Syed Waqas Hassan

Plastid expression of a double‐pentameric vaccine candidate containing human papillomavirus‐16 L1 antigen fused with LTB as adjuvant: transplastomic plants show pleiotropic phenotypes

Human papillomavirus (HPV) causes cervical cancer in women worldwide, which is currently prevented by vaccines based on virus-like particles (VLPs). However, these vaccines have certain limitations in their availability to developing countries, largely due to elevated costs. Concerning the highest burden of disease in resource-poor countries, development of an improved mucosal and cost-effective vaccine is a necessity. As an alternative to VLPs, capsomeres have been shown to be highly immunogenic and can be used as vaccine candidate. Furthermore, coupling of an adjuvant like Escherichia coli heat-labile enterotoxin subunit B (LTB) to an antigen can increase its immunogenicity and reduce the costs related to separate co-administration of adjuvants. Our study demonstrates the expression of two pentameric proteins: the modified HPV-16 L1 (L1_2xCysM) and LTB as a fusion protein in tobacco chloroplasts. Homoplasmy of the transplastomic plants was confirmed by Southern blotting. Western blot analysis showed that the LTB-L1 fusion protein was properly expressed in the plastids and the recombinant protein was estimated to accumulate up to 2% of total soluble protein. Proper folding and display of conformational epitopes for both LTB and L1 in the fusion protein was confirmed by GM1-ganglioside binding assay and antigen capture ELISA, respectively. However, all transplastomic lines showed chlorosis, male sterility and growth retardation, which persisted in the ensuing four generations studied. Nevertheless, plants reached maturity and produced seeds by pollination with wild-type plants. Taken together, these results pave the way for the possible development of a low-cost adjuvant-coupled vaccine with potentially improved immunogenicity against cervical cancer.

Plant-derived vaccines: an approach for affordable vaccines against cervical cancer.

Several types of human papillomavirus (HPV) are causatively associated with cervical cancer, which is the second most common cancer in women worldwide. HPV-16 and 18 are among the high risk types and responsible for HPV infection in more than 70% of the cases. The majority of cervical cancer cases occur in developing countries. Currently available HPV vaccines are expensive and probably unaffordable for most women in low and middle income countries. Therefore, there is a need to develop cost-effective vaccines for these countries. Due to many advantages, plants offer an attractive platform for the development of affordable vaccines. These include low cost of production, scalability, low health risks and the potential ability to be used as unprocessed or partially processed material. Among several techniques, chloroplast transformation is of eminent interest for the production of vaccines because of high yield of foreign protein and lack of transgene transmission through pollen. In this commentary, we focus on the most relevant aspects of plant-derived vaccines that are decisive for the future development of cost-effective HPV vaccines.

Expression of HPV-16 L1 capsomeres with glutathione-S-transferase as a fusion protein in tobacco plastids: an approach for a capsomere-based HPV vaccine.

Human Papillomavirus (HPV) is the main cause of cervical cancer, which is the second most severe cancer of women worldwide, particularly in developing countries. Although vaccines against HPV infection are commercially available, they are neither affordable nor accessible to women in low income countries e.g. Africa. Thus, alternative cost-effective vaccine production approaches need to be developed. This study uses tobacco plants to express pentameric capsomeres of HPV that have been reported to generate elevated immune responses against HPV. A modified HPV-16 L1 (L1_2xCysM) protein has been expressed as a fusion protein with glutathione-S-transferase (GST) in tobacco chloroplasts following biolistic transformation. In total 7 transplastomic lines with healthy phenotypes were generated. Site specific integration of the GST-L1_2xCysM and aadA genes was confirmed by PCR. Southern blot analysis verified homogenous transformation of all transplastomic lines. Antigen capture ELISA with the conformation-specific antibody Ritti01, showed protein expression as well as the retention of immunogenic epitopes of L1 protein. In their morphology, GST-L1 expressing tobacco plants were identical to wild type plants and yielded fertile flowers. Taken together, these data enrich knowledge for future development of cost-effective plant-made vaccines against HPV.

Recent progress in bioethanol production from lignocellulosic materials: A review

ABSTRACT Natural energy sources like petrol and diesel are going to be diminished in the coming future which will lead to increase in the prices and demands of fossil fuels. Therefore, it is important to find a sustainable alternate of fossil fuels. Bioethanol is one of the alternatives, which is produced from different feedstocks including sugar-based, starch-based and lignocellulose-based materials through fermentation. Since sugar-based (sugar cane and sugar beet) and starch-based (corn) materials are sources of staple food, therefore, research on lignocellulosic materials for bioethanol production is a subject of recent studies. Ethanol production from lignocellulosic materials involves different steps, such as pretreatment, hydrolysis, followed by fermentation process and finally ethanol purification. In this review, we have summarized the recent progresses in bioethanol production and processing from lignocellulosic materials.

Towards Generation of Transplastomic Tobacco, Expressing a 35 kdaProtein as an Antigen: A Step towards Affordable Plant made Vaccine against Mycobacterium

Plants offer the unique opportunity to be engineered as bio-factories for the production of different antigens, enzymes and vaccines. Recently chloroplast transformation has gained strong interest in the field of Plant Made Vaccines (PMVs). An efficient cross-protective antigen against Mycobacterium is a 35 kDa protein encoded by mmpI gene in Mycobacterium leprae. Although vaccines against Mycobacterium infections are commercially available, they are neither affordable nor available for patients in resource poor countries. Thus, alternative cost-effective vaccine production approaches need to be developed. In the current study we have reported the possibility to generate transplastomic tobacco carrying 35-kDa protein conferring cross-protective resistance against two different pathogens Mycobacterium leprae and Mycobacterium avium. The mmpI gene along with an adjuvant Lymphotoxin-beta (LTB) was successfully transformed into tobacco chloroplast by the Polyethylene glycol (PEG) mediated transformation method. The PEG transformation method provides an effective and cost efficient transformation procedure and can easily be adopted in resource-poor countries in comparison to biolistic transformation. Integration of LTB and mmpI genes into transplastomic plant genome was confirmed by PCR analysis. In total four transplastomic lines were generated which were healthy and normal. All plants had regular growth pattern, they were able to reach maturity and produce viable seeds. Taken together, the data presented in the study is a valuable step forward to pave the way in the development of cost effective and easily administrable PMVs for resource-poor countries.

New areas of plant-made pharmaceuticals

On October 15 2010 the meeting ‘Recombinant Pharmaceutical Manufacturing from Plants – The Future of Molecular Farming’ hosted by EuroScicon was held at BioPark Hertfordshire, Welwyn Garden city, UK. The scientific program of this very eventful meeting was wide ranging and covered diverse aspects of biopharming. The highlights presented included: safety issues in biopharming; coexpression of multiple proteins; steps towards vaccine generation; and engineering of secondary metabolites and medicinal plants. This article summarizes the stimulating scientific presentations and fruitful panel discussions that subsequently arose during and after this event.

Stable expression of pentameric capsomeres, HPV-16 L1 with glutathione-S-transferase in transplastomic tobacco: a step towards affordable capsomere-based HPV vaccine

Human Papillomavirus (HPV) is a single major source in causing cervical cancer worldwide which is the most common cause of death in females. Around 85% of the cervical cancer cases occur in developing countries where existing vaccines are not available or unaffordable. Hence, alternative platforms are considered necessary for the development of cost-effective vaccines against HPV for their availability in developing countries. Plants offer unique advantages for vaccine production over fermenter-based systems. Pentameric capsomeres ofHPVhave been reported to generate elevated immune responses against HPV. In present study a modified HPV-16 L1 (L1 2xCysM) protein has been expressed as a fusion protein with glutathione-S-transferase (GST) into tobacco plastids. Seven transplastomic lines of Nicotiana benthamiana were generated using biolistic transformation technique. Site specific integration of the GST-L1 2xCysM and aadA genes was confirmed by PCR. Southern blot analysis verified homoplasmy of all transplastomic lines.AntigencaptureELISAwith theconformationspecific antibody Ritti01, showed protein expression as well as the retention of immunogenic epitopes of L1 protein. GST-L1 expressing tobacco plants were completely normal in their morphology. Taken together, this data contribute another step forward towards thedevelopmentof cost-effectiveplant-madevaccinesagainstHPV for resource poor countries.