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

Plant-Made Vaccine Antigens and Biopharmaceuticals

Plant cells are ideal bioreactors for the production and oral delivery of vaccines and biopharmaceuticals, eliminating the need for expensive fermentation, purification, cold storage, transportation and sterile delivery. Plant-made vaccines have been developed for two decades but none has advanced beyond Phase I. However, two plant-made biopharmaceuticals are now advancing through Phase II and Phase III human clinical trials. In this review, we evaluate the advantages and disadvantages of different plant expression systems (stable nuclear and chloroplast or transient viral) and their current limitations or challenges. We provide suggestions for advancing this valuable concept for clinical applications and conclude that greater research emphasis is needed on large scale production, purification, functional characterization, oral delivery and preclinical evaluation

The Environmental Agency's Assessment of the Post-Closure Safety Case for the BNFL DRIGG Low Level Radioactive Waste Disposal Facility

The Environment Agency is responsible, in England and Wales, for authorization of radioactive waste disposal under the Radioactive Substances Act 1993. British Nuclear Fuels plc (BNFL) is currently authorized by the Environment Agency to dispose of solid low level radioactive waste at its site at Drigg, near Sellafield, NW England. As part of a planned review of this authorization, the Environment Agency is currently undertaking an assessment of BNFL's Post-Closure Safety Case Development Programme for the Drigg disposal facility. This paper presents an outline of the review methodology developed and implemented by the Environment Agency specifically for the planned review of BNFL's Post-Closure Safety Case. The paper also provides an overview of the Environment Agency's progress in its on-going assessment programme

Tissue-Print Immunodetection of Transgene Products in Endosperm for High-Throughput Screening of Seeds

This method allows high-throughput qualitative screening to identify seeds containing a transgene product in endosperm tissue. It is particularly useful for determining genetic segregation ratios or identifying seeds to be advanced in a breeding program. Tissue printing is used to avoid time-consuming extraction steps. Antibody-based detection of the transgene product makes this method suitable to any transgene product for which a specific antibody is available. It is possible to screen thousands of seeds per week using this method

Low Cost Industrial Production of Coagulation Factor IX Bioencapsulated in Lettuce Cells for Oral Tolerance Induction in Hemophilia B

Antibodies (inhibitors) developed by hemophilia B patients against coagulation factor IX (FIX) are challenging to eliminate because of anaphylaxis or nephrotic syndrome after continued infusion. To address this urgent unmet medical need, FIX fused with a transmucosal carrier (CTB) was produced in a commercial lettuce (Simpson Elite) cultivar using species specific chloroplast vectors regulated by endogenous psbA sequences. CTB-FIX (~1mg/g) in lyophilized cells was stable with proper folding, disulfide bonds and pentamer assembly when stored ~2 years at ambient temperature. Feeding lettuce cells to hemophilia B mice delivered CTB-FIX efficiently to the gut immune system, induced LAP+ regulatory T cells and suppressed inhibitor/IgE formation and anaphylaxis against FIX. Lyophilized cells enabled 10-fold dose escalation studies and successful induction of oral tolerance was observed in all tested doses. Induction of tolerance in such a broad dose range should enable oral delivery to patients of different age groups and diverse genetic background. Using Fraunhofer cGMP hydroponic system, ~870 kg fresh or 43.5 kg dry weight can be harvested per 1000 ft2 per annum yielding 24,000–36,000 doses for 20-kg pediatric patients, enabling first commercial development of an oral drug, addressing prohibitively expensive purification, cold storage/transportation and short shelf life of current protein drugs

Breadth of CD8 T-cell mediated inhibition of replication of diverse HIV-1 transmitted-founder isolates correlates with the breadth of recognition within a comprehensive HIV-1 Gag, Nef, Env and Pol potential T-cell epitope (PTE) peptide set.

Full characterisation of functional HIV-1-specific T-cell responses, including identification of recognised epitopes linked with functional antiviral responses, would aid development of effective vaccines but is hampered by HIV-1 sequence diversity. Typical approaches to identify T-cell epitopes utilising extensive peptide sets require subjects' cell numbers that exceed feasible sample volumes. To address this, CD8 T-cells were polyclonally expanded from PBMC from 13 anti-retroviral naïve subjects living with HIV using CD3/CD4 bi-specific antibody. Assessment of recognition of individual peptides within a set of 1408 HIV-1 Gag, Nef, Pol and Env potential T-cell epitope peptides was achieved by sequential IFNγ ELISpot assays using peptides pooled in 3-D matrices followed by confirmation with single peptides. A Renilla reniformis luciferase viral inhibition assay assessed CD8 T-cell-mediated inhibition of replication of a cross-clade panel of 10 HIV-1 isolates, including 9 transmitted-founder isolates. Polyclonal expansion from one frozen PBMC vial provided sufficient CD8 T-cells for both ELISpot steps in 12 of 13 subjects. A median of 33 peptides in 16 epitope regions were recognised including peptides located in previously characterised HIV-1 epitope-rich regions. There was no significant difference between ELISpot magnitudes for in vitro expanded CD8 T-cells and CD8 T-cells directly isolated from PBMCs. CD8 T-cells from all subjects inhibited a median of 7 HIV-1 isolates (range 4 to 10). The breadth of CD8 T-cell mediated HIV-1 inhibition was significantly positively correlated with CD8 T-cell breadth of peptide recognition. Polyclonal CD8 T-cell expansion allowed identification of HIV-1 isolates inhibited and peptides recognised within a large peptide set spanning the major HIV-1 proteins. This approach overcomes limitations associated with obtaining sufficient cell numbers to fully characterise HIV-1-specific CD8 T-cell responses by different functional readouts within the context of extreme HIV-1 diversity. Such an approach will have useful applications in clinical development for HIV-1 and other diseases

A Stronger Innate Immune Response During Hyperacute Human Immunodeficiency Virus Type 1 (HIV-1) Infection Is Associated With Acute Retroviral Syndrome

Background: Acute retroviral syndrome (ARS) is associated with human immunodeficiency virus type 1 (HIV-1) subtype and disease progression, but the underlying immunopathological pathways are poorly understood. We aimed to elucidate associations between innate immune responses during hyperacute HIV-1 infection (hAHI) and ARS. // Methods: Plasma samples obtained from volunteers (≥18.0 years) before and during hAHI, defined as HIV-1 antibody negative and RNA or p24 antigen positive, from Kenya, Rwanda, Uganda, Zambia, and Sweden were analyzed. Forty soluble innate immune markers were measured using multiplexed assays. Immune responses were differentiated into volunteers with stronger and comparatively weaker responses using principal component analysis. Presence or absence of ARS was defined based on 11 symptoms using latent class analysis. Logistic regression was used to determine associations between immune responses and ARS. // Results: Of 55 volunteers, 31 (56%) had ARS. Volunteers with stronger immune responses (n = 36 [65%]) had increased odds of ARS which was independent of HIV-1 subtype, age, and risk group (adjusted odds ratio, 7.1 [95% confidence interval {CI}: 1.7–28.8], P = .003). Interferon gamma-induced protein (IP)-10 was 14-fold higher during hAHI, elevated in 7 of the 11 symptoms and independently associated with ARS. IP-10 threshold >466.0 pg/mL differentiated stronger immune responses with a sensitivity of 84.2% (95% CI: 60.4–96.6) and specificity of 100.0% (95% CI]: 90.3–100.0). // Conclusions: A stronger innate immune response during hAHI was associated with ARS. Plasma IP-10 may be a candidate biomarker of stronger innate immunity. Our findings provide further insights on innate immune responses in regulating ARS and may inform the design of vaccine candidates harnessing innate immunity

What shapes attitudes on gender roles among adolescents in Bangladesh

Background In Bangladesh, large gender differentials exist in outcomes in almost all spheres of life, stemming from conservative norms and attitudes around gender. Adolescence is a crucial period for social-emotional learning that can shape gender norms and attitudes.ObjectiveThe aim of the paper is to investigate the extent to which adolescents hold egalitarian attitudes toward gender roles, and to examine the factors that influence egalitarian gender attitudes. Methods The paper uses data from a nationally representative sample survey of 7,800 unmarried girls and 5,523 unmarried boys ages 15–19 years. Adolescents were considered to have egalitarian attitudes on gender role if they disagreed with all the following four unequal gender role statements with regards to socio-economic participation, while respondents who agreed with any one of the four statements were considered to have non-egalitarian attitudes: (1) It is important that sons have more education than daughters, (2) Outdoor games are only for boys, not girls, (3) Household chores are for women only, not for men, even if the woman works outside the home, and (4) Women should not be allowed to work outside the home. Multivariable linear probability regression analysis was implemented to identify the factors shaping attitudes on gender roles. Results Unmarried girls and boys differ hugely in their views on gender roles regarding socio economic participation-girls were much more egalitarian than boys (58 vs. 19%). The multivariate linear probability model results show girls and boys who completed at least grade 10 were 31% points and 15% points more likely to have egalitarian views on gender roles respectively, compared to girls and boys with primary or less education. Having strong connection with parents is associated with having egalitarian views on gender roles among girls but not boys. Adolescents' individual attitude on gender role is highly associated with the views of their community peers for both girls and boys. Girls and boys who had participated in adolescent programs were 6–7% points more likely to have egalitarian attitude than those who were not exposed to these programs. Egalitarian views were also significantly higher, by 5% points among girls and 6% points among boys, who were members of social organizations compared to those who were not. Watching television had positive influence on egalitarian attitudes among girls but not among boys. To create a more egalitarian society, both men and women need to hold progressive attitudes toward gender roles. The interventions must be multilevel, influencing adolescents at the personal, interpersonal, communal, and societal levels