HEVAg-PLGA纳米颗粒疫苗小鼠体内免疫学研究

研究重组戊型肝炎抗原(HEVAg)-乳酸/乙醇酸共聚物(PLGA)纳米颗粒抗原能否在动物体内诱导产生免疫应答。制备HEVAg-PLGA纳米颗粒抗原后,通过皮下、滴鼻、口服途径接种Balb/c小鼠,每隔4周加强免疫两次,HEVAg与铝盐佐剂(铝佐剂疫苗Al_2O_3-Ag)为对照组,一定时间内检测抗体及细胞因子的应答水平。结果HEVAg-PLGA纳米颗粒抗原在小鼠体内诱导产生有效的体液免疫、细胞免疫。滴鼻、口服途径黏膜系统中诱导产生较高滴度的IgA抗体,ELISPOT结果显示鼻腔、唾液腺中IgA ASCs数量显著增加;皮下途径诱导产生较高滴度的IgG抗体;常规铝佐剂疫苗相比于HEVAg-PLGA纳米颗粒抗原诱导较强的IgG抗体水平,未诱导产生黏膜免疫应答;HEVAg-PLGA纳米颗粒抗原诱导产生较强细胞免疫应答,皮下接种途径IFN-γ、IL-4生成细胞数量显著高于其它免疫组。与铝佐剂疫苗相比,HEVAg-PLGA纳米颗粒抗原能有效诱导产生系统免疫及黏膜免疫应答,显示HEVAg-PLGA有潜力成为备选HEV黏膜疫苗抗原,同时展示PLGA颗粒作为黏膜系统抗原递送载体及黏膜佐剂的优越性。     

提高黏膜免疫的方法及研究进展

黏膜是很多病原体入侵机体的重要入口,黏膜疫苗能诱导产生黏膜保护性免疫应答和系统性免疫应答,阻止病原微生物黏附、入侵和繁殖。但多数候选黏膜疫苗的安全性、稳定性、免疫效力及保护作用还无法达到理想的效果,佐剂或载体的使用改善了黏膜疫苗存在的不足,使黏膜疫苗有了广阔的发展前景。文章综述了提高黏膜免疫的方法及研究进展。     

重组腺病毒载体疫苗黏膜免疫机制与途径研究

腺病毒作为载体具有许多优点,因此被广泛地应用于体外基因转导、体内接种疫苗和基因治疗等领域。近年来,国内外学者已经构建了表达不同抗原的腺病毒载体,如人(猿)免疫缺陷病毒Gag,pol,Nef,Env,狂犬病毒糖蛋白,登革热病毒包膜蛋白,乙型肝炎病毒表面抗原,丙型肝炎病毒E1、E2、core、NS3,麻疹病毒核衣壳、凝血素,呼吸道合胞病毒糖蛋白,2型单纯疱疹病毒糖蛋白B、炭疽杆菌保护性抗原等。其中有不少载体疫苗通过黏膜免疫接种,诱导机体产生具有保护作用的免疫反应。本文就重组腺病毒载体疫苗黏膜免疫机制和黏膜免疫途径的研究作一回顾。     

表面表达猪流行性腹泻病毒核蛋白蛋白的重组干酪乳杆菌诱导产生的免疫应答

为探讨表达猪流行性腹泻病毒(PEDV)核蛋白(N)基因的重组干酪乳杆菌口服免疫小鼠后诱导特异性免疫应答,本研究制备表达流行性腹泻病毒核蛋白的重组干酪乳杆菌,应用Western blotting、间接免疫荧光和全细胞ELISA鉴定目的蛋白的表达。然后用该重组干酪乳杆菌口服免疫BALB/c小鼠,分别测定了免疫后不同时间血清中特异性IgG、粪便中特异性的sIgA水平以及血清的中和活性;并测定免疫小鼠脾淋巴细胞增殖情况和细胞因子水平。结果显示,目的蛋白表达在细胞表面,可被阳性血清所识别。免疫小鼠后,可分别在血清中和粪便中检测到较高水平特异性IgG、sIgA(P<0.01),但血清并没有中和活性;淋巴细胞增殖试验和细胞因子测定结果显示,免疫组可产生明显的细胞免疫应答。结果表明,该重组干酪乳杆菌表达系统可诱导小鼠产生黏膜免疫应答和系统免疫应答,具有作为口服疫苗潜在的应用价值。     

乳酸菌用作口服疫苗传递载体的研究

乳酸菌是食品级安全菌,利用乳酸菌为表达载体制成的口服疫苗安全无毒,能诱导机体产生有效的免疫应答和免疫耐受。乳酸菌口服疫苗通过胃肠粘膜进行抗原呈递,使用方便,而且较传统注射途径的免疫效果和依从性好,是理想的疫苗,有广阔的发展前景。     

Prion疾病疫苗研究策略

朊病毒病是一类侵袭人类及多种动物中枢神经系统的致死性退行性脑病,目前缺乏有效的预防和治疗方法。朊病毒病的重组蛋白亚单位疫苗、DNA疫苗、合成肽疫苗、病毒样颗粒疫苗、树突状细胞疫苗、黏膜免疫疫苗等已取得一定进展,但现有的免疫策略仅能部分克服免疫耐受,诱导较低或中等滴度的抗体,对PrPSc感染动物模型只能提供部分保护,Prion疫苗研究任重而道远。     

植物口服疫苗的动物和临床实验*

利用转基因植物生产亚单位疫苗用于口服主动免疫具有安全、廉价和方便等优点。植物可以正确地表达细菌和病毒抗原基因,对动物及人类的临床实验研究表明：食用表达某种抗原的转基因植物可在实验动物或人群体内激起系统免疫和粘膜免疫,产生相应的特异性抗体,这些结果表明了植物口服疫苗的可行性。此外,在治疗自身免疫疾病以及癌症等方面,植物口服疫苗也具有值得关注的作用。     

生物源性基因疫苗的运送系统

基因疫苗具有很多独特的优点,已经成为疫苗研究领域的热点。但由于其免疫原性相对较弱,限制了基因疫苗的广泛应用。人们一直在寻求一种理想的基因疫苗运送系统,它不仅能将基因疫苗导入体内,还能提高基因疫苗的免疫原性,诱导机体产生持续高水平的免疫应答反应。     

细菌菌影疫苗的研究进展

细菌菌影疫苗(Vaccine of bacterial ghost)作为一种新型疫苗,既可诱导机体产生细胞免疫,又能诱导机体产生体液免疫和黏膜免疫。近年来,利用细菌菌影递送载体的特性,构建重组多价疫苗、DNA疫苗及药物递送的应用越来越受到人们的重视。     

Mucosal immunization using recombinant plant-based oral vaccines

The induction of mucosal immunity is very important in conferring protection against pathogens that typically invade via mucosal surfaces. Delivery of a vaccine to a mucosal surface optimizes the induction of mucosal immunity. The apparent linked nature of the mucosal immune system allows delivery to any mucosal surface to potentially induce immunity at others. Oral administration is a very straightforward and inexpensive approach to deliver a vaccine to the mucosal lining of the gut. However, vaccines administered by this route are subject to proteolysis in the gastrointestinal tract. Thus, dose levels for protein subunit vaccines are likely to be very high and the antigen may need to be protected from proteolysis for oral delivery to be efficacious. Expression of candidate vaccine antigens in edible recombinant plant material offers an inexpensive means to deliver large doses of vaccines in encapsulated forms. Certain plant tissues can also stably store antigens for extensive periods of time at ambient temperatures, obviating the need for a cold-chain during vaccine storage and distribution, and so further limiting costs. Antigens can be expressed from transgenes stably incorporated into a host plant's nuclear or plastid genome, or from engineered plant viruses infected into plant tissues. Molecular approaches can serve to boost expression levels and target the expressed protein for appropriate post-translational modification. There is a wide range of options for processing plant tissues to allow for oral delivery of a palatable product. Alternatively, the expressed antigen can be enriched or purified prior to formulation in a tablet or capsule for oral delivery. Fusions to carrier molecules can stabilize the expressed antigen, aid in antigen enrichment or purification strategies, and facilitate delivery to effector sites in the gastrointestinal tract. Many antigens have been expressed in plants. In a few cases, vaccine candidates have entered into early phase clinical trials, and in the case of farmed animal vaccines into relevant animal trials.     

Dendritic cell‐targeting DNA‐based nasal adjuvants for protective mucosal immunity to Streptococcus pneumoniae

To develop safe vaccines for inducing mucosal immunity to major pulmonary bacterial infections, appropriate vaccine antigens (Ags), delivery systems and nontoxic molecular adjuvants must be considered. Such vaccine constructs can induce Ag‐specific immune responses that protect against mucosal infections. In particular, it has been shown that simply mixing the adjuvant with the bacterial Ag is a relatively easy means of constructing adjuvant‐based mucosal vaccine preparations; the resulting vaccines can elicit protective immunity. DNA‐based nasal adjuvants targeting mucosal DCs have been studied in order to induce Ag‐specific mucosal and systemic immune responses that provide essential protection against microbial pathogens that invade mucosal surfaces. In this review, initially a plasmid encoding the cDNA of Flt3 ligand (pFL), a molecule that is a growth factor for DCs, as an effective adjuvant for mucosal immunity to pneumococcal infections, is introduced. Next, the potential of adding unmethylated CpG oligodeoxynucleotide and pFL together with a pneumococcal Ag to induce protection from pneumococcal infections is discussed. Pneumococcal surface protein A has been used as vaccine for restoring mucosal immunity in older persons. Further, our nasal pFL adjuvant system with phosphorylcholine‐keyhole limpet hemocyanin (PC‐KLH) has also been used in pneumococcal vaccine development to induce complete protection from nasal carriage by Streptococcus pneumoniae . Finally, the possibility that anti‐PC antibodies induced by nasal delivery of pFL plus PC‐KLH may play a protective role in prevention of atherogenesis and thus block subsequent development of cardiovascular disease is discussed.

     

Do we need nasal vaccines against COVID 19 to suppress the transmission of infections?

Covid-19 vaccines have within the first year prevented about 14 million deaths but did not induce a strong mucosal immune response. Data from US, UK, Singapore and Israel showed a variable and mostly modest effects of vaccination on virus excretion during breakthrough infections. Contact studies showed decreased transmission of infection from vaccinated index cases, but the effect varied according to dominant virus type, with study type and the nature of the contact group and diminished with time after vaccination. Some researchers suspect that it is unlikely to stop the pandemic with injected vaccines alone. Promising animal experiments were conducted with mucosal vaccines. Mice nasally immunized with a chimpanzee adenovirus vector mounted a mucosal immune response, were protected against viral challenge after a single vaccine dose and suppressed nasal replication of the challenge virus. Phage T4 expressing SARS-CoV-2 spike and nucleocapsid induced a sterilizing lung immunity in nasally vaccinated mice. Also hamsters intranasally immunized with the prefusion-stabilized spike protein showed no infectious virus in nasal turbinates upon challenge. Other studies showed that intranasal vaccination with an adenovirus vaccine reduced but did not eliminated viral transmission from infected to naïve hamsters. Intranasal vaccination of rhesus macaques with adenovirus vaccines also substantially reduced or even suppressed viral replication in the upper and lower respiratory tract. Human data on mucosal SARS-CoV-2 vaccines are so far limited to safety and immunogenicity studies. Aerosolized adenovirus vaccines given either as a booster or as primary immunization were safe and induced similar or superior immune response than injected vaccines while an aerosolized influenza vectored vaccine induced only a weak humoral and cellular immune response. Overall 100 mucosal SARS-CoV-2 vaccines are in development and 20 are in clinical trials. First human trials demonstrate that this will not be an easy task.     

Trans-sialidase recombinant protein mixed with CpG motif-containing oligodeoxynucleotide induces protective mucosal and systemic trypanosoma cruzi immunity involving CD8+ CTL and B cell-mediated cross-priming

The Trypanosoma cruzi trans-sialidase (TS) is a unique enzyme with neuraminidase and sialic acid transfer activities important for parasite infectivity. The T. cruzi genome contains a large family of TS homologous genes, and it has been suggested that TS homologues provide a mechanism of immune escape important for chronic infection. We have investigated whether the consensus TS enzymatic domain could induce immunity protective against acute and chronic, as well as mucosal and systemic, T. cruzi infection. We have shown that: 1) TS-specific immunity can protect against acute T. cruzi infection; 2) effective TS-specific immunity is maintained during chronic T. cruzi infection despite the expression of numerous related TS superfamily genes encoding altered peptide ligands that in theory could promote immune tolerization; and 3) the practical intranasal delivery of recombinant TS protein combined with a ssDNA oligodeoxynucleotide (ODN) adjuvant containing unmethylated CpG motifs can induce both mucosal and systemic protective immunity. We have further demonstrated that the intranasal delivery of soluble TS recombinant Ag combined with CpG ODN induces both TS-specific CD4(+) and CD8(+) T cells associated with vaccine-induced protective immunity. In addition, optimal protection induced by intranasal TS Ag combined with CpG ODN requires B cells, which, after treatment with CpG ODN, have the ability to induce TS-specific CD8(+) T cell cross-priming. Our results support the development of TS vaccines for human use, suggest surrogate markers for use in future human vaccine trials, and mechanistically identify B cells as important APC targets for vaccines designed to induce CD8(+) CTL responses.     

Systemic and mucosal immunity induced by oral somatic transgene vaccination against glycoprotein B of pseudorabies virus using live attenuated Salmonella typhimurium

Glycoprotein B mediates the absorption and penetration of the pseudorabies virus in the form of an immunodominant Ag, and represents a major target for the development of new vaccines. This study evaluated the efficiency of live attenuated Salmonella typhimurium SL7207 for the oral delivery of DNA vaccine encoding the pseudorabies virus glycoprotein B (pCI-PrVgB) in vivo, leading to the generation of both systemic and mucosal immunity against the pseudorabies virus Ag. An oral transgene vaccination of pCI-PrVgB using a Salmonella carrier produced a broad spectrum of immunity at both the systemic and mucosal sites, whereas the intramuscular administration of a naked DNA vaccine elicited no mucosal immunoglobulin (Ig)A response. Interestingly, the Salmonella-mediated oral transgene vaccination of the pseudorabies virus glycoprotein B biased the immune responses to the Th2-type, as determined by the IgG2a/IgG1 ratio and the cytokine production profile. However, oral vaccination mediated by Salmonella harbouring pCI-PrVgB showed inferior protection to systemic immunization against virulent pseudorabies virus infection. The expression of transgene delivered by Salmonella bacteria in antigen-presenting cells of both the systemic and mucosal-associated lymphoid tissues was further demonstrated. These results highlight the potential use of live attenuated S. typhimurium for an oral transgene pseudorabies virus glycoprotein B vaccination to induce broad immune responses.     

Oral bacillus Calmette-Guérin vaccine against tuberculosis: why not?

The bacillus Calmette-Guérin (BCG) vaccine is the only licensed vaccine for human use against tuberculosis (TB). Although controversy exists about its efficacy, the BCG vaccine is able to protect newborns and children against disseminated forms of TB, but fails to protect adults against active forms of TB. In the last few years, interest in the mucosal delivery route for the vaccine has been increasing owing to its increased capacity to induce protective immune responses both in the mucosal and the systemic immune compartments. Here, we show the importance of this route of vaccination in newly developed vaccines, especially for vaccines against TB.     

Vaccine delivery: lipid-based delivery systems

Needle-free delivery of vaccines should not only increase compliance, but should also prove to be a safer and less traumatic method of vaccine delivery. One of the potential ways to achieve needle-free delivery is with the use of lipid-based delivery systems. To demonstrate the utility of these systems, we have shown them to be effective with proteins produced by recombinant DNA technology, plasmid-based vaccines, as well as conventional vaccines. Furthermore, these lipid-based delivery systems were shown to be effective in inducing mucosal immunity if delivered to mucosal surfaces or systemic immunity if different transdermally. These approaches have the potential to revolutionize vaccine delivery in humans and animals.     

Oral tolerance revisited: prior oral tolerization abrogates cholera toxin-induced mucosal IgA responses

Oral delivery of a large dose or prolonged feeding of protein Ags induce systemic unresponsiveness most often characterized as reduced IgG and IgE Ab- and Ag-specific CD4(+) T cell responses. It remains controversial whether oral tolerance extends to diminished mucosal IgA responses in the gastrointestinal tract. To address this issue, mice were given a high oral dose of OVA or PBS and then orally immunized with OVA and cholera toxin as mucosal adjuvant, and both systemic and mucosal immune responses were assessed. OVA-specific serum IgG and IgA and mucosal IgA Ab levels were markedly reduced in mice given OVA orally compared with mice fed PBS. Furthermore, when OVA-specific Ab-forming cells (AFCs) in both systemic and mucosa-associated tissues were examined, IgG AFCs in the spleen and IgA AFCs in the gastrointestinal tract lamina propria of mice given OVA orally were dramatically decreased. Furthermore, marked reductions in OVA-specific CD4(+) T cell proliferative and cytokine responses in spleen and Peyer's patches were seen in mice given oral OVA but were unaffected in PBS-fed mice. We conclude that high oral doses of protein induce both mucosal and systemic unresponsiveness and that use of mucosal adjuvants that induce both parenteral and mucosal immunity may be a better way to assess oral tolerance.     

Upper respiratory tract mucosal immunity for SARS-CoV-2 vaccines

SARS-CoV-2 vaccination significantly reduces morbidity and mortality, but has less impact on viral transmission rates, thus aiding viral evolution, and the longevity of vaccine-induced immunity rapidly declines. Immune responses in respiratory tract mucosal tissues are crucial for early control of infection, and can generate long-term antigen-specific protection with prompt recall responses. However, currently approved SARS-CoV-2 vaccines are not amenable to adequate respiratory mucosal delivery, particularly in the upper airways, which could account for the high vaccine breakthrough infection rates and limited duration of vaccine-mediated protection. In view of these drawbacks, we outline a strategy that has the potential to enhance both the efficacy and durability of existing SARS-CoV-2 vaccines, by inducing robust memory responses in the upper respiratory tract (URT) mucosa.     

Evaluation of Mucosal and Systemic Immune Responses Elicited by GPI-0100- Adjuvanted Influenza Vaccine Delivered by Different Immunization Strategies

Vaccines for protection against respiratory infections should optimally induce a mucosal immune response in the respiratory tract in addition to a systemic immune response. However, current parenteral immunization modalities generally fail to induce mucosal immunity, while mucosal vaccine delivery often results in poor systemic immunity. In order to find an immunization strategy which satisfies the need for induction of both mucosal and systemic immunity, we compared local and systemic immune responses elicited by two mucosal immunizations, given either by the intranasal (IN) or the intrapulmonary (IPL) route, with responses elicited by a mucosal prime followed by a systemic boost immunization. The study was conducted in BALB/c mice and the vaccine formulation was an influenza subunit vaccine supplemented with GPI-0100, a saponin-derived adjuvant. While optimal mucosal antibody titers were obtained after two intrapulmonary vaccinations, optimal systemic antibody responses were achieved by intranasal prime followed by intramuscular boost. The latter strategy also resulted in the best T cell response, yet, it was ineffective in inducing nose or lung IgA. Successful induction of secretory IgA, IgG and T cell responses was only achieved with prime-boost strategies involving intrapulmonary immunization and was optimal when both immunizations were given via the intrapulmonary route. Our results underline that immunization via the lungs is particularly effective for priming as well as boosting of local and systemic immune responses.