Prospects and achievements of genetic engineering in development of antiviral vaccines

Proceeding from the known data various theoretical and experimental approaches to the construction of gene-engineering vaccines are considered. Gene-engineering subunit vaccines of the first generation are based on isolation of the genes coding for the synthesis of full length capsid proteins with the main antigenic determinants and their subsequent expression in suitable recipient cells. Initial idea of the microbiological synthesis as the main way for production of any antiviral vaccines was not confirmed by the later development. Now for this type of vaccines eucaryotic systems are widely employed using the animal virus vectors and the animal cell cultures. Gene-engineering subunit vaccine of the second generation appears to be a chimeric protein with built-in antigenic determinants of different viruses and maximal immunogenicity in monomeric form. The last point reopens the perspective to use a microbiological synthesis for the production of antiviral vaccines. Besides that the chemically synthesized polypeptide antiviral vaccine will be used widely. In gene-engineering subunit vaccines of the third generation it is possible to use not the natural antigenic determinants which often are characterized by high level of the primary structure changes but artificial (non-natural) antigens, that are the capsid protein conservative regions which under natural conditions of infection or immunization do not induce the protective antiviral antibodies. The recombinant DNA technology in addition to subunit type vaccine allows to construct living vaccines which represent a DNA-containing attenuated virus with build-in natural or synthetic gene of the capsid or chimeric protein with antigenic determinants of another viral species.     

Prevention of disease induced by a partially heterologous AIDS virus in rhesus monkeys by using an adjuvanted multicomponent protein vaccine

Recombinant protein subunit AIDS vaccines have been based predominantly on the virus envelope protein. Such vaccines elicit neutralizing antibody responses that can provide type-specific sterilizing immunity, but in most cases do not confer protection against divergent viruses. In this report we demonstrate that a multiantigen subunit protein vaccine was able to prevent the development of disease induced in rhesus monkeys by a partially heterologous AIDS virus. The vaccine was composed of recombinant human immunodeficiency virus type 1 (HIV-1) gp120, NefTat fusion protein, and simian immunodeficiency virus (SIV) Nef formulated in the clinically tested adjuvant AS02A. Upon challenge of genetically unselected rhesus monkeys with the highly pathogenic and partially heterologous SIV/HIV strain SHIV(89.6p) the vaccine was able to reduce virus load and protect the animals from a decline in CD4-positive cells. Furthermore, vaccination prevented the development of AIDS for more than 2.5 years. The combination of the regulatory proteins Nef and Tat together with the structural protein gp120 was required for vaccine efficacy.     

新型冠状病毒亚单位疫苗研制及其高效免疫增强剂的筛选

旨为推动新型冠状病毒（2019-nCoV）亚单位疫苗的开发并探索筛选适用于该疫苗的高效免疫增强剂,本试验分别诱导表达2019-nCoV- S和2019-nCoV- N两个蛋白,经纯化后测定目的蛋白含量,并分别加入不同浓度的松花粉多糖（PPPS）（200、400、800 mg/mL）作为佐剂制备基因工程亚单位疫苗,黄芪多糖佐剂作为对照。选取100只SPF级BALB/c小鼠随机分为10组,每种亚单位疫苗使用5组,免疫疫苗后检测各组小鼠免疫指标,探讨PPPS对2019-nCoV- S和2019-nCoV- N两种亚单位疫苗的免疫增强效果。结果显示,重组表达并纯化的目的蛋白分别在55.68 kD和45.64 kD处出现单一条带,经鉴定表达正确,蛋白质量浓度分别为1.12 mg/mL和0.66 mg/mL。用制作成的含佐剂亚单位疫苗免疫小鼠后发现400 mg/mL PPPS对两种疫苗的免疫效果均有显著的提升效果,并且效果优于黄芪多糖佐剂。综上所述,两种重组蛋白均能诱导较高的抗体水平,PPPS可以作为2019-nCoV亚单位疫苗的候选佐剂。     

Vaccination of dogs against Echinococcus granulosus: a means to control hydatid disease?

Hydatid disease continues to be a substantial cause of morbidity and mortality worldwide. Elimination is difficult with current control options, but reducing egg production by Echinococcus granulosus (Eg) in canines might help to reduce transmission in areas where the parasite is endemic. Recently obtained data using recombinant protein-based and live attenuated Salmonella vaccines are preliminary but encouraging and auger well for the future development of an effective dog vaccine against Eg, although much additional work is required before this becomes a reality. New approaches to control and for the prevention of hydatidosis have been described recently, and important additional gains should be expected if the efficacy of the dog vaccines is confirmed and leads to their incorporation into future control options.     

全球结核病疫苗研究进展

本文旨在对全球结核病疫苗研究进展进行系统综述,描述国际上目前进入临床试验不同阶段的新型疫苗,包括重组卡介苗、亚单位疫苗、治疗性疫苗等,分析我国结核病疫苗研究现状,介绍国际研究发展趋势,如人类疫苗计划、全细胞疫苗、多阶段疫苗等,并对存在的问题和挑战进行讨论,展望未来发展趋势。     

SARS亚基疫苗-突刺蛋白受体结合区RBD在烟草叶绿体中的高效表达

叶绿体表达系统为植物源重组药用蛋白和亚基疫苗的生产提供了一个有效的途径。为验证SARS亚基疫苗在叶绿体中表达的可行性,以及为植物源SARS亚基疫苗的生产提供一套高效、低成本的技术平台,本研究将人工优化合成的SARS-CoV突刺蛋白(S蛋白)受体结合区序列RBD与载体分子CTB融合基因导入烟草叶绿体基因组中。PCR和Southern杂交分析表明,外源融合基因已整合到烟草叶绿体基因组中,并获得同质化。Western杂交分析表明,重组融合蛋白CTB-RBD在叶绿体转基因烟草中获得表达,且主要以可溶性单体形式存在。ELISA分析表明,在不同生长阶段、不同生长部位和不同时间点烟草叶片中,重组融合蛋白CTB-RBD的表达水平呈现明显的变化。重组蛋白在成熟叶片中的表达水平最高可以达到10.2%TSP。本研究通过SARS亚基疫苗RBD在烟草叶绿体中的高效表达,有望为植物源SARS亚基疫苗的生产以及SARS血清抗体的检测提供一个有效的技术平台。     

Identification of antigens for the development of a subunit vaccine against Photobacterium damselae ssp. piscicida

Photobacterium damselae ssp. piscicida (Ph.d.p.), the causative agent of photobacteriosis, is among the most important pathogens affecting finfish aquaculture globally. With the emergence of recombinant technology, subunit vaccines have been actively pursued, but mostly for viral diseases. Bacterial subunit vaccines are more difficult to develop since the bacterial genome is more complex, with numerous candidate antigens, leading to a lengthy and laborious screening process. Immunoproteomics, using western blotting on protein analyzed with 2DE and LC-MS/MS to isolate immune-reactive proteins and acquire amino acid sequences, followed by recombinant technology to clone the candidate gene, identified eight candidate antigens from Ph.d.p., which have been cloned and expressed in Escherichia coli BL21(DE3). These proteins were purified and used as antigens in an efficacy trial. Three, rHSP60, rENOLASE, and rGAPDH proteins, elicited higher specific antibody titers and stronger protective immunity than the other five and an inactivated Ph.d.p. whole bacterial vaccine. These three antigens may be candidates for the development of a subunit vaccine against Ph.d.p.     

结核病免疫机制及疫苗研究进展

结核病是一种棘手的重大传染病.虽然存在一些有一定疗效的治疗药物,亦有预防性疫苗--卡介苗(BCG);但结核病仍在世界范围流行,且发病率和病死率居高不下.结核病的免疫病理机制及疫苗研究近年来取得了一定的进展.结核分枝杆菌通过Toll样受体(TLR)等模式识别受体,激活巨噬细胞的天然免疫反应,清除细菌和调节获得性免疫反应....     

Chemical and biological conjugation strategies for the development of multivalent protein vaccine nanoparticles

The development of subunit vaccine platforms has been of considerable interest due to their good safety profile and ability to be adapted to new antigens, compared to other vaccine typess. Nevertheless, subunit vaccines often lack sufficient immunogenicity to fully protect against infectious diseases. A wide variety of subunit vaccines have been developed to enhance antigen immunogenicity by increasing antigen multivalency, as well as stability and delivery properties, via presentation of antigens on protein nanoparticles. Increasing multivalency can be an effective approach to provide a potent humoral immune response by more strongly engaging and clustering B cell receptors (BCRs) to induce activation, as well as increased uptake by antigen presenting cells and their subsequent T cell activation. Proper orientation of antigen on protein nanoparticles is also considered a crucial factor for enhanced BCR engagement and subsequent immune responses. Therefore, various strategies have been reported to decorate highly repetitive surfaces of protein nanoparticle scaffolds with multiple copies of antigens, arrange antigens in proper orientation, or combinations thereof. In this review, we describe different chemical bioconjugation methods, approaches for genetic fusion of recombinant antigens, biological affinity tags, and enzymatic conjugation methods to effectively present antigens on the surface of protein nanoparticle vaccine scaffolds.     

对研发新型结核病疫苗的分析和展望

结核病是公共卫生当前面临的重要问题。由于BCG预防效果不佳,研究和开发新型结核病疫苗显得必须且急迫。新型结核病疫苗的研究开发路径和观念也经历了变迁,当前主流的研发路径有重组BCG或重组结核菌、重组痘病毒或重组腺病毒载体疫苗、蛋白质亚单位或重组融合蛋白质亚单位疫苗三类,它们在疫苗效力前景,抗原选型、配方、剂型,免疫应答,疫苗生产,疫苗质量控制,临床前研究动物试验,临床试验和使用,对结核病公共卫生政策的影响等方面各有优劣。新型结核病疫苗的成功研发,还需要病原学、发病机制、免疫学和疫苗研发科学的进一步努力。     

A review of combination adjuvants for malaria vaccines: a promising approach for vaccine development

It is obvious that there is a critical need for an efficient malaria vaccine to accelerate malaria eradication. Currently, recombinant subunit vaccination against malaria using proteins and peptides is gaining attention. However, one of the major drawbacks of this approach is the lack of an efficient and durable immune response. Therefore, subunit vaccines require adjuvants to make the vaccine sufficiently immunogenic. Considering the history of the RTS,S vaccine, it seems likely that no single adjuvant is capable of eliciting all the protective immune responses required in many malarial subunit vaccines and the use of combination adjuvants will be increasingly important as the science of malaria vaccines advances. In light of this, it appears that identifying the most effective mixture of adjuvants with minimal adverse effects offers tremendous opportunities in improving the efficacy of vaccines against malaria. Owing to the importance of a multi-adjuvanted approach in subunit malaria vaccine development, this review paper outlines some of the best known combination adjuvants used in malaria subunit vaccines, focusing on their proposed mechanisms of action, their immunological properties, and their notable results. The aim of the present review is to consolidate these findings to aid the application of these combination adjuvants in experimental malaria vaccines.     

Research Advances on Transgenic Plant Vaccines

In recent years, with the development of genetics molecular biology and plant biotechnology, the vaccination (e.g. genetic engineering subunit vaccine, living vector vaccine, nucleic acid vaccine) programs are taking on a prosperous evolvement. In particular, the technology of the use of transgenic plants to produce human or animal therapeutic vaccines receives increasing attention. Expressing vaccine candidates in vegetables and fruits open up a new avenue for producing oral/edible vaccines. Transgenic plant vaccine disquisitions exhibit a tempting latent exploiting foreground. There are a lot of advantages for transgenic plant vaccines, such as low cost, easiness of storage, and convenient immune-inoculation. Some productions converged in edible tissues, so they can be consumed directly without isolation and purification. Up to now, many transgenic plant vaccine productions have been investigated and developed. In this review, recent advances on plant-derived recombinant protein expression systems, infectious targets, and delivery systems are presented. Some issues of high concern such as biosafety and public health are also discussed. Special attention is given to the prospects and limitations on transgenic plant vaccines.     

新型冠状病毒亚单位疫苗研究进展及现状*

自新型冠状病毒肺炎在2019年年末暴发以来,如何高效防控疫情一直是紧急的全球公共安全事件。疫苗是有效阻止病毒感染人体、保护高危人群免于疾病快速进展以及遏制疫情进一步扩大的手段之一,其中亚单位疫苗的主要成分为特定的病毒抗原蛋白或多肽,通过加入疫苗佐剂提高抗原的免疫原性。由于机体仅针对重组蛋白表面的特定抗原表位进行识别并产生抗体,因此亚单位疫苗具有较高的保护能力和安全性。通过对目前已上市及处于临床阶段的各类新型冠状病毒亚单位疫苗进行梳理,介绍了各类亚单位疫苗的抗原设计策略和佐剂选择、整体保护能力及研究进展,并对亚单位疫苗的应用及技术优势进行分析,期望能为亚单位疫苗研发及全球疫情防控提供参考。     

转基因植物疫苗的研究进展

近些年,随着遗传技术和植物基因工程的发展进步,疫苗（亚单位疫苗、活载体疫苗和核酸疫苗等）的研究迅速发展起来。尤其是利用转基因植物技术生产植物疫苗的研究受到了广泛的关注,在转基因植物（蔬菜、水果、农作物）的可食用部位表达抗原生产人或动物治疗用重组蛋白和疫苗的技术为可食性疫苗的研制开辟了新途径,展现了诱人的开发前景。植物来源的疫苗具有很多优势,如生产成本低、易于保存、免疫接种方便、甚至不需提取纯化等处理而直接食用。目前已有很多转基因植物疫苗产品投入开发和生产。文章综述了近几年转基因植物疫苗在表达系统、生产、生物安全／管理、公众健康等方面的研究进展,对转基因植物疫苗存在的问题进行了分析,并对其研究前景提出了展望。     

Virus-like particles as immunogens

Subunit vaccines based on recombinant proteins can suffer from poor immunogenicity owing to incorrect folding of the target protein or poor presentation to the immune system. Virus-like particles (VLPs) represent a specific class of subunit vaccine that mimic the structure of authentic virus particles. They are recognized readily by the immune system and present viral antigens in a more authentic conformation than other subunit vaccines. VLPs have therefore shown dramatic effectiveness as candidate vaccines. Here, we review the current status of VLPs as vaccines, and discuss the characteristics and problems associated with producing VLPs for different viruses.     

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.     

Recombinant vaccines against infectious hematopoietic necrosis virus: production by the Caulobacter crescentus S-layer protein secretion system and evaluation in laboratory trials

We report the development of an IHNV vaccine produced by a new protein production system based on the bacterium Caulobacter crescentus. The subunit vaccines that were tested contain a 184 amino acid segment of the IHNV glycoprotein in different fusion arrangements with the C. crescentus S-layer protein. Relative percent survival of 26 to 34% was demonstrated in rainbow trout fry for a vaccine that contained the 184 amino acid segment of the IHNV glycoprotein fused to the C-terminal one-quarter of the S-layer protein. Inclusion of the universal mammalian T-cell epitopes developed from the measles fusion protein or the tetanus toxin protein did not increase the effectiveness of the IHNV-G/S-layer recombinant protein.     

Transient Gene Expression in Serum-Free Suspension-Growing Mammalian Cells for the Production of Foot-and-Mouth Disease Virus Empty Capsids

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals. It produces severe economic losses in the livestock industry. Currently available vaccines are based on inactivated FMD virus (FMDV). The use of empty capsids as a subunit vaccine has been reported to be a promising candidate because it avoids the use of virus in the vaccine production and conserves the conformational epitopes of the virus. In this report, we explored transient gene expression (TGE) in serum-free suspension-growing mammalian cells for the production of FMDV recombinant empty capsids as a subunit vaccine. The recombinant proteins produced, assembled into empty capsids and induced protective immune response against viral challenge in mice. Furthermore, they were recognized by anti-FMDV bovine sera. By using this technology, we were able to achieve expression levels that are compatible with the development of a vaccine. Thus, TGE of mammalian cells is an easy to perform, scalable and cost-effective technology for the production of a recombinant subunit vaccine against FMDV.     

Anti-CD25 antibody enhancement of vaccine-induced immunogenicity: increased durable cellular immunity with reduced immunodominance

An efficacious vaccine strategy must be capable of inducing strong responses of an appropriate phenotype that are long lasting and sufficiently broad to prevent pathogen escape mechanisms. In the present study, we use anti-CD25 mAb to augment vaccine-induced immunity in mice. We demonstrate that coformulation of Ab and poxviral- or adenoviral-vectored vaccines induces significantly increased T cell responses to a malaria Ag; prior anti-CD25 Ab administration was not required for this effect. Furthermore, this vaccination approach subverts immunodominant epitope hierarchies by enhancing responses to subdominant epitopes induced by recombinant modified vaccinia virus Ankara immunization. Administration of anti-CD25 with a vaccine also induces more durable immunity compared with vaccine alone; significantly higher T cell responses were observed 100 days after the primary immunization. Enhanced immunogenicity is observed for multiple vaccine types with enhanced CD4+ and CD8+ T cell responses induced by bacillus Calmette-Guérin and a recombinant subunit protein vaccine to hepatitis B virus and with multiple Ags of tumor, viral, bacterial, and parasitic origin. Vaccine strategies incorporating anti-CD25 lead to improved protection against pre-erythrocytic malaria challenge. These data underpin new strategies for the design and development of more efficacious vaccines in clinical settings.     

Enhanced immunogenicity of gp120 protein when combined with recombinant DNA priming to generate antibodies that neutralize the JR-FL primary isolate of human immunodeficiency virus type 1

Strategies are needed for human immunodeficiency virus type 1 vaccine development that improves the neutralizing antibody response against primary isolates of the virus. Here we examined recombinant DNA priming followed by subunit protein boosting as a strategy to generate neutralizing antibodies. Both plasmid-based and recombinant protein envelope (Env) glycoprotein immunogens were derived from a primary viral isolate, JR-FL. Serum from rabbits immunized with either gp120 or gp140 DNA vaccines delivered by gene gun inoculation followed by recombinant gp120 protein boosting was capable of neutralizing JR-FL. Neither the DNA vaccines alone nor the gp120 protein alone generated a detectable neutralizing antibody response against this virus. Neutralizing antibody responses using gp120 DNA and gp140 DNA for priming were similar. The results suggest that Env DNA priming followed by gp120 protein boosting provides an advantage over either approach alone for generating a detectable neutralizing antibody response against primary isolates that are not easily neutralized.