Mitigating the looming vaccine crisis: production and delivery of plasmid-based vaccines

The exponentially growing human population and the emergence of new diseases are clear indications that the world can no longer depend solely on conventional vaccine technologies and production schemes. The race to find a new vaccine technology is crucial to help speed up and complement the World Health Organization (WHO) disease elimination program. The ultimate goal is to uncover fast and efficient production schemes in the event of a pandemic, and also to effectively fight deadly diseases such as malaria, bird flu, hepatitis, and human immunodeficiency virus (HIV). Plasmid DNA vaccines, if properly formulated, offer specific priming of the immune system and similar or even better prophylactic effects than conventional vaccines. This article discusses many of the critical issues that need to be considered when developing fast, effective, and reliable plasmid DNA vaccine manufacturing processes. Different modes of plasmid production via bacterial fermentation are compared. Plasmid purification by chromatography is specifically discussed as it is the most commercially viable bioprocess engineering technique for continuous purification of supercoiled plasmid DNA. Current techniques and progress covering the area of plasmid DNA vaccine design, formulation, and delivery are also put forward.     

Industrial scale production of plasmid DNA for vaccine and gene therapy: plasmid design, production, and purification

The past several years have witnessed a rapidly increasing number of reports on utilizing plasmid DNA as a vector for the introduction of genes into mammalian cells for use in both gene therapy and vaccine applications. “Naked DNA vaccines” allow the foreign genes to be transiently expressed in transfected cells, mimicking intracellular pathogenic infection and triggering both the humoral and cellular immune responses. While considerable attention has been paid to the potential of such vaccines to mitigate a number of infections, substantially less consideration has been given to the practical challenges of producing large amounts of plasmid DNA for therapeutic use in humans, for both clinical studies and, ultimately, full-scale manufacturing. Doses of naked DNA vaccines are on the order of milligrams, while typical small-scale Escherichia coli fermentations may routinely yield only a few mg/l of plasmid DNA. There have been many investigations towards optimizing production of heterologous proteins over the past three decades, but in these cases, the plasmid DNA was not the final product of interest. This review addresses the current state-of-the-art means for the production of plasmid DNA at large scale in compliance with existing regulatory guidelines. The impact of the nature of the plasmid vector on the choice of fermentation protocols is presented, along with the effect of varying cultivation conditions on final plasmid content. Practical considerations for the large-scale purification of plasmid DNA are also discussed.     

Mutation detection in plasmid-based biopharmaceuticals

As the number of applications involving therapeutic plasmid DNA (pDNA) increases worldwide, there is a growing concern over maintaining rigorous quality control through a panel of high-quality assays. For this reason, efficient, cost-effective and sensitive technologies enabling the identification of genetic variants and unwanted side products are needed to successfully establish the identity and stability of a plasmid-based biopharmaceutical. This review highlights several bioinformatic tools for ab initio detection of potentially unstable DNA regions, as well as techniques used for mutation detection in nucleic acids, with particular emphasis on pDNA.     

基因工程疫苗的病毒载体

病毒基因工程疫苗是以活病毒为载体将一段外源基因导入机体细胞内,并使外源基因维持较高水平的表达。通过使用复制型或复制缺陷型载体能使表达的抗原诱生机体产生相应的体液抗体,并能引起机体产生细胞介导的免疫反应及粘膜免疫反应。本文主要介绍有可能用于基因工程疫苗的DNA及RNA病毒载体构建及其应用。     

Plasmid DNA vaccines

DNA vaccination is a novel approach for inducing an immune response. Purified plasmid DNA containing an antigen’s coding sequences and the necessary regulatory elements to expres them is introduced into the tissue via intramuscular injection or particle bombardment. Once the DNA reaches the tissue, the antigen is expressed in enough quantity to induce a potent and specific immune response and to confer protection against further infections. The effectiveness of DNA vaccines against viruses, parasites, and cancer cells has been demonstrated in numerous animal models. This new approach comes as an aid for the prevention of infectious diseases for which the conventional vaccines have failed. DNA vaccine research is providing new insights into some of the basic immunological mechanisms of vaccination such as antigen presentation, the role of effector cells, and immunoregulatory factors. In addition, DNA vaccines may enable us to manipulate the immune system in situations where the response to agents is inappropriate or ineffective. The study of the potential deleterious effects of DNA vaccines is furthering our knowledge regarding the relationship between bacterial DNA and the immune system, as well as its potential application for the study of neonatal tolerance and autoimmunity.     

Enhancement of immune responses by DNA vaccination through targeted gene delivery using mannosylated cationic liposome formulations following intravenous administration in mice

The present study investigated the potency of the mannosylated cationic liposomes (Man liposomes) that we have developed in novel DNA vaccine carrier. Ovalbumin (OVA) was selected as a model antigen for vaccination; accordingly, OVA-encoding pDNA (pCMV-OVA) was constructed to evaluate DNA vaccination. The potency of the Man liposome/pCMV-OVA complex was compared with naked pCMV-OVA and that complexed with DC-Chol liposomes. In cultured mouse peritoneal macrophages, MHC class I-restricted antigen presentation of the Man liposome/pCMV-OVA complex was significantly higher than that of naked pCMV-OVA and that complexed with DC-Chol liposomes. After intravenous administration, OVA mRNA expression and MHC class I-restricted antigen presentation on CD11c+ cells and inflammatory cytokines, such as TNF-alpha, IL-12, and IFN-gamma, that can enhance the Th1 response of the Man liposome/pCMV-OVA complex were higher than that of naked pCMV-OVA and that complexed with DC-Chol liposomes. Also, the spleen cells from mice immunized by intravenous administration of the Man liposome/pCMV-OVA complex showed the highest proliferation response and IFN-gamma secretion. These findings suggest that the targeted delivery of DNA vaccine by Man liposomes is a potent vaccination method for DNA vaccine therapy.     

Plasmid DNA vaccine vector design: Impact on efficacy,safety and upstream production

Critical molecular and cellular biological factors impacting design of licensable DNA vaccine vectors that combine high yield and integrity during bacterial production with increased expression in mammalian cells are reviewed. Food and Drug Administration (FDA), World Health Organization (WHO) and European Medical Agencies (EMEA) regulatory guidance's are discussed, as they relate to vector design and plasmid fermentation. While all new vectors will require extensive preclinical testing to validate safety and performance prior to clinical use, regulatory testing burden for follow-on products can be reduced by combining carefully designed synthetic genes with existing validated vector backbones. A flowchart for creation of new synthetic genes, combining rationale design with bioinformatics, is presented. The biology of plasmid replication is reviewed, and process engineering strategies that reduce metabolic burden discussed. Utilizing recently developed low metabolic burden seed stock and fermentation strategies, optimized vectors can now be manufactured in high yields exceeding 2 g/L, with specific plasmid yields of 5% total dry cell weight.     

Cell-penetrating peptides: Application in vaccine delivery

Recent years have seen a surge in interest in cell-penetrating peptides (CPP) as an efficient means for delivering therapeutic targets into cellular compartments. The cell membrane is impermeable to hydrophilic substances yet linking to CPP can facilitate delivery into cells. Thus the unique translocatory property of CPP ensures they remain an attractive carrier, with the capacity to deliver cargoes in an efficient manner having applications in drug delivery, gene transfer and DNA vaccination. Fundamental for an effective vaccine is the delivery of antigen epitopes to antigen-presenting cells, ensuing processing and presentation and induction of an immune response. Vaccination with proteins or synthetic peptides incorporating CTL epitopes have proven limited due to the failure for exogenous antigens to be presented efficiently to T cells. Linking of antigens to CPP overcomes such obstacles by facilitating cellular uptake, processing and presentation of exogenous antigen for the induction of potent immune responses. This review will encompass the various strategies for the delivery of whole proteins, T cell epitopes and preclinical studies utilizing CPP for cancer vaccines.     

红细胞伪装纳米颗粒:一个具有潜力的药物及疫苗递送系统

红细胞伪装纳米颗粒是一种以红细胞或红细胞膜纳米囊泡为载体在体内递送药物、酶、多肽和抗原等物质的系统,具有生物相容性好、循环周期长、靶向性强等优势。本文从红细胞载体的种类、发展历程、递送策略应用以及其局限性和未来的挑战等方面进行了详细阐述,并展望了其未来的发展方向。     

Augmentation of antigen-specific immune responses using DNA-fusogenic liposome vaccine

In an attempt to enhance the immunological efficacy of genetic immunization, we investigated a new biological means for delivering antigen gene directly to the cytoplasm via membrane fusion. In this context, we investigated fusogenic liposome (FL) encapsulating DNA as a possible genetic immunization vehicle. RT-PCR analysis indicated that a FL could introduce and express encapsulating OVA gene efficiently and rapidly in vitro. Consistent with this observation, an in vitro assay showed that FL-mediated antigen-gene delivery can induce potent presentation of antigen via the MHC class I-dependent pathway. Accordingly, immunization with FL containing the OVA-gene induced potent OVA-specific Th1 and Th2 cytokine production. Additionally, OVA-specific CTL responses and antibody production were also observed in systemic compartments including the spleen, upon immunization with the OVA-gene encapsulating FL. These findings suggest that FL is an effective genetic immunization carrier system for the stimulation of antigen-specific immune responses against its encoding antigen.     

Humans and viruses determine the nature of virus vaccine production processes

Vaccine production processes result from the interaction between humans with a particular cell and virus system. The factors that control progress lie not only in the nature of the virus and animal cell but also in the history of the environment in which the process is to be developed. This latter constraint strongly influences the nature of the technical process that is chosen for the production of the vaccine rather than the achievement of efficiency based on one or other of the many possible engineering parameters of the virus production process. In addition to this it is also clear that we have much to learn about the production of viruses from animal cells in culture and that we may be aided by changing our present paradigm of the virus as a cellular enemy to that of the viruses are the cell's best friend.Paper presented at Cell Engineering III Meeting, Florida, 1992.     

Oral delivery of DNA vaccines using attenuated Salmonella typhimurium as carrier

The efficacious delivery of eukaryotic expression plasmids to inductive cells of the immune system constitutes a key prerequisite for the generation of effective DNA vaccines. Here, we have explored the use of bacteria as vehicles to orally deliver expression plasmids. Attenuated Salmonella typhimurium aroA harbouring eukaryotic expression plasmids that encoded virulence factors of Listeria monocytogenes were administered orally to BALB/c mice. Strong cytotoxic and helper T cell responses as well as antibody production were elicited even after a single administration. Mice immunised four times with Salmonella that carried a eukaryotic expression plasmid encoding the secretory listerial protein listeriolysin were protected against a subsequent lethal challenge with this pathogen. A single dose was already partially protective. The efficiency of this vaccination procedure was due to transfer of the expression plasmid from the bacterial carrier to the mammalian host. Evidence for such an event could be obtained in vivo and in vitro. Expression of the desired antigen in various lymphoid tissues was already detectable 1 day after administration of the DNA vaccine and persisted for at least 1 month in spleen and mesenteric lymph nodes. Induction of cytotoxic and helper T cell responses was observed in all mouse strains tested including outbred strains whereas antibodies were mainly detected in BALB/c. Furthermore, we could show that immunogenicity could be improved by increasing the invasiveness of the bacterial carrier.     

Formulation of a dry powder influenza vaccine for nasal delivery

The purpose of this research was to prepare a dry powder vaccine formulation containing whole inactivated influenza virus (VIIV) and a mucoadhesive compound suitable for nasal delivery. Powders containing WIIV and either lactose or trehalose were produced by lyophilization. A micro-ball mill was used to reduce the lyophilized cake to sizes suitable for nasal delivery. Chitosan flakes were reduced in size using a cryo-milling technique. Milled powders were sieved between 45 and 125 μm aggregate sizes and characterized for particle size and distribution, morphology, and flow properties. Powders were blended in the micro-ball mill without the ball. Lyophilization followed by milling produced irregularly shaped, polydisperse particles with a median primary particle diameter of ≈21 μm and a yield of ≈37% of particles in the 45 to 125 μm particle size range. Flow properties of lactose and trehalose powders after lyophilization followed by milling and sieving were similar. Cryo-milling produced a small yield of particles in the desired size range (<10%). Lyophilization followed by milling and sieving produced particles suitable for nasal delivery with different physicochemical properties as a function of processing conditions and components of the formulation. Further optimization of particle size and morphology is required for these powders to be suitable for clinical evaluation. Published: March 10, 2006     

Fish DNA vaccine against infectious hematopoietic necrosis virus: efficacy of various routes of immunisation

The DNA vaccine, pIHNVw-G, contains the gene for the glycoprotein (G) of the rhabdovirus infectious hematopoietic necrosis virus (IHNV), a major pathogen of salmon and trout. The relative efficacy of various routes of immunisation with pIHNVw-G was evaluated using 1.8 g rainbow trout fry vaccinated via intramuscular injection, scarification of the skin, intraperitoneal injection, intrabuccal administration, cutaneous particle bombardment using a gene gun, or immersion in water containing DNA vaccine-coated beads. Twenty-seven days after vaccination neutralising antibody titres were determined, and 2 days later groups of vaccinated and control unvaccinated fish were subjected to an IHNV immersion challenge. Results of the virus challenge showed that the intramuscular injection and the gene gun immunisation induced protective immunity in fry, while intraperitoneal injection provided partial protection. Neutralising antibodies were not detected in sera of vaccinated fish regardless of the route of immunisation used, suggesting that cell mediated immunity may be at least partially responsible for the observed protection.     

Intranasal immunization with chitosan microparticles enhances LACK-DNA vaccine protection and induces specific long-lasting immunity against visceral leishmaniasis

Development of a protective vaccine against Leishmania depends on antigen formulation and adjuvants that induce specific immunity and long-lasting immune responses. We previously demonstrated that BALB/c mice intranasally vaccinated with a plasmid DNA encoding the p36/LACK leishmanial antigen (LACK-DNA) develop a protective immunity for up to 3 months after vaccination, which was linked with the systemic expression of vaccine mRNA in peripheral organs. In this study, LACK-DNA vaccine was associated with biocompatible chitosan microparticles cross-linked with glyceraldehyde (CMC) to boost the long-lasting immunity against the late Leishmania infantum challenge. Infection at 7 days, 3 or 6 months after vaccination resulted in significantly lower parasite loads when compared with non-vaccinated controls. Besides, LACK-DNA-chitosan vaccinated mice showed long-time protection observed after the late time point challenge. The achieved protection was correlated with an enhanced spleen cell responsiveness to parasite antigens, marked by increased proliferation and IFN-γ as well as decreased IL-10 production. Moreover, we found diminished systemic levels of TNF-α that was compatible with the better health condition observed in LACK-DNA/CMC vaccinated-infected mice. Together, our data indicate the feasibility of chitosan microparticles as a delivery system tool to extend the protective immunity conferred by LACK-DNA vaccine, which may be explored in vaccine formulations against Leishmania parasite infections.     

Protective action of bacterial melanin against DNA damage in full UV spectrums by a sensitive plasmid-based noncellular system

The purpose of this study was to introduce a simple and sensitive plasmid-based noncellular system to evaluate the photoprotection of bacterial melanin on DNA damage against ultraviolet (UV) radiation. Plasmid DNA was used to assess the role of melanin in different ranges of UV using a series of in vitro assays. Fluorometric measurements suggested that melanin could efficiently scavenge reactive oxygen species (ROS) generated by UVA irradiation in solution, and the scavenging capability was proportional to the pigment concentration. The protective effect of melanin on plasmid DNA under UVB irradiation was confirmed by the transformation efficiency of the protected DNA, which was at least 10-fold higher than that of the non melanin protected DNA. After the UVC irradiation, the DNA damage of strand breaks was quantified by laser-induced fluorescence capillary electrophoresis. The percentage of supercoiled plasmid was reduced from 80% to less than 5% without melanin protection. In contrast, the percentage of supercoiled DNA only decreased to about 40% in the presence of melanin under the same radiation conditions. All these results demonstrated that bacterial melanin did protect DNA from being damaged throughout full UV irradiation. This system, avoiding the potential interference by cellular DNA repair machinery and intracellular substances, may provide a sensitive in vitro means to evaluate the functions of melanin and other photoprotective compounds from different sources.     

恶性疟原虫抗原表位的亚单位疫苗与DNA疫苗的联合免疫

构建了含有恶性疟原虫抗原基因 ( AWTE)的真核表达质粒 p CMV- AWTE,以及能在大肠杆菌中得到分泌性表达的原核表达质粒 p MC0 5 ,表达的蛋白 AWTE保持了疟原虫抗原的抗原性。将 p CMV- AWTE以及 AWTE两者混合各 1 0μg鼻腔免疫小鼠 ,一次后诱导机体产生了较高水平的体液免疫及细胞免疫