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mRNA Vaccine

Overview

Messenger RNA (mRNA) vaccine is a new vaccine technology developed in recent years.  Compared with traditional vaccines, mRNA vaccines have the advantages of high safety, good balanced immunity, short development cycle and low production cost. With the rapid development of mRNA modification and delivery technology, mRNA technology has rapidly matured, showing broad application prospects in tumor treatment, prevention and treatment of viral diseases, etc. SARS-CoV-2 mRNA vaccine has been developed and successfully applied at a record speed, paving the way for the promotion of mRNA technology in the future.

Strategies for mRNA preparation

  • Sequence construction

First, mRNA vaccines require the design of a DNA template to be transcribed in vitro. The DNA template should contain at least an Open Reading Frame (ORF) and a 5' untranslated region (5' UTR) and 3 'UTR on its flanks. A primer binding site containing available RNA polymerase (eg. T7, T3 or SP6 phage RNA polymerase) is then required to initiate in vitro transcription1.

  • In vitro transcription (IVT)

In vitro transcription (IVT) is a process in which designed template DNA strands are transcribed into RNA strands according to the principle of base complementarity. Transcription begins after RNA polymerase recognizes the transcription promoter. Modified nucleotides can be used to further stabilize RNA and reduce immunogenicity during in vitro transcription2.

  • Adding cap

In vitro transcribed RNA has a highly immunogenic 5 'triphosphate portion. Tri-phosphorylated RNA is recognized by pattern recognition receptor (PRR) in the cytoplasm and induces Type-I interferon (IFN I) response3. To prevent RNA from being recognized as foreign, its triphosphate must be removed and a 5 'cap added.

  • Tailing

Poly(A) tails can slow down the degradation process of RNA from RNA exonuclease and improve RNA stability and translation efficiency. Poly (A) tails are typically 100-250 nucleotides long, but the optimal length depends on the target cell type. The use of modified adenosine can further improve the stability of poly(A) tails and make them resistant to cellular ribonuclease degradation4.

  • Purification

After in vitro transcription, the mRNA needs to be purified and its concentration determined to exclude abnormal, truncated and degraded products. Clinically, mRNA is purified by chromatography to remove short template fragments due to transcription failure or double-stranded (ds) RNA due to self-complementary 3 'extensions, both of which are common causes of impurities.

mRNA delivery system

  • Polymeric nanoparticles

Encapsulation of mRNA with polymeric nanoparticles protects them from degradation, and their specific chemical properties facilitate cellular uptake and endosomal escape. Polyethylenimine (PEI) is a cationic polymer widely used in nucleic acid transportation, and jetPEI®, a commercial linear PEI derivative, has been used for mRNA transfection in vivo and in vitro. Low molecular PEI modified by aliphatic chain can reduce toxicity5.

  • Protein derivative-mRNA complex

Protein derivative-mRNA complex refers to the direct conjugation of proteins such as protamine to RNA. This complex enters the cell through endocytosis and is conjugated by toll-like receptor (TLR), Myeloid Differentiation Marker 88 (MyD88) dependent pathway to activate the immune system.

  • Lipid Nanoparticles (LNPs)

LNPs was originally developed for the delivery of small interfering RNA (siRNA), and has become the most widely used material for delivering mRNA in vivo6.

Major delivery methods for mRNA vaccines

Figure 1. Major delivery methods for mRNA vaccines7

Immunogenicity of mRNA vaccine

Innate immune response

Recognition of immune cells

Exogenous mRNA is generally considered to be immunogenic, exhibiting RNA-virus-like characteristics and activating innate immune cells through TLR. TLR belongs to a group of innate immune response PRR that act as "receptors" for pathogen-associated molecular patterns (PAMP) to detect PAMP. mRNA can be recognized by Antigen presenting cell (APC) and activate TLR, such as TLR3, TLR7 and TLR88. Once TLR detects PAMP, it primes the innate immune response by continuously activating the adaptive immune response.

Recognition of non-immune cells

Retinoic acid-inducible gene I (RIG-I) -like receptor (RLR) in non-immune cells and melanoma differentiation-associated gene 5 (MDA5) sense exogenous mRNA and mediate the production of cytokines and chemokines in mice. Innate immune cells (such as DC and macrophages) are recruited to the injection site of mRNA9. Although early induction of powerful cytokines is beneficial for vaccine efficacy, the vaccine cannot be fully effective because cytokines can cause severe systemic side effects such as autoimmunity or impair the immune response of the mRNA vaccine.

Adaptive immune responses

Antigen presentation

After an injection of the mRNA vaccine, the protein it encodes is translated and presented to the immune system, triggering acquired immunity. The proteins encoded by mRNA are translated and taken up by APC (such as DC) through micro-endocytosis, endocytosis or phagocytosis, which may form phagosomes or endosomes containing antigenic proteins10. Major histocompatibility complex I (MHC-I) and major histocompatibility complex II (MHC-II) were presented on DC. APC can deliver exogenous antigens to CD4+T cells via MHC-II and cross present them to CD8+T cells on MHC-I. The resulting induction of cytotoxic T lymphocytes is called cross excitation. CD4+T cells help B cells and CD8+T cells. Finally, clonal amplification of antigen-specific B and T cells results in the elimination of target cells.

Immunological effect

While peptide-based vaccines are MHC limited, mRNA vaccines allow combinations of mRNA that encode different antigens. mRNA electroporated dendritic cells possess a variety of MHC-I and II restrictive peptides and can induce polyclonal CD4+ and CD8+T cell responses. CD4+ helper T cells are very important for effectively inducing cytotoxic T lymphocyte (CTL) and B cell responses11, and mRNA vaccine can further improve the immune response in the presence of helper epitopes. The induction of CD4+T cell response is autophagy mediated by the introduction of mRNA into dendritic cells. Finally, vaccine compositions can include mRNA that encode immunomodulatory proteins, further enhancing their potency.

Analysis of mRNA therapeutics and vaccines

Given the shrinking timelines for drug development, access to faster and higher-throughput analytical methods has become vital. Indeed, it is essential that analytical approaches for use during mRNA product development and quality-control (QC) testing must keep pace to ensure the identity, safety, and efficacy of these new prophylactic and therapeutic modalities.

  • Security risk

Emphasis should be placed on lipid-related toxicity. Non-specific immune reaction, degradation products produced during preparation and storage, the safety of positive polymer material itself, and the accumulated safety risk of various impurities can be considered comprehensively.

  • mRNA

The proportion of mRNA modification, poly A tail adding efficiency, cap adding efficiency, degree of dephosphorylation, mRNA degradation fragment, mRNA integrity and sequence accuracy, dsRNA and mRNA content should be fully studied. Among them, uncapped, phosphorylated mRNA and dsRNA have non-specific immune stimulation, which are the most concerned impurities in immunology. mRNA modification has two sides. On the one hand, it increases the stability of mRNA, but also enhances the non-specific immune response of mRNA. Cap ratio, poly(A) length and mRNA integrity may affect the effectiveness of the product. mRNA capping process, capping structure, capping raw material impurities, in vitro and in vivo expression efficiency of capped mRNA, in vitro and in vivo immunogenicity are also the focus of pharmaceutical research.

  • LNPs

In addition to CQA of conventional nanomaterials, the following aspects should also be paid attention to: (1) charge. Zeta potential not only affects the stability of nanoparticles, but also affects the entry, endosomal escape and adverse reactions of nanoparticles. (2) Particle size distribution; (3) pH. The preparation contained cationic polymer, and pH could affect the composite of the material and mRNA. (4) Encapsulation rate of mRNA by nanoparticles; (5) integrity, function and content of encapsulated mRNA; (6) mRNA release.

  • Impurity

The impurities in quality research mainly include process-related impurities and product-related impurities. High attention should be paid to the impurities affecting safety, and full research should be conducted on them, including the generation/introduction of related impurities, process removal, and the degree of monitoring. (1) Related impurities of poly positively charged materials, including impurities generated by material synthesis and possible impurities generated in the process of mRNA composite; (2) Oxidation and related degradation products of unsaturated lipids; (3) Particulate matter generated by nanoparticle aggregation is also a potential impurity and should be evaluated according to the actual situation; (4) Unassembled lipid molecules, cationic substances and free mRNA. Unassembled lipid molecules affect the stability of LNPs. Free mRNA is easy to degrade, and may also cause non-specific immune stimulation, affecting the safety and efficacy of the product.

References:

  1. Pardi, N.; et al. m RNA vaccines - a new era in vaccinology. Nat Rev Drug Discov, 2018, 17(4): 261-279
  2. Pardi, N.; et al. In vitro transcription of long RNA containing modified nucleosides. Methods Mol Biol, 2013, 969:29-42
  3. Miao, L.; et al. Delivery of mRNA vaccines with heterocyclic lipids increases anti-tumor efficacy by STING-mediated immune cell activation. Nat Biotechnol, 2019, 37(10): 1174-1185
  4. Strzelecka, D.; et al. Phosphodiester modifications in mRNA poly(A) tail prevent deadenylation without compromising protein expression. RNA, 2020, 26(12): 1815-1837
  5. Dahlman, J E.; et al. In vivo endothelial siRNA delivery using polymeric nanoparticles with low molecular weight. Nat Nanotechnol, 2014, 9(8): 648-655
  6. Hou, X.; et al. Lipid nanoparticles for mRNA delivery. Nat Rev Mater, 2021, 1-17
  7. Wang, Y.; et al. mRNA vaccine: a potential therapeutic strategy. Mol Cancer. 2021 Feb 16;20(1):33.
  8. Heil, F.; et al. Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8. Science, 2004, 303(5663): 1526-1529
  9. Kowalczyk, A.; et al. Self-adjuvanted mRNA vaccines induce local innate immune responses that lead to a potent and boostable adaptive immunity. Vaccine, 2016, 34(33): 3882-3893
  10. Iborra, S.; et al. The DC receptor DNGR-1 mediates cross-priming of CTLs during vaccinia virus infection in mice. J Clin Invest, 2012, 122(5): 1628-1643
  11. Borst, J.; et al. CD4 T cell help in cancer immunology and immunotherapy. Nat Rev Immunol, 2018, 18(10): 635-647

Reagents Solutions

Antibodies for mRNA enzyme analysis

Creative Diagnostics now offers a set of antibodies against mRNA enzymes that can be used for analyzing the type and quality of mRNA enzymes used in mRNA production. Target specifications include T7 RNA polymerase, RNase Inhibitor, DNase I, Pyrophosphatase, capping enzyme, RNMT, and MTPAP. These selected antibodies combine high specificity and sensitivity, and are fully characterized by different applications.

Features

  • Broad range of enzymes covered.
  • Suitable for mRNA in-vitro transcription and modification evaluation.
  • Completely characterized by immunoassay.
  • High affinity and specificity.
  • Unlimited customization options including addition of antibody conjugation.

mRNA Transcription

Enzymes Cat. No. Product Name Applications
T7 RNA Polymerase CABT-B8990 Rabbit Anti T7 RNA Polymerase Polyclonal Antibody WB, ELISA
CABT-B8990H Rabbit Anti T7 RNA Polymerase Polyclonal Antibody [HRP] WB, ELISA
RNH1/RNase Inhibitor DPABH-08841 Rabbit Anti RNH1 (424-454) Polyclonal Antibody ICC/IF, WB
CABT-L7840 Mouse Anti RNH1 Monoclonal Antibody, Clone 2I34 WB, ELISA, FC, ICC/IF
DNase I DCABH-11305 Rabbit Anti DNASE1 Monoclonal Antibody ELISA, ICC/IF
CABT-L7841 Mouse Anti DNASE1 Monoclonal Antibody, Clone C5 WB, IP, IF, IHC, ELISA
CABT-L2071 Rabbit Anti DNASE1 Polyclonal Antibody WB, IF
Pyrophosphatase, Inorganic (yeast) CABT-L7842 Rabbit Anti Inorganic Pyrophosphatase Polyclonal Antibody IF, ELISA, DB, IB
CABT-L7843 Rabbit Anti Inorganic Pyrophosphatase Polyclonal Antibody, Biotin IF, ELISA, DB, IB

Enzymatic Capping

Enzymes Cat. No. Product Name Applications
Capping Enzyme CABT-L7844 Rabbit Anti mRNA-capping Enzyme Polyclonal Antibody ICC, WB
CABT-B11251 Mouse Anti mRNA-capping Enzyme Monoclonal Antibody WB
DPABH-28350 Rabbit Anti mRNA-capping Enzyme Polyclonal Antibody WB, IP, IF, ELISA
RNMT/mRNA Cap 2´-O-MethyltransFerase CABT-BL3171 Mouse Anti RNMT Monoclonal Antibody, Clone 4I4-2E23 WB, IP, IF, IHC, ELISA
CABT-BL3170 Rabbit Anti RNMT (30-130) Polyclonal Antibody WB, ICC/IF, IHC
CABT-L7845 Rabbit Anti RNMT Polyclonal Antibody WB, ICC/IF, IP

Adding Poly-(A) tail

Enzymes Cat. No. Product Name Applications
MTPAP/PolyA Polymerase DPABH-04497 Rabbit Anti MTPAP Polyclonal Antibody WB, ICC/IF, IP
CABT-L7846 Mouse Anti MTPAP Monoclonal Antibody, Clone 2E4 WB, ICC/IF, IHC-P
CABT-L7847 Rabbit Anti MTPAP(Center) Polyclonal Antibody WB, IHC-P, ELISA

ß-lactam antibiotics impurity analysis kit

Creative Diagnostics provides a panel of highly sensitive antibiotics ELISA kits to analyze antibiotic residues. These kits are suitable as good impurity detection tools to be used in the bioproduction process.

Products List

Cat. No. Product Name Compatible Samples
DEIA048 Kanamycin ELISA Kit Vaccine, Cell Culture
DEIA-WZ048V High Sensitivity Kanamycin ELISA Test Kit Cell lysate, Urine, Serum, Recombinant protein, Fermentation liquid
DEIA047 Gentamicin ELISA Kit Vaccine, Cell Culture
DEIA-WZ6884 High Sensitivity Gentamicin ELISA Test Kit Cell lysate, Urine, Serum, Recombinant protein, Fermentation liquid
DEIA040 Ampicillin ELISA Kit Vaccine
DEIA-WZ020 High Sensitivity Streptomycin ELISA Test Kit Serum, Urine, Recombinant protein
DEIA6881V2 Chloramphenicol ELISA Kit Vaccine
Kanamycin ELISA Kit High Sensitivity Kanamycin ELISA Kit Gentamicin ELISA Kit High Sensitivity Gentamicin ELISA Kit
Compatible Samples Vaccine, TE buffer, culture supernatant Cell lysate, urine, serum, fermentation liquid, recombinant protein Vaccine, culture supernatant Urine; serum,
fermentation liquid, recombinant protein
LOD 0.5 ng/mL 0.1 ng/mL 0.1 ng/mL 0.02 ng/mL
LOQ 0.5 ng/mL 0.2 ng/mL 0.1 ng/mL 0.02 ng/mL
Assay Range 0.5-40.5 ng/mL 0.2-16.2 ng/mL 0.1-8.1 ng/ml 0.02-1.5 ng/mL
Precision [Intra-assay CV] 0.5 ng/mL - 6%
1.5 ng/mL - 7%
4.5 mg/mL - 7%
13.5 ng/mL - 12%
16.2 ng/mL - 8%
0.2 ng/mL- 10%
0.1 ng/mL- 3%
0.3 ng/mL- 8%
0.9 ng/mL- 4%
2.7ng/mL- 9%
0.02 ng/mL 10%
1.5 ng/mL 9%
Accuracy [Recovery/Matrix Interference] 81-102% 80-120% 85-105% 85-115%
Number of Wash Steps 1 1 2 1
Total time to result 1h 45 mins 75 mins 50 mins

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