Protective immunity against influenza A virus induced by immunization with DNA plasmid containing influenza M gene

DNA vaccination is characterized by its preferential induction of the cytotoxic T cell lymphocyte (CTL) response and is expected to be a useful means of protection against viral infection. We examined the protective effect of an expression plasmid (pME18S-M) containing M1 and M2 genes of influenza A/PR/8/34. We detected the CTL activity by introducing these plasmids into BALB/c mice by either the intramuscular or the intranasal route. The influenza-specific antibody response was also induced, although its neutralizing effect against influenza virus was not observed. From 70 to 80% protection was observed in the mice immunized with the pME18S-M plasmid followed by lethal infection with influenza viruses of the A/WSN/33 and A/PR/8/34 strains, whereas all mice without the plasmid vaccination failed to survive. This protective activity was significantly weakened when the CD8(+) cells of these immunized mice were eliminated by several injections of anti-CD8 antibody. The protective activity was also weakened when anti-CD4 antibody was injected in the early phase of DNA vaccination. These data suggest that the pME18S-M plasmid is useful as a DNA vaccine for overcoming highly mutational influenza viruses.     

Characterization of the humoral and cellular immune responses against hepatitis C virus core induced by DNA-based immunization

Hepatitis C Virus (HCV) causes most cases of posttransfusion hepatitis. Chronic HCV infection is highly related to chronic hepatitis, cirrhosis and hepatocellular carcinoma. Current therapies are only minimally effective and no vaccine has been developed. DNA-based immunization could be of prophylactic and therapeutic value for HCV infection. By intramuscular inoculation in BALB/c mice with an HCV recombinant plasmid pCI-HCV-C, we found significant levels of IgM antibody, but no significant IgG rise. After boost the immunized mice with recombinant HCV-core protein (cp1-10; 1-164aa), the anticore IgG, verified by Western-blotting, rose rapidly, which was two weeks earlier than that with control plasmid. Spleen cells from pCI-HCV-C immunized mice gave higher proliferation index (PI) than control (P < 0.05). The PI of cp1-10 boosted mice was even higher. Proliferation blocking assay with mAb proved the responding cell to be of CD4+ CD8- phenotype, supporting specific priming of T helper cells. A 51Cr-releasing CTL assay specific for HCV-core was developed, and a specific CTL response against HCV-core was demonstrated in both pCI-HCV-C immunized mice and mice boosted with cp1-10. Strong cytotoxic activity against peptide-pulsed p815 cells (H-2d), but not EL-4 cells (H-2b), suggested MHC class I restriction of the CTL activity. Blocking of CTL with mAb proved the effector cells to be of CD4- CD8+. Three CTL epitopes in HCV-core protein were demonstrated. We failed to detect CTL when immunized only with core protein. The results suggested that vaccination with HCV-core derived DNA sequences could be an effective method to induce humoral and cellular immune responses to HCV.     

Enhanced cellular immune responses to SIV Gag by immunization with influenza and vaccinia virus recombinants

An effective vaccination strategy against human immunodeficiency virus type 1 (HIV-1) should include the induction of potent cellular immune responses against conserved HIV-1 antigens. We have generated five replication competent recombinant influenza viruses (rFlu/SIV Gag nos. 1-5) expressing different portions of Gag of simian immunodeficiency virus (SIV). Single intranasal immunizations in mice with each rFlu/SIV Gag viruses resulted in different degrees of protection against a challenge with recombinant vaccinia virus expressing SIV Gag. Immunized BALB/c mice had detectable CD8+ T cell responses specific for Gag peptide 185-199 when mice were vaccinated with rFlu/SIV Gag no. 3 virus, and for Gag peptides 281-295 and 285-299 when vaccinated with rFlu/SIV Gag no. 4 virus. Cellular immune responses against SIV Gag were further enhanced by a booster with a recombinant vaccinia virus expressing SIV Gag in both the spleen and local lymph node tissues, resulting in the induction of robust Gag-specific CD8+ T cell responses at both systemic and mucosal levels. We suggest that a prime-boost immunization regimen using recombinant influenza and vaccinia viruses expressing HIV Gag might represent an effective means to induce potent HIV-specific, protective CD8+ T cell responses.     

Protective immunity by intramuscular injection of low doses of influenza virus DNA vaccines

Dose—response relationships were investigated between dose of influenza virus haemagglutinin (HA) or nucleoprotein (NP) DNA vaccines, and immunogenicity and protective efficacy based on humoral and cellular immunity. In mice, intramuscular (i.m.) injection of HA or NP DNA, at doses of 100 ng to 1 μg, was found to generate haemagglutination inhibiting (HI) antibodies and cytotoxic T-lymphocytes, respectively, and provide protection in influenza virus challenge models. A direct correlation between the amount of DNA injected and the level of HI antibody was observed. In non-human primates, high-titre neutralizing antibodies were induced in animals vaccinated with as little as 10 μg of HA DNA. These results indicate that low doses of DNA administered by i.m. injection provide protective efficacy against influenza.     

Protective immunity against heterologous challenge with encephalomyocarditis virus by VP1 DNA vaccination: effect of coinjection with a granulocyte-macrophage colony stimulating factor gene

For DNA vaccination studies, recombinant VP1 protein of encephalomyocarditis virus (EMCV) was produced from Escherichia coli, and eukaryotic VP1 expression vector, pCT-Gs-VP1, was generated and used as a DNA vaccine. Mice were immunized intramuscularly (i.m.) with pCT-Gs-VP1 in the presence or absence of plasmid DNA expressing granulocyte-macrophage colony stimulating factor (GM-CSF), and were subsequently analyzed for their anti-VP1 immune responses with recombinant VP1 in ELISA. Immunization of mice with pCT-Gs-VP1 resulted in VP1-specific immune response and 43% protection from subsequent lethal heterologous challenge of EMCV. Coinjection of mice with pCT-Gs-VP1 and plasmid DNA encoding GM-CSF was shown to increase the seroconversion rate of the immunized mice with a single DNA injection, and enhanced to a higher degree VP1-specific immunity, which appeared to result in better protection (about 80%) from lethal virus challenge. Thus, our results provide evidence for the potential use of GM-CSF to induce better immune response and resistance against viral infection in DNA vaccination.     

Enhancement of the immunity to foot-and-mouth disease virus by DNA priming and protein boosting immunization.

Subunit vaccination is effective in eliciting humoral responses to a variety of viral antigens, however, it has not generated persistent protective immunity to foot-and-mouth disease virus (FMDV). In this study, we observed that priming mice with a DNA plasmid encoding VP1 of the FMDV O/Taiwan/97 capsid protein followed by boosting with a VP1 peptide conjugate (P29-KLH) resulted in production of not only high titers of antibodies but also antibodies with FMDV neutralizing activities. Moreover, the mice immunized in this manner cleared the virus from their sera in FMDV challenge experiments. Mice subjected to DNA plasmid priming and P29-KLH protein boosting had relatively higher ratio of IgG2a/IgG1 than those primed and boosted with P29-KLH conjugate. Addition of an oligodeoxynucleotide (ODN) containing immunostimulatory cytosine-phosphate-guanosine (CpG) motifs to P29-KLH conjugate also induced a higher ratio of IgG2a/IgG1 and significantly higher titer of neutralizing antibodies. These results indicate that treating animals with DNA plasmids priming and FMDV antigen(s) boosting may elicit immunity to FMD and this immune response may be augmented by CpG ODN.     

Enhanced induction of the IgA response in pigs by calcitriol after intramuscular immunization

In this study, the immunomodulating effect of two steroid hormones namely 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] and dehydroepiandrosterone (DHEA) was examined on the antigen-specific antibody responses by intramuscular immunization of pigs with human serum albumin alone (HSA) or supplemented with 2 microg of 1,25(OH)(2)D(3,) 40 microg of DHEA or the combination of both steroids. 1,25(OH)(2)D(3) significantly enhanced the antigen-specific IgA and IgM serum response. Higher HSA-specific IgA titers were also found in the mucosal secretions (saliva, feces and nasal) of the steroid treated animals, especially in the 1,25(OH)(2)D(3) group. Furthermore, 1,25(OH)(2)D(3) and DHEA increased the number of antigen-specific IgA and IgG antibody-secreting cells in the local draining lymph nodes, but only low numbers were detected in lymph nodes draining the mucosa. DHEA decreased the IgM serum response and had a tendency to enhance the IgG2 and IgG serum responses. Strong and comparable IgG, IgG1 and IgG2 serum responses were seen in all groups. Combining both steroids did not result in a higher IgA serum response. On the contrary DHEA seems to neutralize the effect of 1,25(OH)(2)D(3) on the IgA response. In conclusion, 1,25(OH)(2)D(3) significantly enhanced the antigen-specific IgA and IgM response in serum and the number of antigen-specific IgA and IgG ASC in the local draining lymph nodes following intramuscular immunization.     

Enhancement of humoral and cellular immunity in chickens against reticuloendotheliosis virus by DNA prime-protein boost vaccination

Reticuloendotheliosis virus (REV) causes an oncogenic, immunosuppressive and runting syndrome in multiple avian hosts worldwide. In this study, an optimal vaccination strategy was developed to enhance the immune responses against REV infection. Chickens were vaccinated twice intramuscularly with plasmid pCAGgp90 encoding gp90 protein of REV, or with recombinant gp90 protein, or vaccinated with plasmid pCAGgp90 and then boosted with recombinant gp90 protein. The humoral immune responses were monitored by ELISA and virus neutralizing test. In addition, lymphocyte proliferation response, cytokine production and protection effectiveness against REV infection were also evaluated. Although all vaccinated groups developed immune responses, chickens primed with pCAGgp90 plasmid and boosted with rgp90 protein developed higher levels of antibodies compared with those immunized with pCAGgp90 plasmid or rgp90 protein alone. Furthermore, enhanced cellular immune responses were induced following priming with the pCAGgp90 plasmid and boosting with the rgp90 protein. In addition, the DNA prime-protein boost vaccine yielded 100% protection of chickens from REV viremia caused by challenge infection. These findings demonstrated that a DNA prime-protein boost vaccination strategy could enhance both humoral and cellular immune responses in chickens, highlighting the potential value of such an approach in the prevention of REV infection.     

Engineering of N-glycosylation of hepatitis C virus envelope protein E2 enhances T cell responses for DNA immunization

N-linked oligosaccharides are known to be important in modulating immunogenicity of some viral proteins. However, the roles of N-glycans of hepatitis C virus (HCV) E2 envelope glycoprotein in specific cellular immune responses remain elusive. Previous studies showed that the epitopes aa481-500 and aa551-570 of E2 might be important for immunoreactivity and that the binding site of E2 for hCD81 is located at aa480-493 and aa544-551 within the E2 protein. Here, we made plasmids containing genes encoding either wild type or mutant E2 proteins in which N-glycosylation sites (N560NT and N576ST) close to these important regions were mutated separately or in combination. The immunogenicities of wild type E2 and three mutated E2 proteins were analyzed in BALB/c mice using DNA vaccination. The E2-M2 mutant (at N576ST) significantly enhanced (compared to control) E2-specific CTL activity (P<0.05), expression of IFN-gamma (P<0.05) and suppression of tumor growth (P<0.05). Also, high IgG2a/IgG1 ratios were elicited in a Th1-type response. These results indicate that engineering of the N-glycosylation site N576ST of HCV E2 protein enhances specific cellular immune responses, providing insights into the development of E2-based DNA vaccines with enhanced immunogenicity.     

紫外交联实验筛选HCV RNA结合蛋白的应用

目的 选择一种用于筛选与丙型肝炎病毒（HCV）RNA核心区结合的细胞蛋白质分子较佳方法。方法 用体外转录的方法制备地高辛标记（DIG）-HCV RNA；从HepG2细胞中提取细胞蛋白质分子。分别用紫外交联实验（UV）和凝胶迁移实验（EMSA）筛选HepG2细胞中可与HCV RNA结合的蛋白质分子。结果 2种实验方法均可检测到细胞蛋白质分子与HCV RNA结合。但凝胶迁移实验结果所见DIG—RNA信号比较弥散，而紫外交联实验的结果可见比较清晰的RNA-结合蛋白条带。结论 紫外交联实验是用于筛选HCV—RNA结合蛋白的首选方案。     

DNA vaccine expressing the non-structural proteins of hepatitis C virus diminishes the expression of HCV proteins in a mouse model

Most of the people infected with hepatitis C virus (HCV) develop chronic hepatitis, which in some cases progresses to cirrhosis and ultimately to hepatocellular carcinoma. Although various immunotherapies against the progressive disease status of HCV infection have been studied, a preventive or therapeutic vaccine against this pathogen is still not available. In this study, we constructed a DNA vaccine expressing an HCV structural protein (CN2), non-structural protein (N25) or the empty plasmid DNA as a control and evaluated their efficacy as a candidate HCV vaccine in C57BL/6 and novel genetically modified HCV infection model (HCV-Tg) mice. Strong cellular immune responses to several HCV structural and non-structural proteins, characterized by cytotoxicity and interferon-gamma (IFN-γ) production, were observed in CN2 or N25 DNA vaccine-immunized C57BL/6 mice but not in empty plasmid DNA-administered mice. The therapeutic effects of these DNA vaccines were also examined in HCV-Tg mice that conditionally express HCV proteins in their liver. Though a reduction in cellular immune responses was observed in HCV-Tg mice, there was a significant decrease in the expression of HCV protein in mice administered the N25 DNA vaccine but not in mice administered the empty plasmid DNA. Moreover, both CD8⁺ and CD4⁺ T cells were required for the decrease of HCV protein in the liver. We found that the N25 DNA vaccine improved pathological changes in the liver compared to the empty plasmid DNA. Thus, these DNA vaccines, especially that expressing the non-structural protein gene, may be an alternative approach for treatment of individuals chronically infected with HCV.     

乙型肝炎病毒C基因多态性及其蛋白特性分析

目的 调查中学生人群乙型肝炎病毒(HBV)C基因多态性及其乙型肝炎病毒C蛋白(HBc)特性变化,为了解乙型肝炎病毒核心抗原(HBcAg)变异提供理论依据.方法 采用HBV-C基因特异性引物对23份义乌地区大学生HBV携带者(血清学检测为大三阳患者)血清标本进行PCR扩增鉴定,并取21份PCR检测阳性产物直接测序;进一步用Vector NTI 8.0和DNA Star软件分析HBV-C基因多态性及HBc蛋白特性的变化.结果 23份标本中,HBV-C DNA阳性率91.30％;与标准基因序列(Gen Bank X01587)比较,HBV-C基因同源性为93.3％～95.1％;分别形成54种突变模式,其中一处由于核苷酸的改变,翻译的氨基酸也发生了相应的改变,此处氨基酸序列的第130处脯氨酸(P)突变为苏氨酸(T),其余标本均为同义突变;但突变处编码蛋白在亲水性、表面可及性及抗原性与标准株相比较无明显变化.结论 义乌市大学生HBV携带者HBV-C基因序列有一定程度的变异.     

Improved HIV-1 specific T-cell responses by short-interval DNA tattooing as compared to intramuscular immunization in non-human primates

The new intradermal DNA delivery technique, termed DNA tattooing might overcome the discrepancy between the encouraging immunogenicity results obtained with DNA vaccines in murine studies and the poor results obtained in non-human primates and humans, the so called "simian barrier". Here, we demonstrate a 10- to 100-fold increase in the magnitude of vaccine specific T-cell responses in peripheral blood from DNA tattooed rhesus macaques, as compared to T-cell responses in animals immunized via intramuscular (IM) route. A marked increase in the magnitude of the antigen specific T-cell responses as well as an increase in the number of animals responding to the immunogens was observed. These findings in non-human primates suggest that similar results may be observed in humans. Clinical trials are planned to validate tattooing as an optimal method of DNA vaccine delivery in humans.     

A truncated variant of the hepatitis C virus core induces a slow but potent immune response in mice following DNA immunization

Vaccination of BALB/c mice with pIDKCo, a plasmid containing the coding sequence for the first 176 amino acids of the hepatitis C virus (HCV) core protein, induced both humoral and cellular specific immune responses. Particularly, the level of anti-core antibodies increased slowly with time up to a mean value above 1:8000 that was generally superior than that found in anti-HCV positive individuals. Six out of nine anti-HCV positive human sera were able to inhibit at different extent the binding of mouse anti-core sera to a recombinant capsid protein. Our results show that it is possible to elicit a potent humoral and cellular immune response against the HCV core antigen in mice following DNA immunization.     

Oral immunization with attenuated Salmonella carrying a co-expression plasmid encoding the core and E2 proteins of hepatitis C virus capable of inducing cellular immune responses and neutralizing antibodies in mice

Hepatitis C virus (HCV) core protein has long been considered an attractive candidate for inclusion in a protective vaccine. However, this protein may hamper the development of systemic immune responses because of its immune suppressive properties. We previously reported that immune responses to HCV core protein could be efficiently induced by attenuated Salmonella carrying the HCV core protein, but not the HCV core DNA vaccine. To optimize the combination of the core protein and envelope protein 2 (E2) into a vaccine formulation to induce cellular immune responses and neutralizing antibodies, we constructed a plasmid containing two expression cassettes. One expression cassette was included to regulate the expression of HCV core protein by an inducible in vivo-activated Salmonella promoter, the other was included to regulate the expression of HCV E2 protein by the cytomegalovirus enhancer/promoter. Oral immunization of BALB/c mice with the attenuated Salmonella strain SL7207 carrying this plasmid efficiently induced HCV core and E2-specific cellular immune responses and antibodies. IgG purified from immunized mice could neutralize the infectivity of HCV pseudoparticles (HCVpp) of both the autologous Con 1 isolate and the heterologous H77 isolate, and cell culture produced HCV (HCVcc) of Con1-JFH1 chimera. These results indicated that this vaccine strategy can effectively deliver core and E2 protein to the immune system and provide a promising approach for the development of prophylactic and therapeutic vaccines against HCV infection.     

Induction of protective immunity against toxoplasmosis in mice by DNA immunization with a plasmid encoding Toxoplasma gondii GRA4 gene

GRA4 is a dense granule protein of Toxoplasma gondii that is a candidate for vaccination against this parasite. We have inserted the entire coding sequence of GRA4 into an eukaryotic expression vector to determine whether DNA immunization can elicit protective immune response to T. gondii. Susceptible C57BL/6 mice were then vaccinated intramuscularly with GRA4 DNA and orally challenged with a lethal dose of 76 K T. gondii strain cysts. Immunization with pGRA4 resulted in a 62% survival of C57BL/6 infected mice. Mice immunized with GRA4 DNA developed high levels of serum anti-GRA4 immunoglobulin G antibodies as well as a cellular immune response, as assessed by splenocyte proliferation, in response to recombinant GRA4 protein restimulation in vitro. The cellular immune response was associated with IFN-gamma and IL-10 synthesis, suggesting a modulated Th1-type response. Splenocyte proliferation was strongly enhanced and protection slightly higher by inoculation with GRA4 DNA combined with a granulocyte-macrophage colony-stimulating factor expressing vector. This is the first report that demonstrates the establishment of a DNA vaccine-induced protective immunity against the acute phase of T. gondii infection.     

Antigenicity of hepatitis C virus envelope proteins expressed in Chinese hamster ovary cells

A putative second envelope glycoprotein (E2) of hepatitis C virus (HCV) was constitutively produced in a Chinese hamster ovary cell line stably transformed with a plasmid expressing E2 protein under the control of an exogenous promoter and a signal sequence. E2 protein that lacked part of the C-terminal hydrophobic region was glycosylated with high-mannose type oligosaccharides and retained in the cells. On the other hand, E2 protein lacking the entire C-terminal hydrophobic region was glycosylated with complex type oligosaccharides (complex form) and excreted into the culture medium. Immunoreactivity of the high-mannose and complex forms of E2 proteins against sera from HCV infected patients were analyzed. We found that the antigenicity of the complex form of E2 protein was greater than that of the high-mannose form of E2 protein. This result indicated that the complex form of the E2 protein is superior for use in diagnosing HCV infection.     

Targeted expression of HTLV-I envelope proteins in muscle by DNA immunization of mice

We have compared two types of plasmids for DNA immunization against HTLV-I envelope glycoproteins. One type of plasmid contains the coding DNA of the complete envelope gene of HTLV-I under the control of the CMV promoter with (CMVenvLTR) or without (CMVenv) the tax/rex genes. The second type contains the coding DNA of the complete env gene of HTLV-I under the control of the human desmin muscle specific promoter (DesEnv). These plasmids were inoculated into mice and the humoral response was studied by flow cytometry, ELISA and neutralization assays. Inoculation of the DesEnv construct elicited a higher humoral response with better neutralization properties than the injection of CMVenvLTR or CMVenv plasmids. The choice of vectors will be important for the design of genetic HTLV-I vaccines.     

Protection induced by intramuscular immunization with DNA vaccines of pseudorabies in mice, rabbits and piglets.

Glycoprotein gene gB, gC and gD of pseudorabies virus (PrV) strain Ea, which was isolated locally in Wuhan, were cloned from the viral genome DNA and expressed in vitro controlled by the major immediately-early promotor/enhancer of HCMV. In the presented paper, Balb/c mice, rabbits and piglets were vaccinated intramuscularly two times at 2-week interval with those eukaryotic expression plasmid pcDB, pcDC and pcDD, respectively. The animals injected with pcDB, pcDC, pcDD or mix DNA developed anti-PrV antibodies. Neutralizing antibody titers obtained 2-5log(2), 2 weeks after the second vaccination. Cellular immune responses were also detected by lymphoproliferation assay and cytotoxic T lymphocyte (CTL) activity assay in all groups vaccinated with DNA. Immune responses elicited by DNA vaccines provided protections with different degrees against lethal dose PrV challenge. In mice, protections induced by pcDC, pcDD or mix DNA were 100%, similar to that by inactivated vaccine. Protections were more than 50% induced by pcDC, pcDD or mix DNA in rabbits. Protections induced by pcDB were the lowest among DNA immunization in mice or rabbits. However, pcDB could elicit the higher cellular responses in rabbits or piglets. In piglets, body temperatures of animals injected with pcDB, pcDC, pcDD or mix DNA did not change significantly after challenge with 2x10(5) pfu of PrV strain Ea, and the means daily growth post-challenge of those animals were higher than those injected with inactivated vaccine or parental plasmid. Neither DNA vaccines nor inactivated vaccine could prevent or delay virus excretion after challenge. Our experiments in experimental animals and natural hosts suggested the efficiency and potential application of DNA vaccines for pseudorabies in pigs.     

A single immunization with a plasmid encoding hepatitis C virus (HCV) structural proteins under the elongation factor 1-alpha promoter elicits HCV-specific cytotoxic T-lymphocytes (CTL).

Recent studies have raised the possibility that DNA-based vaccination may prove useful for generating virus-specific cytotoxic T-lymphocytes (CTL) responses. Recently, a plasmid containing the human elongation factor 1alpha(EF1-alpha) promoter, pEF321, was reported to be a versatile expression vector for gene expression in mammalian cells in vitro. In the present study, we assessed the capability of a novel plasmid, pEFCE1E2, encoding hepatitis C virus (HCV) structural proteins (core, E1 and E2) under the EF1-alpha promoter to generate CTL against HCV in vivo. BALB/c mice were immunized with the pEFCE1E2 but not with a plasmid possessing the same cDNA under the cytomegalovirus developed HCV-specific effector cells by a single immunization. These effector cells elicited by pEFCE1E2 immunization were CD8(+) and major histocompatibility complex class I restricted. These studies provided evidence for the potential utility of the EF1-alpha promoter for development of DNA vaccines against HCV infections.