Elicitation of strong immune responses by a DNA vaccine expressing a secreted form of hepatitis C virus envelope protein E2 in murine and porcine animal models

INTRODUCTION Hepatitis C continues to be a severe health threat to a large population with about 123 million people being affected globally[1]. The etiologic agent, hepatitis C virus (HCV)[2] is able to establish persistent infections in up to 85% of infe…     

Comparative efficacy of the Schistosoma mansoni nucleic acid vaccine,Sm23, following microseeding or gene gun delivery

Sm23 is an integral membrane protein expressed widely in the human parasitic worm Schistosoma mansoni. Sm23 has already been shown to elicit protective immune responses following immunization with peptides or DNA constructs. In this study, we evaluated the immunogenicity and the protective efficacy of the Sm23 DNA vaccine using two different intradermal DNA delivery methods: microseeding and gene gun. Using both techniques, all mice immunized with the Sm23-pcDNA construct generated Sm23-specific immunoglobulin (Ig)G antibody, while mice immunized with the control plasmid, pcDNA, did not. Antibody isotypes analysis revealed that microseeding elicited mainly IgG2a and IgG2b antibodies, with relatively low levels of IgG1 and IgG3. The relative IgG1/IgG2a ratio was 0.03, indicative of a Th1 type immune response. In contrast, gene gun immunization resulted in significantly higher levels of IgG1 and IgG3. The relative IgG1/IgG2a ratio in this case was 11, indicative of a Th2 type immune response. No significant difference in the levels of IgG2b was observed. Coimmunization with plasmid DNA encoding either interleukin (IL)-12 or IL-4 by microseeding did not affect the levels of IgG1, while the levels of IgG2a and IgG2b were reduced. On the other hand, the levels of IgG3 were significantly increased by IL-4, but unchanged by IL-12. Importantly, in all experiments, the Sm23-pcDNA vaccine provided statistically significant levels of protection against challenge infection. Microseeding immunizations resulted in higher levels of protection (31-34% protection) than gene gun immunization (18% protection). This suggests that the Th1 type immune response elicited by microseeding immunization was responsible for the higher protection levels. However, the protective effect of the vaccine was not affected by coadministering plasmids encoding either IL-12 or IL-4 using the microseeding technique.     

DNA immunization with Onchocerca volvulus genes, Ov-tmy-1 and OvB20: serological and parasitological outcomes following intramuscular or GeneGun delivery in a mouse model of onchocerciasis

The objective of this study was to evaluate whether the distinct immune responses invoked by epidermal and intramuscular DNA immunization could be harnessed to improve upon the levels of protection to Onchocerca volvulus infective larvae achieved previously by recombinant protein immunization. Intramuscular (IM) and epidermal (GeneGun) routes of DNA immunization generally drive T helper1 and Th2 dominant responses, respectively. This dichotomy was used in an attempt to further define the nature of host-protective immunity in a mouse model of onchocerciasis. Mice were immunized with DNA plasmids expressing the O. volvulus antigens, Ov-TMY-1 (tropomyosin) and OvB20 (a nematode specific gene product). While, IM and GeneGun immunization of mice with Ov-tmy-1 induced expected Th1/Th2-associated IgG isotype profiles, mice responded to OvB20 immunization with a Th2 dominant response, irrespective of the delivery route. Despite inducing potent serological responses, neither DNA construct promoted statistically significant levels of protection to L3 challenge infection. We conclude that DNA immunization has good potential for induction of humoral responses against nematode infections and that serological responses alone do not predict vaccination efficacy under the conditions used here to measure host resistance to parasite challenge.     

汉滩病毒核酸疫苗滴鼻及皮肤划痕免疫小鼠的比较研究

目的观察汉滩病毒的DNA疫苗滴鼻及皮肤划痕免疫诱导机体产生的免疫应答,探索汉滩病毒DNA疫苗新的免疫途径。方法采用不同剂量50μg、10μg、1μg的裸露质粒pcDNA3.1B-S1,3分别进行滴鼻及皮肤划痕免疫小鼠,采用ELISA方法分别检测血清及粪便中特异性抗体变化,观察其诱导的系统和黏膜免疫反应的差异。结果用汉滩病毒核酸疫苗免疫小鼠,通过表皮划痕方式其诱导体液免疫在50μg剂量时与滴鼻途径相当,在10μg及1μg低剂量时优于滴鼻途径,但在黏膜免疫方面,其明显不如滴鼻途径。结论滴鼻免疫对特异的黏膜免疫激发作用明显优于皮肤划痕,疫苗的滴鼻免疫途径较皮肤划痕有着明显的优势。     

致肾盂肾炎大肠埃希菌粘附素重组蛋白诱导小鼠免疫应答

目的获得UPECP菌毛粘附素PapG重组蛋白纯化产物,研究其诱导小鼠的免疫应答,为进一步的PapG疫苗及免疫学研究创造条件。方法GST-PapG重组蛋白经诱导表达和亲和层析纯化后接种于BALB/c小鼠;ELISA法检测小鼠免疫血清的抗体效价,以血凝抑制试验测定其免疫反应性。结果重组蛋白纯化产物可诱导小鼠产生针对PapG粘附素的特异性免疫应答,免疫小鼠血清抗体的效价显著升高,而且具有较强的血凝抑制作用。结论GST-PapG重组蛋白纯化产物具有良好的免疫原性,可用于UPEC抗粘附候选疫苗的筛选和进一步的免疫学研究。     

日本脑炎病毒DNA疫苗和基因工程亚单位颗粒疫苗的动物保护实验研究

目的　评价 3种日本脑炎病毒 (JEV)新型疫苗 ,即E蛋白病毒样颗粒 (VLPs)、DNA疫苗 (pV/JE)以及E蛋白颗粒 (Eps)的动物免疫效果。方法　BALB/c小鼠经 2次接种疫苗后 ,测定其病毒特异性的CTL活性、结合抗体、中和抗体 ,并以10LD5 0JEV攻击免疫小鼠 ,观察其保护效果。结果　各新型疫苗组的CTL活性为 33%～ 5 6 % ,灭活疫苗组为 2 2 % ,而载体和PBS对照组的CTL活性分别为 17%和 18%。一次免疫后 ,实验组小鼠血清结合抗体阳转率为 90 %。除DNA疫苗组外 ,各组小鼠的二次免疫血清抗体效价高于一次免疫的抗体效价 (P <0 0 5 ) ,且以VLPs加佐剂组小鼠血清抗体效价最高 ,Eps加佐剂组次之 ,均高于 pV/JE组和灭活疫苗组 (P <0 0 5 )。三种疫苗可诱发小鼠产生中和抗体。各实验组小鼠能够抵抗JEV的攻击 ,而对照组小鼠的死亡率为 37 5 %。结论　三种新型疫苗可以正确表达JEV的E蛋白表位 ,可诱发免疫小鼠产生抗JEV的中和抗体并有保护性作用 ,免疫效果优于灭活疫苗 ,有可能成为候选的JEV新型疫苗。     

乙型肝炎病毒核心区DNA疫苗接种后H-2d小鼠的特异性免疫应答

目的观察乙型肝炎病毒（ＨＢＶ）核心区ＤＮＡ疫苗接种后ＢＡＬＢ／ｃ（－２ｄ）小鼠的特异性免疫应答。方法构建ＨＢＶ核心区ＤＮＡ疫苗（ＰＪＷ４３０３／ＨＢｃ）;用基因枪法和肌肉注射祛将该ＤＮＡ疫苗接种ＢＡＬＢ／ｃ小鼠;ＥＬＩＳＡ法检测小鼠血清抗－ＨＢＣ（ＩｇＧ）及ＩｇＧ亚类（ＩｇＧ１,ＩｇＧ２ａ）;５１铬释放法检测小鼠脾细胞ＨＢｃＡｇ特异性ＣＴＬ活性。结果该ＤＮＡ疫苗在体外转染细胞中可良好表达ＨＢｃＡｇ。血清抗－ＨＢｃ终点滴度在ＤＮＡ疫苗基因枪组和肌肉注射接种组小鼠分别为１：３２８０５０和１：１０９３５０．两组小鼠的抗－ＨＢｃＩｇＧ亚类均以ＩｇＧ２ａ略占优势。两组小鼠的ＨＢＣＡｔｈｅ异性ＣＴＬ杀伤活性分别达到５１．１％和５５．２％。结论ＨＢＶ核心区ＤＮＡ疫苗在小鼠实验中具有良好的细胞免疫和体液免疫原性。     

HIV-1CN54合成gp120基因(syngp120)的DNA疫苗鼻内免疫小鼠诱导全身的和粘膜的免疫应答

目的 初步探讨HIV-1CN54合成gp120基因的DNA疫苗（pcDNA3.1-syngp120）鼻内接种小鼠是否诱发免疫应答。方法　DNA疫苗免疫后,制备脾和肠系膜淋巴结（MLN）淋巴细胞,在体外测其增殖应答和CD8+CTL应答。间接ELISA法测血清和粘膜洗液抗原-特异的IgG和IgA抗体滴度。中和实验测免疫血清和阴道洗液是否中和 HIV-1SF33。结果 在 DNA疫苗未次免疫后,小鼠第 1周检测到脾和 MLN CD8+CTL应答较弱,而第 5周未检测到。另外,在末次免疫后的第1、5周检测到MLN而未检测到脾淋巴细胞发生增殖应答。并且检测到特异的血清IgG抗体和粘膜的（包括粪便和阴道洗液）IgA抗体,但未检测到血清的IgA抗体和粘膜的（包括粪便和阴道洗液）IgG抗体。中和实验发现末次免疫后第5周的血清能中和实验室毒株HIV-1SF33（B亚型）,而阴道洗液则没有。结论 该DNA疫苗鼻内免疫小鼠可诱导粘膜免疫应答,包括 MLN淋巴细胞增殖应答和 CD8+CTL应答,同时诱导较弱的粘膜IgA抗体应答。此外,能诱导脾CD8+CTL应答和血清IgG抗体应答。免疫血清中和HIV-1S F33,而阴道洗液不能。     

DNA-Based immunization produces Th1 immune responses to hepatitis delta virus in a mouse model

Hepatitis delta virus (HDV) superinfection is one of the major causes of fulminant hepatitis in endemic areas of hepatitis B virus (HBV) infection. Currently, there is no effective treatment or vaccine against HDV superinfection. DNA-based immunization is a promising antiviral strategy to prevent or treat persistent viral infections. In this study, we investigated the immunological effects of DNA vaccines against HDV in BALB/c mice. Plasmid (pD) encoding large hepatitis D antigen (L-HDAg), or plasmid (pS/pD) coexpressing hepatitis B surface antigen (HBsAg) and L-HDAg, were injected into mice intramuscularly. The seroconversion rate, anti-HBs levels, anti-HDV titers, T-cell proliferation responses, and T-helper (Th)-release cytokine profiles were analyzed. Mice immunized with plasmids, pS/pD or pD, produced low, but significant, titers of anti-HDV antibodies. In contrast, pS/pD induced much stronger anti-HBs titers in the immunized animals. Interestingly, splenic lymphocytes derived from pS/pD-inoculated mice demonstrated significant proliferation responses to recombinant HBsAg and HDAg, and resulted in a Th1-like immune response as suggested by the production of interferon gamma (INF-gamma) and interleukin-2 (IL-2), but not IL-4. The splenic lymphocyte derived from the pD-inoculated mice showed a similar Th1 response to the stimulation of HDAg, but not to HBsAg. In conclusion, our results suggest that DNA vaccines against HDV can induce significant cellular immune responses with a Th1 preference. HBV and HDV coimmunization can be performed by DNA vaccines. These results are promising for the future development of prophylactic and therapeutic HDV vaccines.     

汉滩病毒核酸疫苗滴鼻免疫小鼠效果的实验

目的　观察汉滩病毒DNA疫苗滴鼻免疫诱导机体产生的免疫应答 ,探索汉滩病毒DNA疫苗新的免疫途径。方法　用重组质粒PcDNA3 1B -S1 3 经滴鼻接种BALB/c小鼠 ,采用ELISA、淋巴细胞转化试验及流式细胞仪等方法检测了其诱导的系统和粘膜免疫反应。结果　免疫小鼠血清IgG及粪便IgA明显增高 (P <0 0 1) ,且IgA增幅较大 ;实验组淋巴细胞增殖反应明显 ,刺激指数 (SI)显著增大 (P <0 0 5 ) ;免疫后CD+ 4 、CD+ 8T细胞均增加 (P <0 0 1) ,而CD+ 4 /CD+ 8T细胞比值无显著变化 (P >0 0 5 )。结论　汉滩病毒重组质粒滴鼻免疫能诱导机体产生了特异的系统免疫和较强的粘膜免疫 ,滴鼻的疫苗免疫途径有着潜在的应用价值。     

Nasal immunization induces Haemophilus influenzae-specific Th1 and Th2 responses with mucosal IgA and systemic IgG antibodies for protective immunity.

To determine the efficacy of a mucosal vaccine against nontypeable Haemophilus influenzae (NTHi), mice were immunized nasally, orally, intratracheally, or intraperitoneally with NTHi antigen together with cholera toxin. Antigen-specific IgA antibody titers in nasal washes and the numbers of antigen-specific IgA-producing cells in nasal passages showed the greatest increases in mice immunized nasally. Cytokine analysis showed that interferon-gamma, interleukin (IL)-2, IL-5, IL-6, and IL-10 were induced by nasal immunization, suggesting that Th2- and Th1-type cells were generated. Furthermore, bacterial clearance of a homologous strain of NTHi from the nasal tract was significantly enhanced in the nasal immunization group. These findings suggest that nasal immunization is an effective vaccination regimen for the induction of antigen-specific mucosal immune responses, which reduce the colonization of NTHi in the nasal tract.     

DNA immunization encoding the secreted nonstructural protein 3 (NS3) of hepatitis C virus and enhancing the Th1 type immune response

To induce a sustained and specific cellular immune response to hepatitis C virus (HCV), DNA immunization of mice was performed using plasmids containing the HCV nonstructural gene 3 (HCV/NS3). Plasmids were constructed such that the NS3 gene was expressed in a secreted form, a nonsecreted form or as a membrane-bound antigen. The plasmid encoding the secreted antigen induced the strongest humoral and cellular immunity and favoured the T-helper type 1 (Th1) pathway as shown by cytokine profiles and switching of antibody subclasses. Our study indicates that DNA immunization with a secreted form of HCV/NS3 is an effective means of inducing primary Th1 immune responses in the murine model.     

CpG DNA can induce strong Th1 humoral and cell-mediated immune responses against hepatitis B surface antigen in young mice

Successful neonatal immunization of humans has proven difficult. We have evaluated CpG-containing oligonucleotides as an adjuvant for immunization of young mice (1–14 days old) against hepatitis B virus surface antigen. The protein-alum-CpG formulation, like the DNA vaccine, produced seroconversion of the majority of mice immunized at 3 or 7 days of age, compared with 0–10% with the protein-alum or protein-CpG formulations. All animals, from neonates to adults, immunized with the protein-alum vaccine exhibited strong T helper (Th)2-like responses [predominantly IgG1, weak or absent cytotoxic T lymphocytes (CTL)]. Th2-type responses also were induced in young mice with protein-CpG (in 1-, 3-, and 7-day-old mice) and protein-alum-CpG (in 1- and 3-day-old mice) but immunization carried out at older ages gave mixed Th1/Th2 (Th0) responses. DNA vaccines gave Th0-like responses when administered at 1 and 7 days of age and Th1-like (predominantly IgG2a and CTL) responses with 14-day-old or adult mice. Surprisingly, the protein-alum-CpG formulation was better than the DNA vaccine for percentage of seroconversion, speed of appearance, and peak titer of the antibody response, as well as prevalence and strength of CTL. These findings may have important implications for immunization of human infants.     

Potential advantages of DNA immunization for influenza epidemic and pandemic planning.

Immunization with purified DNA is a powerful technique for inducing immune responses. The concept of DNA immunization involves insertion of the gene encoding the antigen of choice into a bacterial plasmid and injection of the plasmid into the host where the antigen is expressed and where it induces humoral and cellular immunity. The most effective routes and methods for DNA immunization are bombardment with particles coated with DNA ("gene gun" technique), followed by the intramuscular and intradermal routes. DNA immunization technology has the potential to induce immunity to all antigens that can be completely encoded in DNA, which therefore include all protein, but not carbohydrate, antigens. DNA immunization results in presentation of antigens to the host's immune system in a natural form, like that achieved with live-attenuated vaccines. The DNA immunization strategy has the potential to rapidly provide a new vaccine in the face of an emerging influenza pandemic.     

Preferential induction of a Th1 immune response and inhibition of specific IgE antibody formation by plasmid DNA immunization.

We compared the antigen-specific antibody isotypes and lymphokine secretion by CD4+ T cells in BALB/c mice immunized intradermally with either Escherichia coli beta-galactosidase (beta-gal) or plasmid DNA (pDNA) encoding beta-gal in a cytomegalovirus-based expression vector (pCMV-LacZ). pCMV-LacZ induced mainly IgG2a, whereas beta-gal in saline or alum induced IgG1 and IgE beta-gal-specific antibodies. In addition, splenic CD4+ T helper (Th) cells isolated from pDNA-immunized mice secreted interferon-gamma but not interleukin (IL)-4 and IL-5, whereas Th cells from beta-gal-injected mice secreted IL-4 and IL-5 but not interferon-gamma after in vitro stimulation with antigen. Together these data demonstrate that pDNA immunization induced a T helper type 1 (Th1) response, whereas protein immunization induced a T helper type 2 (Th2) response to the same antigen. Interestingly, priming of mice with pCMV-LacZ prevented IgE antibody formation to a subsequent i.p. beta-gal in alum injection. This effect was antigen-specific, because priming with pCMV-LacZ did not inhibit IgE anti-ovalbumin antibody formation. Most importantly, intradermal immunization with pCMV-LacZ (but not pCMV-OVA) of beta-gal in alum-primed mice caused a 66-75% reduction of the IgE anti-beta-gal titer in 6 weeks. Also, pCMV-LacZ induced specific IgG2a antibody titers and interferon-gamma secretion by Th cells in the beta-gal in alum-primed mice. The data demonstrate that gene immunization induces a Th1 response that dominates over an ongoing protein-induced Th2 response in an antigen-specific manner. This suggests that immunization with pDNA encoding for allergens may provide a novel type of immunotherapy for allergic diseases.     

Protective efficacy against respiratory syncytial virus following murine neonatal immunization with BBG2Na vaccine: influence of adjuvants and maternal antibodies

Alum-adsorbed BBG2Na, a recombinant vaccine derived in part from the respiratory syncytial virus (RSV) subgroup A G protein, induced moderate antibody titers after 1 immunization in 1-week-old mice but conferred complete lung protection upon RSV challenge. The anti-BBG2Na IgG1-IgG2a neonatal isotype profile was suggestive of dominant Th2 responses compared with those in adults. Formulation of BBG2Na with a Th1-driving adjuvant efficiently shifted neonatal responses toward a more balanced and adultlike IgG1-IgG2a profile without compromising its protective efficacy. BBG2Na-induced protective immunity was maintained even after early life immunization in the presence of high titers of maternal antibodies. Under these conditions, the protective efficacy (86%-100%) reflected the high capacity of the nonglycosylated G2Na immunogen to escape inhibition by RSV-A-induced maternal antibodies. Thus, immunization with BBG2Na protected against viral challenge despite neonatal immunologic immaturity and the presence of maternal antibodies, two major obstacles to neonatal RSV vaccine development.     

Synergistic effect of combined HIV/HCV immunogens: a combined HIV-1/HCV candidate vaccine induces a higher level of CD8+ T cell-immune responses in HLA-A2.1 mice

Dual infections with HIV-1 and Hepatitis C virus (HCV) may proceed in concert to cause severe disease. HIV positive individuals that become infected with HCV advance more rapidly to AIDS than those that are infected with HIV-1 alone. In this study, HLA-A2.1 mice were immunized with a combination vaccine including HIV and HCV immunogens (polycistronic DNA + proteins) or vaccine containing either HIV or HCV immunogens. Mice immunized with the combined HIV/HCV regimen had similar antibody titers as the group receiving either the HIV-1 or HCV only regimen. Proliferative immune responses showed that mice receiving the combined HIV/HCV vaccine exhibited a three fold higher stimulation index (SI) to gp120 than mice immunized with the vaccine containing HIV alone. To determine whether our vaccine strategy induced Th1 or Th2 immune responses, IFN-gamma and IL-4/IL-5 were measured. The combined HIV/HCV vaccine induced a higher level of Th1 responses to HIV-1 gag protein compared with the other groups, as measured by IFN-gamma production. Interestingly, detection of IFN-gamma by ELISPOT assay demonstrated that the combined HIV/HCV vaccine group had increased numbers of spot forming cells (SFC) to HIV-gp120 peptides when compared to that of the HIV-1 only vaccine group. The combined HIV/HCV vaccine group also showed an increase in SFC to HCV-core peptides in comparison with the group receiving the HCV only vaccine. Intracellular IFN-gamma staining confirmed the ELISPOT results and demonstrated that the combined HIV/HCV group had significantly higher percentages of HIV and HCV-specific CD8+ T cells in comparison to the groups receiving the HIV or HCV vaccines. These results suggest a new approach to maximize vaccine efficacy against HIV and HCV.     

Capric acid and hydroxypropylmethylcellulose increase the immunogenicity of nasally administered peptide vaccines

Immunization by the nasal route is an established method for the induction of mucosal and systemic humoral and cell-mediated antigen-specific responses. However, the effectiveness of nasal immunization is often hampered by the need for increased doses of antigen. Bioadhesives and absorption enhancers were investigated for their ability to enhance immune responses in mice after nasal immunization with model HIV-1 peptide and protein immunogens. Two additives, hydroxypropylmethylcellulose (HPMC) and capric acid, consistently enhanced antigen-specific serum IgG endpoint titers under conditions in which antigen dose was limiting. Nasal immunization of mice with 20 microg of an HIV-1 peptide immunogen plus cholera toxin (CT) as adjuvant induced serum antipeptide IgG titers of 1:9.5log2 after four immunizations while the addition of CA or HPMC to the vaccine formulation increased serum antipeptide IgG titers to 1:15.4log2 and 1:17.6log2, respectively. When 5 microg recombinant HIV-1 gp41 was used as the immunogen, the addition of CA or HPMC to the vaccine formulation increased serum anti-gp41 IgG titers to 1:11.6log2 and 1:8.8log2, respectively, compared to 1:5.2log2 after three nasal immunizations with 5 microg gp41 + CT alone. Thus, HPMC and capric acid may be useful additives that increase the immunogenicity of nasally administered vaccines and permit less antigen to be used with each immunization.