNA vaccine of SARS-Cov S gene induces antibody response in mice

The spike (S) protein, a main surface antigen of SARS-coronavirus (SARS-CoV), is one of themost important antigen candidates for vaccine design. In the present study, three fragments of the truncated S protein were expressed in E. coli, and analyzed with pooled sera of convalescence phase of SARS patients. The full length S gene DNA vaccine was constructed and used to immunize BALB/c mice. The mouse serum IgG antibody against SARS-CoV was measured by ELISA with E. coli expressed truncated S protein or SARS-CoV lysate as diagnostic antigen. The results showed that all the three fragments of S protein expressed by E. coli was able to react with sera of SARS patients and the S gene DNA candidate vaccine could induce the production of specific IgG antibody against SARS-CoV efficiently in mice with seroconversion ratio of 75% after 3 times of immunization. These findings lay some foundations for further understanding the immunology of SARS-CoV and developing SARS vaccines.     

核酸疫苗初免-蛋白疫苗加强的免疫策略提高日本血吸虫核酸疫苗免疫效果

为研制抗血吸虫疫苗提供实验依据,探讨了抗血吸虫SjGST-32核酸疫苗与蛋白疫苗联合免疫的免疫增强效应及免疫应答特征。将日本血吸虫DNA疫苗VR1012-SjGST-32与重组蛋白疫苗rSjGST-32分别在第0、2和4周免疫小鼠,在第6周攻击感染日本血吸虫尾蚴,攻击感染45 d后剖杀小鼠,计算减虫率、检卵率以及检测肝脏病理变化,观察免疫保护效果;检测小鼠血清中特异性IgG抗体滴度,T细胞增殖反应和抗原特异性CD4+IFN-γ+、CD4+IL-4+和CD4+IL-10+的数量,探讨免疫应答特征。结果显示,DNA初免-蛋白加强的联合免疫组的保护作用优于单独免疫组,显著提高了减虫率(42.3%)和减卵率(59.6%),并且能够显著减轻血吸虫虫卵对肝脏的病理损害;进一步发现,DNA疫苗和蛋白疫苗联合应用增强了机体T淋巴细胞增殖反应、抗体IgG滴度以及抗原特异性CD4+IFN-γ+的产生。这些研究为新型血吸虫疫苗的优化设计和合理应用提供了依据。     

Toxoplasma gondii: protective immunity against experimental toxoplasmosis induced by a DNA vaccine encoding the perforin-like protein 1

Toxoplasma gondii is an important zoonotic parasite infecting about one third of the world population, causing congenital infections and eye disease. T. gondii perforin-like protein 1 (TgPLP1) is believed to be involved in the acute virulence of T. gondii in mice, and is therefore of interest as a vaccine candidate. In this study, we constructed a DNA vaccine expressing TgPLP1, and evaluated the immune response in Kunming mice. The gene sequence encoding TgPLP1 was inserted into the eukaryotic expression vector pVAX I, and Kunming mice were immunized intramuscularly with the plasmid. After immunization, we evaluated the immune response using lymphoproliferative assay, cytokine and antibody measurements, and the survival times of mice challenged lethally with 1 × 10³ tachyzoites of the virulent T. gondii RH strain. The results showed that pVAX/TgPLP1 alone or with pVAX/IL-18 developed specific anti-TLA (T. gondii lysate antigen) antibodies and specific lymphocyte proliferative responses. Co-injection of pVAX/IL-18 significantly increased the production of IFN-γ and IL-2. Further, challenge experiments showed that co-immunization of pVAX/TgPLP1 with pVAX/IL-18 significantly (P < 0.05) increased survival time (12.7 ± 1.2 days) of immunized mice, compared with pVAX/TgPLP1 alone (11.3 ± 0.9 days). These results demonstrate that TgPLP1 is a potential vaccine candidate against toxoplasmosis, worth further evaluation in other animal hosts. IL-18 could enhance the immune effect of TgPLP1, prolonging the survival time of immunized mice.     

Protective efficacy of a DNA vaccine encoding capsid protein of porcine circovirus‐like virus P1 against porcine circovirus 2 in mice

The capsid protein is the major immunogenic protein of porcine circovirus 2 (PCV2). The nucleotide sequence of porcine circovirus‐like virus P1 shares high homology with open reading frame (ORF) 2 of PCV2, and ORF1 of P1 encodes its structural protein. Mice were vaccinated twice intramuscularly with a plasmid expressing the P1 ORF1 protein (pcDNA3.1(+)‐ORF1) at 2‐week intervals. All animals vaccinated with pcDNA3.1(+)‐ORF1 developed higher specific anti‐P1 antibody levels, and had less PCV2 viremia and milder histopathological changes than PCV2‐challenged mice in the control group. Our results show that the P1 DNA vaccine elicited immune responses against PCV2 infection in a mouse model.

     

Plant‐produced Zika virus envelope protein elicits neutralizing immune responses that correlate with protective immunity against Zika virus in mice

The global Zika virus (ZIKV) outbreak and its link to foetal and newborn microcephaly and severe neurological complications in adults call for the urgent development of ZIKV vaccines. In response, we developed a subunit vaccine based on the ZIKV envelope (E) protein and investigated its immunogenicity in mice. Transient expression of ZIKV E (zE) resulted in its rapid accumulation in leaves of Nicotiana benthamiana plants. Biochemical analysis revealed that plant‐produced ZIKV E (PzE) exhibited specific binding to a panel of monoclonal antibodies that recognize various zE conformational epitopes. Furthermore, PzE can be purified to >90% homogeneity with a one‐step Ni²⁺ affinity chromatography process. PzE are found to be highly immunogenic, as two doses of PzE elicited both potent zE‐specific antibody and cellular immune responses in mice. The delivery of PzE with alum induced a mixed Th1/Th2 immune response, as the antigen‐specific IgG isotypes were a mixture of high levels of IgG1/IgG2c and splenocyte cultures from immunized mice secreted significant levels of IFN‐gamma, IL‐4 and IL‐6. Most importantly, the titres of zE‐specific and neutralizing antibodies exceeded the threshold that correlates with protective immunity against multiple strains of ZIKV. Thus, our results demonstrated the feasibility of plant‐produced ZIKV protein antigen as effective, safe and affordable vaccines against ZIKV.     

Protective immunity against Bordetella pertussis by a recombinant DNA vaccine and the effect of coinjection with a granulocyte-macrophage colony stimulating factor gene

A recombinant pertussis DNA vaccine was described here with its immunogenicity and the ability to induce protection against B. pertussis infection in mice. Three immunodominant antigen gene fragments of pertussis, pertussis toxin subunit 1 (pts1), fragments of pertactin (prn) and filamentous hemagglutinin (fha), were recombined as fragment pts1-prn-fha named ppf, and it was cloned to plasmid pVAX1 as pVAX1/ppf. Compared to those injected with pVAX1, the mice injected with pVAX1/ppf significantly elicited more antigen specific antibody anti-PTS1, anti-PRN, anti-FHA and cytokine IL-10, IFN-gamma. When pGM-CSF was coinjected with pVAX1/ppf, the mice showed significantly increases of the three antibodies and cytokine IL-10, IL-4, IFN-gamma and TNF-alpha compared to those injected with pVAX1 only. The mice in group pVAX1/ppf & pGM-CSF, in particular; induced much more anti-PTS1, IL-4 and TNF-alpha than those in group pVAX1/ppf. In the intracerebral mouse protection test, the mice immunized with pVAX1/ppf or pVAX1/ppf & pGM-CSF induced protection to a lethal dose of B. pertussis. The results indicate that recombinant DNA vaccine and pGM-CSF coinjection can induce protective immunity against B. pertussis, demonstrating a valuable method to prevent pertussis.     

抗动脉粥样硬化核酸疫苗安全性的初步研究

目的：研究抗动脉粥样硬化核酸疫苗的安全性。方法：观察该质粒在小鼠组织中的分布和急性毒性,井在正常给药的情况下对新西兰大白兔进行6个月毒性试验。采用肌肉注射20μg/只和100μg/只,用P（温法检测外源基因在小鼠组织的分布和存留时间;同样采用肌肉注射,对最高剂量达到到50kg/kg体重的小鼠进行急性毒性试验。结果：实验证明该核酸疫苗在注射部位可存留时间为8周,其他组织如心、肝、脾、肺、肾、脑和生殖器官也有低水平分布,急毒试验显示,在剂量达到有效荆量的60倍的情况下,无可观察到的毒性反应发生,小鼠血液学和生化指标亦无异常;新西兰大白兔毒性观察,体内生化指标以及解剖学研究结果表明,该药无可观察到的毒性反应发生。结论：该核酸疫苗无可观察到的明显毒性反应,安全性良好。