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Development of anti-COVID-19 mRNA vaccine incorporating two proline residues has its origin in the structural studies of human Respiratory Syncytial Virus and its relevance to cancer community

 For those who closely follow the development of mRNA vaccines targeting COVID-19 coronavirus, one cannot help but notice the artificial insertion of two proline residues to the encoded spike protein.  As with other advances in science, there have been multiple layers of discoveries that preceded the innovation.  To dissect the underlying rationale for adding the residues, one may need to revisit the molecular pathology of human Respiratory Syncytial Virus (hRSV).

hRSV is a medium-sized enveloped, negative-sense RNA virus (as opposed to coronavirus with positive-sense RNA genome), which was first isolated from chimpanzee in 1955 and subsequently isolated in human infants.  It is a common causative agent for respiratory infection in infants/children and potentially the elderly or immune compromised individuals.  Similar to COVID-19, hRSV mediates fusion with the cell membrane using its fusion protein F, which is comprised of N-terminal F2 subunit and C-terminal F1 subunit.  In the pre-fusion conformation, the hydrophobic fusion peptide stays buried (Huang et al., 2019).  Upon activation, it undergoes unfolding to transform from a globular to linear conformation, allowing the fusion peptide to insert into the host cell membrane and form a bridge, which then undergoes further conformational change to fuse the viral and cell membranes (to allow the transfer of hRSV genome into the infected cell).

 Until recently, the structural information of hRSV's F protein was not available.  To resolve its structure through X-ray diffraction measurement, one must first obtain a sufficient amount of the protein to form a crystal.  The structure of pre-fusion F protein is quite unstable though it adopts a rigid post-fusion structure upon engaging the host cell membrane.  Its unstable structure has hampered the attempt to obtain a sufficient quantity via expressing the recombinant F protein in transfected cells.

To stabilize its 3D conformation, various adjustments or modifications had to be introduced to the F protein.  For the post-fusion conformation, it required removing the fusion peptide from the construct to achieve a moderate level of expression.  For the pre-fusion conformation, an antibody was added to trap the conformation or disulfide bonds were incorporated artificially to stabilize the structure.  Alternatively, Langedijk and colleagues at Janssen Infectious Diseases and Vaccines (The Netherlands) stabilized the refolding regions of F protein via the substitution of proline residues (Krarup et al., 2015).

                    

Likewise, through proline substitutions, the pre-fusion conformation of the spike (S) protein was stabilized to achieve expression in the case of MERS-CoV coronavirus (Wang et al., 2019).   MERS-CoV is a coronavirus that emerged in Saudi Arabia in 2012 exhibiting a significantly higher rate of mortality than SARS (or COVID-19) coronavirus.  For COVID-19, a similar technique was used to stabilize the pre-fusion conformation of the S protein by substituting two proline residues (in the C-terminal S2 fusion apparatus), which yielded a higher level of expression.  It allowed J. McLellan (University of Texas at Austin, USA), B. Graham (National Institute of Allergy and Infectious Diseases, USA) and their colleagues as well as D. Veesler and colleagues (University of Washington, USA) to obtain the cryo-electron microscopic image of COVID-19 spike protein (Wrapp et al., 2020; Walls et al., 2020).  An X-ray diffraction image of a complex of COVID-19 spike protein bound to an antibody was later obtained by I. Wilson and colleagues (Scripps Research Institute, USA) (Yuan et al., 2021).

As with the structural work, the mRNA vaccines targeting COVID-19 also require protein expression in vivo after the injection.  Hence, for vaccine development, the authors suggested that the stabilized structure of the pre-fusion COVID-19 S protein may "maintain the most neutralization sensitive epitopes when used as candidate vaccine antigens" (Wrapp et al., 2020).   Yet, based on the prior data obtained concerning the convalescent cases that have subsequently recovered, it appears those infected with COVID-19 were capable of mounting the antibody-based or cellular based immunity against the coronavirus (despite the 'unstable' pre-fusion S protein structure in vivo).  Assuming that the antibody generated using the stabilized pre-fusion S protein could recognize the unstable version of the same protein found in vivo, Moderna Therapeutics and Pfizer/BioNTech opted to utilize the mRNA sequence encoding the COVID-19 spike protein containing the two proline substitutions for the vaccine, which has been administered to the public recently (Polack et al., 2020; Keech et al., 2020).  The efficacy of the vaccine remains a grave concern for cancer patients as the pandemic increased their mortality 5-fold.

 The key to preventing epidemic is the ability to diagnose the infected early to preempt further propagation. For this, Bio-Synthesis, Inc. provides primers and probes (as well as synthetic RNA control) for COVID-19 diagnosis via RT-PCR assay.  It specializes in oligonucleotide modification and provides an extensive array of chemically modified nucleoside analogues (over ~200) including bridged nucleic acid (BNA). A number of options are available to label oligonucleotides (DNA or RNA) with fluorophoreseither terminally or internally as well as to conjugate to peptidesor antibodies. It recently acquired a license from BNA Inc. of Osaka, Japan, for the manufacturing and distribution of BNANC, a third generation of BNA oligonucleotides. To meet the demands of therapeutic application, its oligonucleotide products are approaching GMP grade. Bio-Synthesis, Inc. has recently entered into collaborative agreement with Bind Therapeutics, Inc. to synthesize miR-21 blocker using BNA for triple negative breast cancer. The BNA technology provides superior, unequalled advantages in base stacking, binding affinity, aqueous solubility and nuclease resistance. It also improves the formation of duplexes and triplexes by reducing the repulsion between the negatively charged phosphates of the oligonucleotide backbone. Its single-mismatch discriminating power is especially useful for diagnosis (ex. FISH using DNA probe). For clinical application, BNA oligonucleotide exhibits lesser toxicity than other modified nucleotides. 

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References

Huang J, Diaz D, Mousa JJ.  Antibody Epitopes of Pneumovirus Fusion Proteins.  Front Immunol.  29;10:2778 (2019).  PMID: 31849961

Keech C, Albert G, et al. Phase 1-2 Trial of a SARS-CoV-2 Recombinant Spike Protein Nanoparticle Vaccine.  N Engl J Med. 383:2320-2332 (2020).  PMID: 32877576

Krarup A, Truan D, et al. A highly stable prefusion RSV F vaccine derived from structural analysis of the fusion mechanism.  Nat Commun. 6:8143 (2015).  PMID: 26333350

Polack FP, Thomas SJ, et al. Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine.  N Engl J Med. 383:2603-2615 (2020).  PMID: 33301246

Walls AC, Veesler D, et al. Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein.    Cell. 181:281-292.e6 (2020).  PMID: 32155444

Wang N, Graham BS, McLellan JS et al. Structural Definition of a Neutralization-Sensitive Epitope on the MERS-CoV S1-NTD.   Cell Rep. 28:3395-3405.e6 (2019). PMID: 31553909

Wrapp D, Wang N, et al.  Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation.  Science. 367:1260-1263 (2020).   PMID: 32075877

Yuan M, Wilson IA, et al. A highly conserved cryptic epitope in the receptor binding domains of SARS-CoV-2 and SARS-CoV.   Science.  368(:630-633 (2020.  PMID: 32245784