Socioeconomic, demographic and environmental determinants of infant mortality in Nepal

The Nepal Fertility and Family Planning Survey of 1986 demonstrated that demographic variables, previous birth interval and survival of preceding child, still predominated as determinants of infant mortality, particularly in rural areas of Nepal. However, in urban Nepal, where the level of socioeconomic development is higher, an environmental variable, along with previous birth interval and survival of preceding child emerges as important in determining infant mortality. Separate policy measures for child survival prospects in rural and urban Nepal are suggested.     

The maize α-zein promoter can be utilized as a strong inducer of cellulase enzyme expression in maize kernels

Transgenic Research - Expression of recombinant proteins in plants is a technology for producing vaccines, pharmaceuticals and industrial enzymes. For the past several years, we have produced...     

Engineered chloroplasts as vaccine factories to combat bioterrorism

Bacillus anthracis is ideal for making biological weapons, but the licensed anthrax vaccine is unsuitable for widespread public administration. Recombinant subunit-vaccine candidates offer potential alternatives, and plant-based production systems facilitate the inexpensive bulking of target antigens. A recent report demonstrates expression of anthrax protective antigen in tobacco chloroplasts--this material is immunogenic and protective when injected into mice. Provided an economic purification scheme can be developed, this technology holds promise for an improved vaccine.     

Production of recombinant proteins in clonal root cultures using episomal expression vectors

We have developed a fully contained system for expressing recombinant proteins that is based on clonal root cultures and episomal expression vectors. Clonal root lines expressing green fluorescent protein (GFP) or human growth hormone were generated from Nicotiana benthamiana leaves infected with the tobacco mosaic virus-based vector 30B after exposure to Agrobacterium rhizogenes. These lines accumulated GFP at over 50 mg per kg fresh tissue, a level that is comparable with other plant production systems in early stage development. Accumulation of both hGH and GFP in the clonal root lines was sustained over a 3-year period, and in the absence of antibiotic selection. This technology shows promise for commercial production of vaccine antigens and therapeutic proteins in contained facilities.     

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.     

Development of an edible subunit vaccine in corn against enterotoxigenic strains of escherichia coli

Summary Advances in the development of subunit vaccines and in the production of foreign proteins in plants together offer the prospect of stable and inexpensive vaccine delivery systems. Various bacterial and viral proteins stably produced in plants have been shown to elicit immune responses in feeding trials. We have extended this approach by using Zea mays as the plant production system. Corn has several advantages as a vaccine delivery vehicle, most notably established technologies to generate transgenic plants, to optimize traits through breeding and to process the seed into a palatable form. Here we report on the production in corn seed of the GM₁ receptor binding (B) subunit of the heat-labile toxin (Lt) from enterotoxigenic strains of Escherichia coli. Versions of the Lt-B gene were synthesized to give optimum codon usage for corn and to target the protein to either the cell surface or the cytoplasm. These synthetic genes were fused to a strong promoter and transformed into corn. Lt-B was highly expressed in corn seed at up to 1.8% of the total soluble protein and this was further increased approximately five-fold through plant breeding. As in E. coli. Lt-B produced in corn forms a functional pentamer that can bind to the GM₁ receptor. Furthermore, Lt-B pentamer stored in corn seed is much more resistant to heat than is the pure protein, allowing the transgenic corn to be readily processed into an edible form. This work demonstrates the potential of using products derived from transgenic corn seed as delivery vehicles for subunit vaccines.     

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.     

Medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants

The use of plants for medicinal purposes dates back thousands of years but genetic engineering of plants to produce desired biopharmaceuticals is much more recent. As the demand for biopharmaceuticals is expected to increase, it would be wise to ensure that they will be available in significantly larger amounts, on a cost-effective basis. Currently, the cost of biopharmaceuticals limits their availability. Plant-derived biopharmaceuticals are cheap to produce and store, easy to scale up for mass production, and safer than those derived from animals. Here, we discuss recent developments in this field and possible environmental concerns.