Plants as biofactories: Production of pharmaceutical recombinant proteins

Abstract Plants can be used for the large-scale production of a variety of recombinant proteins, destined for agroindustrial, biomedical and pharmaceutical applications. In fact, blood components, hormones, enzymes, cytokines, antibodies and vaccines have been successfully produced in plants. Plant systems offer unique advantages in comparison with conventional techniques (bacteria, mammal or yeast cells): i) lower production costs; ii) synthesis of functional proteins, similar to those produced in animal cells, absolutely free of animal pathogens; iii) easy scale-up and purification technology. Furthermore, the engineering of edible plants may allow for the delivery of the recombinant protein (e.g. vaccines) through fruits, tubers, leaves or seeds. In this way the cold chain, necessary for the storage and the transport of purified recombinant products could be avoided, as well as the administration procedures by injection. This review describes gene transfer methods (including stable and transient transformation), plant species used and strategies to obtain high yields of protein, with attention focused towards plant-derived antibodies and vaccines, known as "plantibodies" or "plantigens". Up to now, several groups are working in this promising field of research demonstrating that plants are able to produce proteins derived from different kingdoms, with highly complex structures (e.g. immunoglobulins A and G) and the different, post-translational, modification patterns of plants do not dramatically affect the properties or the biological activity of the recombinant protein. Using plants as biofactories to produce "green" therapeutic proteins is not only a proof of concept but a reality as an approach for agroindustry

Engineering stable cytoplasmic intrabodies with designed specificity

Many attempts have been made to develop antibody fragments that can be expressed in the cytoplasm ("intrabodies") in a stable and functional form. The recombinant antibody fragment scFv(F8) is characterised by peculiarly high in vitro stability and functional folding in both prokaryotic and eukaryotic cytoplasm. To dissect the relative contribution of different scFv(F8) regions to cytoplasmic stability and specificity we designed and constructed five chimeric molecules (scFv-P1 to P5) in which several groups of residues important for antigen binding in the poorly stable anti-hen egg lysozyme (HEL) scFv(D1.3) were progressively grafted onto the scFv(F8) scaffold. All five chimeric scFvs were expressed in a soluble form in the peri-plasm and cytoplasm of Escherichia coli. All the periplasmic oxidised forms and the scFv(P3) extracted from the cytoplasm in reducing conditions had HEL binding affinities essentially identical (K-d = 15 nM) to that of the cognate scFv(D1.3) fragment (K-d = 16 nM). The successful grafting of the antigen binding properties of D1.3 onto the scFv(F8) opens the road to the exploitation of this molecule as a scaffold for the reshaping of intrabodies with desired specificities to be targeted to the cytoplasm. (C) 2003 Elsevier Science Ltd. All rights reserved

<i>Wolbachia</i> Density and Cytoplasmic Incompatibility in <i>Aedes albopictus</i>: Concerns with Using Artificial <i>Wolbachia</i> Infection as a Vector Suppression Tool

<div><p>The mosquito <i>Aedes albopictusi</i> is a competent vector of harmful human pathogens, including viruses causing dengue and chikungunya. Cytoplasmic incompatibility (CI) induced by endosymbiotic <i>Wolbachia</i> can be used to produce functionally sterile males that can be released in the field as a suppression tool against this mosquito. Because the available sexing methods are not efficient enough to avoid unintentional release of a few transinfected females, we assessed the CI pattern in crosses between <i>w</i>Pip <i>Wolbachia</i>-transinfected (AR<i>w</i>P) females and wild-type males of <i>Ae</i>. <i>albopictus</i> in this study. Quantitative polymerase chain reaction was used to monitor the titer of the <i>Wolbachia</i> strains that naturally infect <i>Ae</i>. <i>albopictus</i>, that is, <i>w</i>AlbA and <i>w</i>AlbB, in age-controlled males and females. Data were coupled with incompatibility level detected when the above-mentioned males were crossed with AR<i>w</i>P females. <i>Wolbachia</i> infection titer was also monitored in samples of wild caught males. Incompatibility level was positively correlated only with <i>w</i>AlbA density. Crosses between wild-type males having very low <i>w</i>AlbA density (<0.001 <i>w</i>AlbA/actin copy numbers) and AR<i>w</i>P females were partially fertile (CI_corr = 68.06 ± 6.20). Individuals with low <i>w</i>AlbA titer were frequently found among sampled wild males (30%–50% depending on the site and period). AR<i>w</i>P males can be as considered as a very promising tool for suppressing <i>Ae</i>. <i>albopictus</i>. However, crosses between wild males having low <i>w</i>AlbA density and AR<i>w</i>P females may be partially fertile. In the case of local establishment of the transinfected mosquito line, this occurrence may favor the replacement of the wild-type mosquitoes with the AR<i>w</i>P line, thus reducing the long-term efficacy of incompatible insect technique. Various alternative strategies have been discussed to prevent this risk and to exploit <i>Wolbachia</i> as a tool to control <i>Ae</i>. <i>albopictus</i>.</p></div

Humanization of a highly stable single-chain antibody by structure-based antigen-binding site grafting

The murine single-chain variable fragment F8 (scFv(F8)) is endowed with high intrinsic thermodynamic stability and can be functionally expressed in the reducing environment of both prokaryotic and eukaryotic cytoplasm. The stability and intracellular functionality of this molecule can be ascribed mostly to its framework regions and are essentially independent of the specific sequence and structure of the supported antigen-binding site. Therefore, the scFv(F8) represents a suitable scaffold to construct stable scFv chimeric molecules against different antigens by in vitro evolution or antigen-binding site grafting. Thanks to the favourable pharmacokinetic properties associated to a high thermodynamic stability of antibody fragments, such scFv(F8) variants may be exploited for a wide range of biomedical applications, from in vivo diagnosis to therapy, as well as to interfere with the function of intracellular proteins and pathogens, and for functional genomics studies. However, the potential immunogenicity of the murine framework regions represents a limitation for their exploitation in therapeutic applications. To overcome this limitation, we humanized a derivative of the scFv(FS), the anti-lysozyme scFv(11E), which is endowed with even higher thermodynamic stability than the parent antibody. The humanization was carried out by substituting the framework residues differing from closely related V-H and V-L domains of human origin with their human counterparts. Site-directed mutagenesis generated the fully humanized product and four intermediate scFvs, which were analyzed for protein expression and antigen binding. We found that the substitution Tyr 90 -> Phe in the V-H domain dramatically reduced the bacterial expression of all mutants. The back-mutation of Phe H90 to Tyr led to the final humanized variant named scFv(H5)H90Tyr. This molecule comprises humanized V-H and V-L framework regions and is endowed with HEL-binding affinity, stability in human serum and functionality under reducing conditions comparable to the murine cognate antibody. Consequently, the humanized scFv(H5)H90Tyr represents a suitable scaffold onto which new specificities towards antigens of therapeutic interest can be engineered for biomedical applications. (C) 2008 Elsevier Ltd. All rights reserved

Combining Wolbachia-induced sterility and virus protection to fight Aedes albopictus-borne viruses

International audienceAmong the strategies targeting vector control, the exploitation of the endosymbiont Wolba-chia to produce sterile males and/or invasive females with reduced vector competence seems to be promising. A new Aedes albopictus transinfection (ARwP-M) was generated by introducing wMel Wolbachia in the ARwP line which had been established previously by replacing wAlbA and wAlbB Wolbachia with the wPip strain. Various infection and fitness parameters were studied by comparing ARwP-M, ARwP and wild-type (S ANG population) Ae. albopictus sharing the same genetic background. Moreover, the vector competence of ARwP-M related to chikungunya, dengue and zika viruses was evaluated in comparison with ARwP. ARwP-M showed a 100% rate of maternal inheritance of wMel and wPip Wolba-chia. Survival, female fecundity and egg fertility did not show to differ between the three Ae. albopictus lines. Crosses between ARwP-M males and S ANG females were fully unfertile regardless of male age while egg hatch in reverse crosses increased from 0 to about 17% with S ANG males aging from 3 to 17 days. When competing with S ANG males for S ANG females, ARwP-M males induced a level of sterility significantly higher than that expected for an equal mating competitiveness (mean Fried index of 1.71 instead of 1). The overall Wolbachia density in ARwP-M females was about 15 fold higher than in ARwP, mostly due to the wMel infection. This feature corresponded to a strongly reduced vector competence for chikungunya and dengue viruses (in both cases, 5 and 0% rates of transmission at 14 and 21 days post infection) with respect to ARwP females. Results regarding Zika virus did not highlight significant differences between ARwP-M and ARwP. However, none of the tested ARwP-M females was capable at transmitting ZIKV. These findings are expected to promote the exploitation of Wolbachia to suppress the wild-type Ae. albopictus populations. PLOS Neglected Tropical Diseases | https://doi

Density of <i>w</i>AlbA <i>Wolbachia</i> in wild caught <i>Ae</i>. <i>albopictus</i> males from two Italian sites (Crevalcore, CRE, Bologna; Anguillara Sabazia, ANG, Rome) and two periods (September 2013; July 2014).

<p>Density values have been clustered in three density classes. The individuals belonging to each density class are reported as percentages of the whole amount. CI level is expected to decrease to about 68% in crosses between AR<i>w</i>P females and males with <i>w</i>AlbA density values <0.001 <i>w</i>AlbA/act.</p

Age dependent variation of <i>w</i>AlbA density in <i>Ae</i>. <i>albopictus</i> males (a) and females (b) belonging to two Italian populations (S_ANG from Anguillara Sabazia, Rome: empty squares; S_CRE from Crevalcore, Bologna: full circles).

<p>Data trend is displayed via a polynomial trend-line together with the associated function (dashed line for S_ANG, solid line for S_CRE).</p

Correlation between <i>w</i>AlbA (a) or <i>w</i>AlbB (b) <i>Wolbachia</i> density and expressed CI levels (CI_corr index) in <i>Ae</i>. <i>albopictus</i> males when crossed with AR<i>w</i>P females at three different ages (3 ± 1; 11 ± 1; 19 ± 1).

<p>Data trend is displayed via a polynomial trend-line together with the associated function.</p

Density of <i>w</i>AlbA <i>Wolbachia</i> and associated CI level expressed by <i>Ae</i>. <i>albopictus</i> males when crossed with AR<i>w</i>P females.

<p>Density values have been clustered in three density classes.</p><p>* − statistically significant difference, by Bonferroni’s multiple comparison test with α = 0.05.</p><p>Density of <i>w</i>AlbA <i>Wolbachia</i> and associated CI level expressed by <i>Ae</i>. <i>albopictus</i> males when crossed with AR<i>w</i>P females.</p