Vaccine preventable viral diseases in developing countries

There are several viral infectious diseases with a high impact on developing countries which can be prevented by immunization with existing vaccines. The most important are poliomyelitis, measles, hepatitis B and yellow fever. Vaccines against poliomyelitis and measles used within the framework of the WHO/Expanded Programme on Immunization prevent about 1.4 million deaths from measles and 360,000 cases of paralytic polio per year in developing countries, but about 1.5 million measles' deaths and 200,000 cases of paralytic polio still occur. Hepatitis B infection and its sequelae are responsible for over 50 million infections and one million deaths annually. Highly effective hepatitis B vaccines are now available and the price of these vaccines for the developing world has fallen dramatically. Despite the availability of a safe and efficacious yellow fever vaccine since 1937, 5400 cases of this disease with 3200 deaths were reported in Africa and South America from 1986 to 1988. Because of the efficacy of existing vaccines and the lack of animal reservoirs or vectors, systematic vaccination programmes within the framework of the Expanded Programme on Immunization (EPI) could theoretically eliminate and even eradicate poliomyelltis, measles and hepatitis B. Many different obstacles need to be overcome before these goals are realized.This paper was presented at the IUMS Symposium on New Developments in Diagnosis and Control of Infectious Diseases held in conjunction with the Eighth International Congress of Virology, Berlin, Germany, 24–31 August 1990.     

Hepatitis vaccines: recent advances

Despite the availability of hepatitis A vaccines that might provide protection for decades, hepatitis B vaccines that provides protection for at least 15 years and the recent introduction of a combined hepatitis A and B vaccine, these infections continue to spread in both the developed and developing world. Hepatitis A vaccine coverage has been limited to high-risk groups: such a selective immunisation policy is unlikely to have a major impact. If adequate immunogenicity in infants is confirmed, dosing schedules can be improved and the costs of vaccination reduced, universal paediatric immunisation with combined hepatitis A and B products is likely to result in the eventual eradication of these infections. In the interim, novel hepatitis A vaccines are being investigated and additional studies on hepatitis A vaccine immunogenicity in infants are in progress. Worldwide use of hepatitis B vaccines for the newborn, young children and high-risk groups should control this infection and obviate the need for a vaccine against hepatitis D. Newer hepatitis B vaccines that may reduce the likelihood of non-responsiveness and have immunotherapeutic value are under study. A recombinant hepatitis E vaccine for use in endemic regions is currently in clinical trials. The development of an effective hepatitis C vaccine has been agonisingly slow and many impediments have been recognised. These include the lack of a susceptible small animal, a high degree of hepatitis C virus (HCV) genomic diversity and failure to produce high quantities of HCV in tissue culture. The development of a novel HCV replicon system may be a major breakthrough. Nonetheless, it may still be exceedingly difficult to produce a vaccine that uniformly provides sterilising immunity; the possibility of developing a hepatitis C vaccine that can prevent chronic infection is an exciting concept that requires further investigation. Advances in recombinant technology, the use of novel genetic (DNA-based) vaccines, expression of hepatitis antigens in plants and improved adjuvants also hold considerable promise.     

Oral hepatitis B vaccine candidates produced and delivered in plant material

Hepatitis B is a major global health problem; approximately two billion people are infected with the virus worldwide, despite the fact that safe and efficacious vaccines have been developed and used for nearly 20 years. Prohibitive costs for vaccine purchase and administration restrict uptake in many developing nations. Agencies such as the Global Alliance for Vaccination and Immunization are helping to make current vaccines more available, but reduced costs would greatly aid this effort. Oral delivery is an option to reduce the expense of administering hepatitis B vaccines. It may also improve compliance, and orally delivered vaccines may be more efficacious among poor responders to current vaccines. However, to induce protective efficacy, oral administration may require encapsulation of antigen and delivery of large doses. Plant-based expression systems offer an oral delivery alternative with low production costs, and they also encapsulate the antigen. Some plant-based systems also stabilize antigen and therefore reduce storage and distribution costs. The hepatitis B major surface antigen has been expressed in several plant systems. A variety of regulatory sequences and subcellular targets have been used to achieve expression suitable for early stage clinical trials. However, further increase in expression will be necessary for practical and efficacious products. Appropriate processing can yield palatable products with uniform antigen concentration. The antigen expressed in plant systems shows extensive disulphide cross-linking and oligomerization and forms virus-like particles. Oral delivery of the antigen in plant material can induce a serum antibody response, prime the immune system for a subsequent injection of antigen and give a boosted response to a prior injection. Small scale clinical trials in which the antigen has been delivered orally in edible plant material indicate safety and immunogenicity.     

The control of viral diseases in the developing countries with the use of existing vaccines]

In developing countries, every year about 70 million measles cases occur with 1.5 million deaths, over 200,000 children contract paralytic poliomyelitis, 50 million people get infected with viral B hepatitis causing over 1 million deaths, and several thousand people perish because of yellow fever according to WHO data. At the present time, there are 12 vaccines against viruses: vaccines against German measles and mumps in addition to the above. The universal immunization program (UIP) of WHO targets measles and polio. In 1989, a WHO resolution envisioned a 90% immunization coverage by the year 2000. Measles vaccination is recommended for children aged 9-23 months, since most children have maternal antibodies during the first 3-13 months of age. The Edmonston-Zagreb vaccine provided seroconversion of 92, 96, and 98% for 18 months vs. the 66, 76, and 91% rate of the Schwarz vaccine. In the US, measles incidence increased from 1497 cases in 1983 to 6382 cases in 1988 to over 14,000 cases in 1989, prompting second vaccination in children of school age. The highest incidence of polio was registered in Southeast Asia, although it declined from 1 case/100,000 population in 1975 to .5/100,000 in 1988. Oral poliomyelitis vaccine (OPV) provides protection: there is only 1 case/2.5 million vaccinations. Hepatitis B has infected over 2 billion people. About 300 million are carriers, with a prevalence of 20% in African, Asian, and Pacific region populations. Plasmatic and bioengineered recombinant vaccine type have been used in 30 million people. The first dose is given postnatally, the second at 1-2 months of age, and the 3rd at 1 year of age. Yellow fever vaccine was 50 years old in 1988, yet during 1986-1988 there were 5395 cases with 3172 deaths in Africa and South America. Vaccination provides 90-95% seroconversion, and periodic follow-up vaccinations under UIP could eradicate these infections and their etiologic agents.     

基因工程乙肝疫苗研究进展

乙肝病毒(hepatitisBvirus,HBV)为嗜肝病毒,全世界有超过3亿人受到乙肝病毒的感染。综述近年来乙肝疫苗的最新研究进展,并对乙肝疫苗的发展历程,应用中出现的问题,克服不应答及低应答的策略,乙肝疫苗的发展方向以及目前最新的DNA疫苗进行了阐述 。     

Isolation,cloning and transgenic expression of hepatitis B surface antigen (HBsAg) in Solanum lycopersicum L

The Hepatitis B virus (HBV) infection is one of the most widespread viral infections of humans. HBV causes acute and chronic hepatitis. Chronic hepatitis leads to hepatocellular carcinoma, which is a significant cause of death. DNA-based immunization programs to control the spread of Hepatitis B in developing countries are costly and require special storage and transportation. The alternative way is to express Hepatitis B surface antigen (HBsAg) in plants to develop oral vaccines. In this study, HBsAg gene was isolated, cloned, and then transformed in tomato plants. The transgenic tomato plants were confirmed through RT-qPCR. HBsAg expression was analysed in mature green and red stages of tomato fruit through quantitative real-time PCR. It was observed that expression of HBsAg was high in matured red tomato as compared to mature green. The present study is the first step to developing Solanum lycopersicum as an edible vaccine production system in this world region.     

植物口服疫苗的研究进展

植物口服疫苗是通过转基因植物生产,通过口服的方式预防疾病的生物制品.作为一种新型疫苗,其研究开始于三十几年前.由于植物口服疫苗可以最大程度地降低传统疫苗的潜在风险,在疫苗生产中具有优势,因此拥有良好的商业生产前景.植物疫苗价格低廉,生产过程安全,可产生与注射疫苗相似效价效果,无论是在控制养殖业抗生素滥用的情况下作为替代...     

乙肝疫苗的研究进展

乙型肝炎是由乙型肝炎病毒(hepatitis B virus,HBV)引起的传染性疾病,流行范围广泛,危害严重。目前,乙型肝炎的预防和治疗主要通过接种乙肝疫苗。现针对乙肝疫苗的类型及其免疫学作总结,为乙肝疫苗的深入研究提供参考。     

Removing N-terminal sequences in pre-s1 domain enhanced antibody and B-cell responses by an HBV large surface antigen DNA vaccine

Although the use of recombinant hepatitis B virus surface (HBsAg) protein vaccine has successfully reduced global hepatitis B infection, there are still a number of vaccine recipients who do not develop detectable antibody responses. Various novel vaccination approaches, including DNA vaccines, have been used to further improve the coverage of vaccine protection. Our previous studies demonstrated that HBsAg-based DNA vaccines could induce both humoral and CMI responses in experimental animal models. However, one form of the the HBsAg antigen, the large S antigen (HBs-L), expressed by DNA vaccine, was not sufficiently immunogenic in eliciting antibody responses. In the current study, we produced a modified large S antigen DNA vaccine, HBs-L(T), which has a truncated N-terminal sequence in the pre-S1 region. Compared to the original HBs-L DNA vaccine, the HBs-L(T) DNA vaccine improved secretion in cultured mammalian cells and generated significantly enhanced HBsAg-specific antibody and B cell responses. Furthermore, this improved HBsL DNA vaccine, along with other HBsAg-expressing DNA vaccines, was able to maintain predominantly Th1 type antibody responses while recombinant HBsAg protein vaccines produced in either yeast or CHO cells elicited mostly Th2 type antibody responses. Our data indicate that HBsAg DNA vaccines with improved immunogenicity offer a useful alternative choice to recombinant protein-based HBV vaccines, particularly for therapeutic purposes against chronic hepatitis infection where immune tolerance led to poor antibody responses to S antigens.     

DNA shuffling and screening strategies for improving vaccine efficacy

The efficacy of vaccines can be improved by increasing their immunogenicity, broadening their crossprotective range, as well as by developing immunomodulators that can be coadministered with the vaccine antigen. One technology that can be applied to each of these aspects of vaccine development is MolecularBreeding directed molecular evolution. Essentially, this technology is used to evolve genes in vitro through an iterative process consisting of recombinant generation followed by selection of the desired recombinants. We have used DNA shuffling and screening strategies to develop and improve vaccine candidates against several infectious pathogens including Plasmodium falciparum (a common cause of severe and fatal human malaria), dengue virus, encephalitic alphaviruses such as Venezuelan, western and eastern equine encephalitis viruses (VEEV, WEEV, and EEEV, respectively), human immunodeficiency virus-1 (HIV-1), and hepatitis B virus (HBV). By recombining antigen-encoding genes from different serovar isolates, new chimeras are selected for crossreactivity; these vaccine candidates are expected to provide broader crossprotection than vaccines based on a single serovar. Furthermore, the vaccine candidates can be selected for improved immunogenicity, which would also improve their efficacy. In addition to vaccine candidates, we have applied the technology to evolve several immunomodulators that when coadministered with vaccines can improve vaccine efficacy by fine-tuning the T cell response. Thus, DNA shuffling and screening technology is a promising strategy to facilitate vaccine efficacy.     

A chloroplast-derived C4V3 polypeptide from the human immunodeficiency virus (HIV) is orally immunogenic in mice

Although the human immunodeficiency virus (HIV) causes one of the most important infectious diseases worldwide, attempts to develop an effective vaccine remain elusive. Designing recombinant proteins capable of eliciting significant and protective mammalian immune responses remain a priority. Moreover, large-scale production of proteins of interest at affordable cost remains a challenge for modern biotechnology. In this study, a synthetic gene encoding a C4V3 recombinant protein, known to induce systemic and mucosal immune responses in mammalian systems, has been introduced into tobacco chloroplasts to yield high levels of expression. Integration of the transgene into the tobacco plastome has been verified by Southern blot hybridization. The recombinant C4V3 protein is also detected in tobacco chloroplasts by confocal microscopy. Reactivity of the heterologous protein with both an anti-C4V3 rabbit serum as well as sera from HIV positive patients have been assayed using Western blots. When administered by the oral route in a four-weekly dose immunization scheme, the plant-derived C4V3 has elicited both systemic and mucosal antibody responses in BALB/c mice, as well as CD4+ T cell proliferation responses. These findings support the viability of using plant chloroplasts as biofactories for HIV candidate vaccines, and could serve as important vehicles for the development of a plant-based candidate vaccine against HIV.     

Recombinant vaccines for hepatitis E

Hepatitis E virus causes epidemics of acute hepatitis in many developing countries. It infrequently causes disease in developed countries, but avirulent strains might circulate. Some evidence suggests that hepatitis E might be a zoonosis. There is probably only a single serotype. A candidate vaccine consisting of baculovirus-expressed recombinant capsid protein protected macaques from hepatitis E--it passed phase I clinical trials and is currently scheduled for phase II/III clinical trials.     

A vectored measles virus induces hepatitis B surface antigen antibodies while protecting macaques against measles virus challenge

Hepatitis B virus (HBV) acute and chronic infections remain a major worldwide health problem. Towards developing an anti-HBV vaccine with single-dose scheme potential, we engineered infectious measles virus (MV) genomic cDNAs with a vaccine strain background and expression vector properties. Hepatitis B surface antigen (HBsAg) expression cassettes were inserted into this cDNA and three MVs expressing HBsAg at different levels generated. All vectored MVs, which secrete HBsAg as subviral particles, elicited humoral responses in MV-susceptible genetically modified mice. However, small differences in HBsAg expression elicited vastly different HBsAg antibody levels. The two vectors inducing the highest HBsAg antibody levels were inoculated into rhesus monkeys (Macaca mulatta). After challenge with a pathogenic MV strain (Davis87), control naive monkeys showed a classic measles rash and high viral loads. In contrast, all monkeys immunized with vaccine or a control nonvectored recombinant vaccine or HBsAg-expressing vectored MV remained healthy, with low or undetectable viral loads. After a single vaccine dose, only the vector expressing HBsAg at the highest levels elicited protective levels of HBsAg antibodies in two of four animals. These observations reveal an expression threshold for efficient induction of HBsAg humoral immune responses. This threshold is lower in mice than in macaques. Implications for the development of divalent vaccines based on live attenuated viruses are discussed.     

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.     

Targeting and expression of antigenic proteins in transgenic plants for production of edible oral vaccines

Summary Exploiting plants as biological bioreactors for production and delivery of edible oral subunit vaccines is a promising application of biotechnology. Efforts to enhance expression levels of transgenes coding for antigenic proteins by exploiting promoters, targeting sequences, and enhancer elements have produced rather low quantities of the antigen in plant tissues, but enough to induce immune responses in feeding studies. This review will cover components of various gene constructs used in developing plant-based vaccines against a myriad of viral and bacterial diseases. Specifically, it will focus on sequences that are involved in targeting the antigen to mucosal tissues of the intestinal tract, thus enhancing the immunogenicity of the plant-based vaccine as well as those components that result in higher accumulation of the protein within the plant.     

The structure of hepatitis B surface antigen and its antigenic sites

Hepatitis B virus infects about 200 million people worldwide yearly. The consequences of the infection range from mild, self-limiting hepatitis with full recovery and immunity to chronic infection with liver disease of varying severity, including fulminant hepatitis and death. Alternatively, individuals may become healthy carriers of the virus and thus serve as a reservoir of infection. In addition, all chronic carriers of the virus are also at risk for development of primary hepatocellular carcinoma later in life. Efforts to combat this disease on a global scale require an inexpensive vaccine. Studies of the hepatitis B viral coat protein (HBsAg) have permitted the development of two generations of vaccine to date, and detailed structural studies of its antigenic sites may eventually allow the development of a completely synthetic (and therefore inexpensive) vaccine in the future.     

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.     

乙型肝炎-卡介苗联合疫苗的研究进展

乙型肝炎疫苗和卡介苗均是我国常年生产和使用的生物制品,其安全性、有效性早已被证实。根据我国计划免疫规定,乙肝疫苗和卡介苗必须在新生儿出生后24h内接种。因诸多原因,不能简单地将现行2种疫苗混合使用,而须分2针接种。制备乙型肝炎-卡介苗联合疫苗,可以减少针次,降低副作用。动物试验显示,联合疫苗的免疫效果不低于2种单价疫苗。     

Prospects for new viral vaccines

Animal virology has made outstanding contributions to preventive medicine by the development of vaccines for the control of infectious disease in man and animals. Cost-benefit analysis indicates substantial savings in health care costs from the control of diseases such as smallpox, poliomyelitis, yellow fever and measels. Areas for further development include vaccines for influenza (living, attenuated virus), the herpes group (varicella: cytomegalovirus), respiratory syncytial virus, rotavirus and hepatitis A, B, and non A/non B. The general options for vaccine formulation are discussed with particular emphasis on approaches with the use of viral genetics to 'tailor make' vaccine viruses with defined growth potential in laboratory systems, low pathogenicity, and defined antigens. Current progress with the development of an inactivated hepatitis B vaccine is reviewed as a case study in vaccine development. The impact of recent experiments in cloning hepatitis B virus DNA in E. coli on the production of a purified viral polypeptide vaccine is assessed.     

丙型肝炎病毒（HCV）实验性疫苗的研究进展

丙型肝炎病毒是引起输血相关肝炎及慢性肝炎、肝硬化、肝癌的主要病原,目前尚无有效的治疗与预防手段。本文将综述HCV感染所引起的机体免疫应答及近年来实验性疫苗（主要是DNA疫苗、病毒载体疫苗及联合疫苗）的研究进展。