Frontiers in the Standardization of the Plant Platform for High Scale Production of Vaccines

Abstract

The recent COVID-19 pandemic has highlighted the value of technologies that allow a fast setup and production of biopharmaceuticals in emergency situations. The plant factory system can provide a fast response to epidemics/pandemics. Thanks to their scalability and genome plasticity, plants represent advantageous platforms to produce vaccines. Plant systems imply less complicated production processes and quality controls with respect to mammalian and bacterial cells. The expression of vaccines in plants is based on transient or stable transformation systems and the recent progresses in genome editing techniques, based on the CRISPR/Cas method, allow the manipulation of DNA in an efficient, fast, and easy way by introducing specific modifications in specific sites of a genome. Nonetheless, CRISPR/Cas is far away from being fully exploited for vaccine expression in plants. In this review, an overview of the potential conjugation of the renewed vaccine technologies (i.e., virus-like particles-VLPs, and industrialization of the production process) with genome editing to produce vaccines in plants is reported, illustrating the potential advantages in the standardization of the plant platforms, with the overtaking of constancy of large-scale production challenges, facilitating regulatory requirements and expediting the release and commercialization of the vaccine products of genome edited plants.

Keywords: genome editing; plant factory system; vaccines; virus-like particles.

Figure 1

Advantages of the CRISPR/Cas9 method with respect to Agrobacterium-mediated transformation. The peculiarities of CRISPR/Cas9 (indicated in the green part) with the corresponding ameliorations (indicated in the red part), in comparison to the conventional Agrobacterium-based transformation, are represented. HDR = homologous-derived repair, CoP = constitutive promoter, sgRNA = single guide RNA, RNP = ribonucleoprotein. Created with Biorender.com (https://biorender.com/; accessed on 20 April 2021).

Figure 2

Alignments of tobacco TVCV-related sequence Ntab-TN90_AYMY-SS16611 and TVCV genomic sequences and the predicted proteins. Alignments were obtained by Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo; accessed on 20 April 2021) and visualized by Multiple Sequence Alignment Viewer 1.20.0 (https://www.ncbi.nlm.nih.gov/projects/msaviewer; accessed on 20 April 2021). The percentage of sequence similarity and gaps are reported for each alignment. The four ORFs in the TVCV genome are highlighted for the DNA alignment: red—CP (coat protein) encoding ORF1, yellow—MP (movement protein) encoding ORF2, green—Pr (peptidase)/RT (reverse transcriptase)/RH (Rnase H) encoding ORF3, blue—TF (transactivator factor) encoding ORF4. Red bars in the DNA alignment indicate sequence differences. The protein domain detected by Pfam (http://pfam.xfam.org/; accessed on 20 April 2021) are indicated in the protein alignments. The function of RasMol amino acid colors has been utilized.

Figure 3

Jalview alignment of the CP encoded by Ntab-TN90_AYMY-SS16611 and TVCV. Conserved residues are indicated in blue in the Jalview alignment [121]. Conservation and quality of the alignment are indicated. Secondary structures, predicted by Jpred Secondary Structure Prediction are indicated in green (β-sheets) and red (α-helices); JNetCONF = the confidence estimate for the prediction; JNetHMM = HMM profile-based prediction; JNETPSSM = PSSM-based prediction; JNETJURY = a ‘*’ in this annotation indicates that the JNETJURY was invoked to rationalize significantly different primary predictions.

Figure 4

Predicted 3D structures of Ntab-TN90_AYMY-SS16611 and TVCV CPs. The 3D structures have been predicted by trRosetta [122] and visualized by RasMol [123] from the pdb files obtained by trRosetta [122].

Figure 5

Steps for the generation of a standardized tobacco host for vaccine production through the association between CVP and genome editing techniques. Genome editing can be used to manipulate the TVCV-like sequences integrated in N. tabacum genome showing high sequence similarity to TVCV CP encoding ORF. Two possible strategies can be postulated depending on the ability of the tobacco CP corresponding sequences to generate VLPs. (1) Functional tobacco TVCV-related CPs: a small epitope encoding sequence can be fused to the tobacco CP-related ORF by prime editing; a constitutive promoter region can be integrated upstream the CP-related ORF by CRISPR-Cas9 taking advantage of HDR. (2) Unfunctional tobacco TVCV-related CP: the tobacco CP-related sequence could be replaced by the TCVC CP ORF carrying a constitutive promoter and the epitope encoding sequence by using CRISPR-Cas9 with sgRNAs targeting sites flanking the tobacco locus. The standardized host plant, able to produce functional CVPs, can be successively modified by replacing the epitope sequence through base editing in order to obtain functional CVPs towards different pathogens/strains. CP = coat protein, Ep1 = epitope 1, Ep2 = epitope 2, CoP = constitutive promoter. Created with Biorender.com (https://biorender.com/; accessed on 20 April 2021).

Figure 6

Summary of the production steps and quality control specification of industrial vaccines production.