Successful boosting of a DNA measles immunization with an oral plant-derived measles virus vaccine

Despite eradication attempts, measles remains a global health concern. Here we report results that demonstrate that a single-dose DNA immunization followed by multiple boosters, delivered orally as a plant-derived vaccine, can induce significantly greater quantities of measles virus-neutralizing antibodies than immunization with either DNA or plant-derived vaccines alone. This represents the first demonstration of an enhanced immune response to a prime-boost vaccination strategy combining a DNA vaccine with edible plant technology.     

Prevention of bubonic and pneumonic plague using plant-derived vaccines

Yersinia pestis, the causative agent of bubonic and pneumonic plague, is an extremely virulent bacterium but there are currently no approved vaccines for protection against this organism. Plants represent an economical and safer alternative to fermentation-based expression systems for the production of therapeutic proteins. The recombinant plague vaccine candidates produced in plants are based on the two most immunogenic antigens of Y. pestis: the fraction-1 capsular antigen (F1) and the low calcium response virulent antigen (V) either in combination or as a fusion protein (F1–V). These antigens have been expressed in plants using all three known possible strategies: nuclear transformation, chloroplast transformation and plant-virus-based expression vectors. These plant-derived plague vaccine candidates were successfully tested in animal models using parenteral, oral, or prime/boost immunization regimens. This review focuses on the recent research accomplishments towards the development of safe and effective pneumonic and bubonic plague vaccines using plants as bioreactors.     

Plants as bioreactors for the production of vaccine antigens

Plants have been identified as promising expression systems for commercial production of vaccine antigens. In phase I clinical trials several plant-derived vaccine antigens have been found to be safe and induce sufficiently high immune response. Thus, transgenic plants, including edible plant parts are suggested as excellent alternatives for the production of vaccines and economic scale-up through cultivation. Improved understanding of plant molecular biology and consequent refinement in the genetic engineering techniques have led to designing approaches for high level expression of vaccine antigens in plants. During the last decade, several efficient plant-based expression systems have been examined and more than 100 recombinant proteins including plant-derived vaccine antigens have been expressed in different plant tissues. Estimates suggest that it may become possible to obtain antigen sufficient for vaccinating millions of individuals from one acre crop by expressing the antigen in seeds of an edible legume, like peanut or soybean. In the near future, a plethora of protein products, developed through ‘naturalized bioreactors’ may reach market. Efforts for further improvements in these technologies need to be directed mainly towards validation and applicability of plant-based standardized mucosal and edible vaccines, regulatory pharmacology, formulations and the development of commercially viable GLP protocols. This article reviews the current status of developments in the area of use of plants for the development of vaccine antigens.     

Low-dose oral immunization with lyophilized tissue of herbicide-resistant lettuce expressing hepatitis B surface antigen for prototype plant-derived vaccine tablet formulation

Efficient immunization against hepatitis B virus (HBV) and other pathogens with plant-based oral vaccines requires appropriate plant expressors and the optimization of vaccine compositions and administration protocols. Previous immunization studies were mainly based on a combination of the injection of a small surface antigen of HBV (S-HBsAg) and the feeding with raw tissue containing the antigen, supplemented with an adjuvant, and coming from plants conferring resistance to kanamycin. The objective of this study was to develop a prototype oral vaccine formula suitable for human immunization. Herbicide-resistant lettuce was engineered, stably expressing through progeny generation micrograms of S-HBsAg per g of fresh weight and formed into virus-like particles (VLPs). Lyophilized tissue containing a relatively low, 100-ng VLP-assembled antigen dose, administered only orally to mice with a long, 60-day interval between prime and boost immunizations and without exogenous adjuvant, elicited mucosal and systemic humoral anti-HBs responses at the nominally protective level. Lyophilized tissue was converted into tablets, which preserved S-HBsAg content for at least one year of room temperature storage. The results of the study provide indications on immunization methodology using a durable, efficacious, and convenient plant-derived prototype oral vaccine against hepatitis B.     

Hepatitis virus vaccines: present status

During the past decade there has been extraordinary progress toward the development of vaccines for the prevention of type A and type B hepatitis. The successful propagation of hepatitis A virus in cell culture in 1979 was followed by the preparation of experimental live attenuated hepatitis A vaccines that have been shown to induce antibody in marmosets and chimpanzees and protect immunized marmosets against challenge with hepatitis A virus. The first human immunization trials will begin in mid-1982. An inactivated hepatitis B vaccine that was licensed in the United States in November 1981 has been shown to be safe, immunogenic, and effective. When this vaccine becomes available for use in July 1982, it will be recommended for persons who are considered to be at increased risk of contracting hepatitis B infection. Future generations of hepatitis B vaccines may be prepared from hepatitis B surface antigen derived from DNA recombinant technology or by in vitro synthesis of HBs Ag determinants by chemical means.     

A combination of intradermal jet-injection and electroporation overcomes in vivo dose restriction of DNA vaccines

ABSTRACT: BACKGROUND: The use of optimized delivery devices has been shown to enhance the potency of DNA vaccines. However, further optimization of DNA vaccine delivery is needed for this vaccine modality to ultimately be efficacious in humans. METHODS: Herein we evaluated antigen expression and immunogenicity after intradermal delivery of different doses of DNA vaccines by needle or by the Biojector jet-injection device, with or without the addition of electroporation (EP). RESULTS: Neither needle injection augmented by EP nor Biojector alone could induce higher magnitudes of immune responses after immunizations with a high dose of a DNA vaccine as compared to immunizations with a considerably lower dose. Biojector delivery followed by EP, however, overcame this observed dose restriction and induced significantly higher cellular and humoral immune responses after immunization with a high dose of DNA. Furthermore, a close correlation between in vivo antigen expression and cell-mediated immune responses was observed. CONCLUSIONS: These results show that two optimized DNA vaccine delivery devices can act together to overcome dose restrictions of plasmid DNA vaccines.     

Carrier-induced epitopic suppression, a major issue for future synthetic vaccines

Synthetic antigens have been shown, in experimental models, to induce protective immunity against a variety of pathogens. These studies have demonstrated that, due to their low immunogenicity, these synthetic antigens required conjugation to carrier molecules. Therefore, the choice of appropriate carriers for human immunization by future synthetic vaccines is a major issue. Tetanus toxoid is generally considered to be an effective potential carrier devoid of side-effects. However, the present study performed in mice with two synthetic vaccine models demonstrates that the immune response against the synthetic epitopes conjugated to tetanus toxoid can be suppressed by pre-existing immunity against this same carrier. Because most humans have been exposed to this antigen, this effect may have important implications for the development of synthetic vaccines.     

Immune responses to hepatitis B surface antigen following epidermal powder immunization

Langerhans cells in the epidermis of skin are potent antigen-presenting cells that trigger the immune system to respond to invading microorganisms. We have previously shown that epidermal powder immunization with a powdered inactivated influenza virus vaccine, by targeting the Langerhans cell-rich epidermis, was more efficacious than deeper tissue injection using a needle and syringe. We now report enhanced humoral and cellular immune responses to recombinant hepatitis B surface antigen following epidermal powder immunization. We observed that epidermal powder immunization with unadjuvanted hepatitis B surface antigen elicited an antibody titre equivalent to that induced by the alum-adjuvanted vaccine delivered by intramuscular injection, suggesting that epidermal powder immunization can overcome the need for adjuvantation. We demonstrated that synthetic CpG oligonucleotides (CpG DNA) could be coformulated with hepatitis B surface antigen and delivered by epidermal powder immunization to further augment the antibody response and modulate T helper cell activities. Epidermal powder immunization of hepatitis B surface antigen formulated with CpG DNA formulations resulted in 1.5-2.0 logs higher IgG antibody titres than alum-adjuvanted commercial vaccines administered by intramuscular injection. Formulation of hepatitis B surface antigen with CpG DNA elicited an augmented IgG2a antibody response and increased frequency of IFN-gamma secreting cells. In addition, CpG DNA was found to activate epidermal Langerhans cells and stimulate the production of TNF-alpha and IL-12 cytokines by epidermal cells, explaining its strong adjuvant activity following epidermal powder immunization. These results show that epidermal powder immunization is a safe and effective method to deliver hepatitis B surface antigen and the addition of new adjuvants, such as CpG DNA, may further enhance the efficacy of this vaccine.     

Heat-shock protein 70 from plant biofactories of recombinant antigens activate multiepitope-targeted immune responses

Although a physiological role of heat-shock proteins (HSP) in antigen presentation and immune response activation has not been directly demonstrated, their use as vaccine components is under clinical trial. We have previously demonstrated that the structure of plant-derived HSP70 (pHSP70) can be superimposed to the mammalian homologue and similarly to the mammalian counterpart, pHSP70-polypeptide complexes can activate the immune system. It is here shown that pHSP70 purified from plant tissues transiently expressing the influenza virus nucleoprotein are able to induce both the activation of major histocompatibility complex class I-restricted polyclonal T-cell responses and antibody production in mice of different haplotypes without the need of adjuvant co-delivery. These results indicate that pHSP70 derived from plants producing recombinant antigens may be used to formulate multiepitope vaccines.     

Immunostimulant adjuvant patch enhances humoral and cellular immune responses to DNA immunization

The focus of this report is on the development of an improved DNA immunization protocol, which takes advantage of the strengths of DNA immunization, as well as those associated with adjuvant delivered by transcutaneous immunostimulatory (IS) patches. Because transcutaneous delivery of adjuvants to the skin at the vaccination site has been shown to amplify the immune response to protein antigens, we hypothesized that the same IS patch when placed on the skin at the site of DNA injection could further enhance the immune response to a DNA influenza vaccine. We have combined an influenza DNA vaccine, hemagglutinin fused with three copies of complement C3d, to enhance uptake and antigen presentation, with an IS patch containing heat-labile enterotoxin from Escherichia coli. Coadministration of a potent adjuvant in IS patches placed on the skin at the site of DNA vaccination dramatically amplifies anti-influenza antibody immune response. Supplementing DNA vaccines with IS patches may be a particularly valuable strategy because DNA vaccines can be rapidly modified in response to mutations in pathogens, and individuals with compromised immune systems such as transplant patients and the elderly will benefit from the enhanced antibody response induced by the IS patches.     

Effectiveness of vaccination with recombinant HpaA from Helicobacter pylori is influenced by host genetic background

Several studies have explored the production and immunogenicity of HpaA as a potential protective antigen against Helicobacter pylori but little is known regarding its protective capabilities. We therefore evaluated the protective efficacy of recombinant HpaA (rHpaA) as a candidate vaccine antigen against H. pylori. To explore the impact of genetic diversity, inbred and outbred mice were prophylactically and therapeutically immunized with rHpaA adjuvanted with cholera toxin (CT). Prophylactic immunization induced a reduction in bacterial colonization in BALB/c and QS mice, but was ineffective in C57BL/6 mice, despite induction of antigen-specific antibodies. By contrast, therapeutic immunization was effective in all three strains of mice. Prophylactic immunization with CT-adjuvanted rHpaA was more effective when delivered via the nasal route than following intragastric delivery in BALB/c mice. However, HpaA-mediated protection was inferior to that induced by bacterial lysate. Hence, protective efficacy is inducible with vaccines containing HpaA, most relevantly shown in an outbred population of mice. The effectiveness of protection induced by HpaA antigen was influenced by host genetics and was less effective than lysate. HpaA therefore has potential for the development of effective immunization against H. pylori but this would probably entail the antigen to be one component of a multiantigenic vaccine.     

A morphine/heroin vaccine with new hapten design attenuates behavioral effects in rats

Heroin use has seriously threatened public heath in many countries, but the existing therapies continue to have many limitations. Recently, immunotherapy has shown efficacy in some clinical studies, including vaccines against nicotine and cocaine, but no opioid vaccines have been introduced in clinical studies. The development of a novel opioid antigen designed specifically for the prevention of heroin addiction is necessary. A morphine-keyhole limpet hemocyanin conjugate was prepared and administered subcutaneously in rats. Antibody titers in plasma were measured using an enzyme-linked immunosorbent assay (ELISA). Competitive ELISA was used to assess the selectivity of the antibodies. Dopamine concentrations in the nucleus accumbens in rats after vaccine administration were determined by high-performance liquid chromatography with electrochemical detection. The effects of the vaccine on the heroin-primed restatement of self-administration and locomotor sensitization were evaluated. A novel hapten, 6-glutarylmorphine, was produced, and the vaccine generated a high antibody titer response. This vaccine displayed specificity for both morphine and heroin, but the anti-morphine antibodies could not recognize dissimilar therapeutic opioid compounds, such as buprenorphine, methadone, naloxone, naltrexone, codeine, and nalorphine. The morphine antibody significantly decreased morphine-induced locomotor activity in rats after immunization. Importantly, rats immunized with this vaccine did not exhibit heroin-primed reinstatement of heroin seeking when antibody levels were sufficiently high. The vaccine reduced dopamine levels in the nucleus accumbens after morphine administration, which is consistent with its behavioral effects. These results suggest that immunization with a novel vaccine is an effective means of inducing a morphine-specific antibody response that is able to attenuate the behavioral and psychoactive effects of heroin.     

增强DNA疫苗免疫应答的新进展

DNA疫苗为编码抗原蛋白的真核表达载体,注入体内后在原位表达所编码的抗原并诱导免疫应答,在预防感染、治疗自身免疫性疾病、过敏性疾病和肿瘤等疫病中有着很好的应用前景。但与灭活疫苗相比,其免疫效价还比较低。有多种策略能够增强或调节DNA疫苗诱导的免疫应答,其中,作为外源基因载体的质粒的组成及插入的有关基因均可直接或间接地影响免疫反应的效果,在构建DNA疫苗质粒时,加入细胞因子、融合信号、泛素等基因以及ISS序列,另外还可以通过设计一些对抗原提成细胞有影响的分子共注射,以及加入转移分子,都可以明显增强DNA疫苗的免疫效果,从而有利于研制更有效的DNA疫苗。     

A Novel Virus-Like Particle Based Vaccine Platform Displaying the Placental Malaria Antigen VAR2CSA

Placental malaria caused by Plasmodium falciparum is a major cause of mortality and severe morbidity. Clinical testing of a soluble protein-based vaccine containing the parasite ligand, VAR2CSA, has been initiated. VAR2CSA binds to the human receptor chondroitin sulphate A (CSA) and is responsible for sequestration of Plasmodium falciparum infected erythrocytes in the placenta. It is imperative that a vaccine against malaria in pregnancy, if administered to women before they become pregnant, can induce a strong and long lasting immune response. While most soluble protein-based vaccines have failed during clinical testing, virus-like particle (VLP) based vaccines (e.g., the licensed human papillomavirus vaccines) have demonstrated high efficacy, suggesting that the spatial assembly of the vaccine antigen is a critical parameter for inducing an optimal long-lasting protective immune response. We have developed a VLP vaccine display platform by identifying regions of the HPV16 L1 coat protein where a biotin acceptor site (AviTag^TM) can be inserted without compromising VLP-assembly. Subsequent biotinylation of Avi-L1 VLPs allow us to anchor monovalent streptavidin (mSA)-fused proteins to the biotin, thereby obtaining a dense and repetitive VLP-display of the vaccine antigen. The mSA-VAR2CSA antigen was delivered on the Avi-L1 VLP platform and tested in C57BL/6 mice in comparison to two soluble protein-based vaccines consisting of naked VAR2CSA and mSA-VAR2CSA. The mSA-VAR2CSA Avi-L1 VLP and soluble mSA-VAR2CSA vaccines induced higher antibody titers than the soluble naked VAR2CSA vaccine after three immunizations. The VAR2CSA Avi-L1 VLP vaccine induced statistically significantly higher endpoint titres compared to the soluble mSA-VAR2CSA vaccine, after 1^st and 2^nd immunization; however, this difference was not statistically significant after 3^rd immunization. Importantly, the VLP-VAR2CSA induced antibodies were functional in inhibiting the binding of parasites to CSA. This study demonstrates that the described Avi-L1 VLP-platform may serve as a versatile system for facilitating optimal VLP-display of large and complex vaccine antigens.     

Composition of acellular pertussis and combination vaccines: a general review.

Since the development and introduction of the acellular pertussis vaccine in Japan in the early eighties, we have come a long way in using this component in combination with other vaccines. However, the basic problem in development of an effective and safe pertussis vaccine is that the antigens to induce complete protection against clinical pertussis and the precise mechanism by which pertussis vaccine confers immunity is yet unknown. Hence, the composition of future acellular pertussis vaccine remains an open issue. Recently, acellular pertussis vaccine has been licensed for the booster doses in the U.S.A. and for primary immunization of infants in Italy and Germany. A multicentric trial has been carried out to compare the serological response and adverse reactions of 13 acellular pertussis vaccines. These vaccines contained one or more of the four components, i.e. FHA, PT, 69 kDa OMP and fimbriae. All vaccines were associated with substantially fewer and less adverse reactions and were more immunogenic with respect to antibodies against the added antigens. DTP vaccines in the near future will have combinations of other components and the key antigen for combination will be acellular pertussis component which is going to replace whole cell pertussis component in DTP vaccines. In view of this, manufacturers like ourselves from the developing countries are still groping in the dark, uncertain whether we should have a single component acellular pertussis vaccine or multicomponent one. This will have a major impact on the cost of production, the final cost of the combination vaccines and the regulatory issues that we will have to tackle in view of the recent thinking on harmonization in the pharmaceutical industry.     

Immunobiological characteristics of impurities in corpuscular influenza vaccines

Admixtures of free antigens have been shown to play the main role in the anaphylactogenic danger of vaccines. The immunogenic and anaphylactogenic action of such antigenic admixtures in corpuscular influenza vaccine can be observed after the immunization of animals in 2 or 3 injections. Host antigens incorporated into viral particles induce no anaphylactic reaction in guinea pigs after their immunization in 3 injections.     

基因疫苗导入技术研究进展

基因疫苗积极的临床结果证明了,基因免疫是一种有效的临床免疫模式。虽然,喷射注射法的精确作用机制还不太清楚,但临床前研究表明,在皮肤内直接打靶抗原呈递细胞可有效地增强免疫反应。另外,局部给药法和树突细胞体外加载抗原的实验结果显示,直接打靶抗原呈递细胞可放大、控制和调节预防及治疗性基因疫苗的免疫结果。尽管基因枪有许多令人鼓舞的优点,但由于价格和便利性的障碍,它是否能商业化还不能确定。利用基因法治疗和预防疾病所涉及的安全性对基因疫苗要求更严格。这要有可控的质粒导入系统和组织特异性表达系统。     

The combined activation of positive costimulatory signals with modulation of a negative costimulatory signal for the enhancement of vaccine-mediated T-cell responses

Blockade of CTLA-4 by monoclonal antibodies (mAb) can mediate regression of tumors and increase the efficacy of tumor antigen specific vaccines. Blockade of CTLA-4 has also been shown to significantly increase the avidity of antigen-specific T cells after immunization with live recombinant viral vector based vaccine. Here, we demonstrate a biological synergy between CTLA-4 blockade and active vaccine therapy consisting of recombinant vaccinia and avipox viruses expressing carcinoembryonic antigen (CEA) and three T cell costimulatory molecules to enhance antitumor effects. However, this synergy was very much dependent on the temporal relationship of scheduling of the two agents. We evaluated the strategies in both a foreign antigen model using β-galactosidase as immunogen, and in a “self” antigen model using CEA as immunogen. For antitumor activity the model used consisted of mice transgenic for human CEA and a murine carcinoma cell line transfected with CEA. The enhanced antitumor activity after vaccine and CTLA-4 blockade did not result in any signs of autoimmunity. These studies form a rational basis for the use of vector-based vaccines with anti-CTLA-4 and demonstrate that both enhancement of positive costimulatory signals and inhibition of negative costimulatory signals can be simultaneously exploited. These studies also underscore the importance of “drug” scheduling in vaccine combination therapies.     

Vaccine manufacturing: challenges and solutions

The recent influenza vaccine shortages have provided a timely reminder of the tenuous nature of the world's vaccine supply and the potential for manufacturing issues to severely disrupt vital access to important vaccines. The application of new technologies to the discovery, assessment, development and production of vaccines has the potential to prevent such occurrences and enable the introduction of new vaccines. Gene-based vaccines, virus-like particles, plant-derived vaccines and novel adjuvants and delivery systems represent promising approaches to creating safer, more potent vaccines. As a consequence, more people will have faster access to more effective vaccines against a broader spectrum of infectious diseases. However, the increased cost of producing new vaccines and regulatory uncertainty remain challenges for vaccine manufacturers.