key: cord-296967-qiil3gqk
authors: Tatlow, Dean; Tatlow, Corinne; Tatlow, Scarlet; Tatlow, Savanah
title: A novel concept for treatment and vaccination against Covid‐19 with an inhaled chitosan‐coated DNA vaccine encoding a secreted spike protein portion
date: 2020-08-08
journal: Clin Exp Pharmacol Physiol
DOI: 10.1111/1440-1681.13393
sha: 
doc_id: 296967
cord_uid: qiil3gqk

A novel concept in DNA vaccine design is the creation of an inhaled DNA plasmid construct containing a portion of the coronavirus spike protein for treatment and vaccination. The secretion of a spike protein portion will function as a competitive antagonist by interfering with the binding of coronavirus to the angiotensin‐converting enzyme 2 (ACE2) receptor. The secreted protein binding to the ACE2 receptor provides a unique mechanism of action for treatment to all strains of coronavirus in naïve patients, by blocking the ACE2 receptor site. An inhaled plasmid DNA vaccine replicates the route of lung infection taken by coronavirus with transfected cells secreting spike protein portions to induce immunity. Unlike most DNA vaccines with intracellular antigen presentation through MHC I, the current vaccine relies on the secreted proteins presentation through MHC II as well as MHC I to induce immunity. Lung specific production of vaccine particles by inhaled plasmid DNA is appealing since it may have limited systemic side‐effects, and may induce both humoral and cytotoxic immunity. Finally, the ease and ability to rapidly produce this plasmid construct makes this an ideal solution for managing the emerging threat of coronavirus.

COVID-19 is a disease which causes a contagious acute respiratory infection in humans by the coronavirus named, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Symptoms of infection varying from asymptomatic to critically ill with acute respiratory distress syndrome. 1 Cases of coronavirus infection have now spread to almost every country, with currently no effective treatment or vaccine available. This highlights the urgent need for the development of an effective treatment and/or vaccine.

The genome of the novel SARS-CoV-2 was first released in late December 2019, and since then a great deal of work has been done identifying the gene involved in infection. The genome of the novel SARS-CoV-2 virus encodes structural proteins that make up the outer layer which include; the spike, envelope, and membrane proteins. 2 The key feature of SARS-CoV-2 is the surface spike protein, which plays a critical role in infection by mediating high specificity viral attachment to the host cell surface ACE2 receptor allowing for viral entry. 3 These findings of the identified spike proteins in the SARS-CoV-2 receptor binding domain and ACE2 region may provide useful information for treatment or vaccine development. 1, 3 Various forms of vaccines have been used to fight against bacterial and viral disease.

Plasmid DNA vaccines are of great interest, since they provide the ability to be rapidly constructed and manufactured. It was first shown that Plasmid DNA could protect mice from

This article is protected by copyright. All rights reserved influenza challenge in the 1990s. 4 The challenge with plasmid DNA is the optimal deliver of DNA into the desired cells for the expression of proteins needed to generate an immune response. 5 In this paper a series of inhaled plasmid DNA vaccine construct containing various forms of the coronavirus spike protein sequence may provide potential treatment and vaccine options as revealed by Yu et al. (Fig 1.) . 6 Plasmid DNA produced spike proteins will function to induce immunity as a vaccine candidate (Fig. 2a. ) and work as a competitive antagonist against coronavirus host cell attachment (Fig.2b) .

DNA vaccination has emerged over the years as a novel approach for the prevention of bacterial and viral diseases. In recent years DNA vaccination has made considerable progress with the development of many DNA vaccines entering into human clinical trials, most notably Zika virus phase 2 and HPV infection phase 1 trials. [7] [8] [9] The DNA vaccine for Zika virus known as VRC5283 was shown to be safe and well tolerated resulting in 100% of the participants generating humoral and cellular responses. 7,8 VRC5283 is a plasmid DNA constructed with Zika virus genes prM, E(envelope) and a Japanese encephalitis virus coding sequence to improve secretion of the Zika vaccine particle. 7 

This article is protected by copyright. All rights reserved A major obstacle in DNA vaccine development is low immunogenicity. 10 Administration of naked DNA vaccines is usually inefficient, since its negative charge prevents it from crossing cell membranes because of charge repulsion with negative charge phospholipids. 10 An important development in DNA vaccination is the improvement in the delivery with nanoparticles of chitosan. Chitosan nanoparticles have provided an effective means for prevention of DNA vaccine degradation while the cationic nature enables binding to the negative charge of DNA. 10 Chitosan has many favourable features for DNA vaccine delivery to the mucosal surface such as mucoadhesion, highly soluble, inert and non-immunogenic. 10 Since mucosal surfaces are a major site for a majority of pathogens that infect humans, 10 This article is protected by copyright. All rights reserved 1. 6 Three DNA constructs are the S1/S2 without the transmembrane (TM) and C-terminal (CT) domains, S1 and recptor binding domain (RBD) with trimerization taq. The other three DNA constructs contain the JEV TM and CT domains added to S1/S2, S1 and RBD. These DNA constructs can be tested for protein secretion quantity, and effectiveness of blocking coronaviurs infection. Other commonly used fusion partners include the constant domain IgG (the Fc region), Maltose-binding protein (MBP), small ubiquitin-like modifier(SUMO), and human serum albumin (HSA) tag. 11 Cells transfected by the DNA construct will also secrete a portion of the spike protein as a competitive antagonist. It has been shown that the S1 protein fragment of the S1 protein binds with high efficiently to the ACE2 receptor revealing the potential of a portion of the spike protein to work as a competitive antagonist once secreted. 12 The mechanism of action of DNA vaccination with these inhaled chitosan DNA constructs involves encoding antigen(s) that are transfected into either antigen presenting cells (APC) or somatic cells. In one scenario intracellular processing of plasmid DNA leads to vaccine derived endogenous peptide presentation on MHC-I molecules. 9, 13, 14 This antigen is expressed to the cell surface with MHC I and presented to cytotoxic CD8+ T cells stimulating cell mediated immunity. 9 In the other scenario antigens of exogenous origin or secreted protein from plasmid DNA are loaded on MHC II resulting in activation of helper CD4 + T cells which in turn contribute to B cell priming to yield a humoral immune response. 9 In mucosal vaccination, the plasmid DNA antigen is secreted into the extracellular space where M cells participate in antigen uptake and transport them to the extracellular space. 9 At this point the antigen is released to APC cells for presentation to immune cells. 9 These antigenic proteins expressed and secreted by plasmid DNA vaccines access both the exogenous and endogenous pathways in the activation of both humoral and cellular mediated immune responses. 15

This article is protected by copyright. All rights reserved There are studies that show a patient's immune response resulting from delivery of a vaccine to the lung may equal or exceeds the immune response created by vaccine injection. 16 It has been proposed that a mucosal immune response may be created via particles of the vaccine material depositing on the upper respiratory tract. 17 A systemic immune response may be created via particles of the vaccine material depositing into the deep lung. 17 In a study by weaver there was a significant differences in antibody and T-cell response with IM (intramuscular)

immunized mice compared to inhaled immunization. 8, 15 However, there was no significant difference between IM and inhaled immunized mice response when challenged with lethal influenza virus amounts. 15 These results suggest that vaccine efficacy of IM and inhaled vaccines are almost identical, and inhaled vaccination may be adequate route of administration even though antibody and T-cell responses are low.

In this paper the primary use of an inhaled plasmid DNA vaccine containing the coronavirus spike protein DNA sequence is for treatment. The small chitosan plasmid DNA size of approximately138.nm 18 can reach deep into the lung, similar to that of the coronavirus with a size of 125nm. 3 Once in the lower respiratory tract or alveolar region of the lung deposited plasmid DNA will be taken up and expression of coronavirus spike proteins by host cells such as pneumocytes will occur. This will create a competitive antagonistic environment whereby coronavirus will have few options available for binding to the ACE2 receptor. Thus, the expression of coronavirus spike proteins provides a protective barrier against coronavirus infection of type 2 pneumocytes. A major concern with using an inhaled chitosan DNA vaccine as a treament is obtaining sufficient production of secretion protein to compete with coronavirus.

Currently, there are few studies available using a DNA vaccine by the respiratory route for treatment, a study by Kodama et al., reveals the practicality of this route by using the luciferase

This article is protected by copyright. All rights reserved gene to show gene expression in lung tissue by fluorescene intensty. 19 This study revealed that high levels of inhaled DNA vaccine expression can occur in lung tissue as seen by the increase in fluorsecence in lung samples, and in microscopic lung sections. 19 These results indicate that high levels of protein expression may be generated by inhaled DNA for competitive antagonism against coronavirus. A large dose of inhaled chitosan DNA construct or multiple doses can easily be given when symptoms first appear when there is a lower viral load early in infection. In comparison to drug treatment approaches, inhaled DNA treatment has many advantages, with a prolonged half-life, site-specific action, no metabolic concerns, and also ease of manufacture, administration and storage.

Another major concern with coronavirus vaccine development is the harmful inflammatory response known as antibody-dependent enhancement in which non neutralizing antibodies bind to coronavirus and enhance entry into cells. 20 A recent study by Yu et al., has shown protective immunity in rhesus monkeys with little side effects of DNA vaccination by using various forms of the coronavirus spike protein. 6 The DNA vaccine constructs in the article by Yu et al., reveals that both secreted and non secreted proteins were able to provide protective immunity. 6 The mechanism of action of the inhaled DNA construct may also work for treatment and vaccination of other respiratory viruses such as influenza where a HA expressed protein prevents binding of any strain of influenza. In addition, this novel method of treatment may be used for delivering DNA encoding proteins to other tissues where secretion of proteins into the extracellular space may provide a therapeutic benefit. A DNA vaccine encoding secretion of interferon beta protein to locally transfected tissue could be inhaled for deliver to lungs or oral deliver to the gastrointestinal tract.

This article is protected by copyright. All rights reserved

This DNA construct has a great deal of versatility for use as a treatment or vaccine. Many studies have proven DNA vaccines to be safe and well tolerated. The use of plasmid DNA provides an easy means of production which should provide a low cost method for delivering vaccines to the lung with some form of nebulizer or metered-dose inhaler.

There may be many cons associated with inhaled DNA vaccination such as autoimmunity and/or unseen problems with the quantity of vaccine particles produced. However, lung specific production of coronavirus spike proteins may be a safer alternative than systemic drug treatment.

Currently, plasmid DNA vaccination is only licensed for veterinary use, hopefully further testing may allow for licensing in humans.

Characterization of the receptor-binding domain (RBD) of 2019 novel coronavirus: implication for development of RBD protein as a viral attachment inhibitor and vaccine

Proteomics analysis unravels the functional repertoire of coronavirus nonstructural protein 3

Coronaviruses: an overview of their replication and pathogenesis

Heterologous protection against influenza by injection of DNA encoding a viral protein

A Comparison of Plasmid DNA and mRNA as Vaccine Technologies

DNA vaccine protection against SARS-CoV-2 in rhesus macaques

Safety, tolerability, and immunogenicity of two Zika virus DNA vaccine candidates in healthy adults: randomised, open-label, phase 1 clinical trials

Zika Virus Vaccine Development: Progress in the Face of New Challenges

Therapeutic DNA Vaccines for Human Papillomavirus and Associated Diseases. Hum

Intranasal DNA vaccine for protection against respiratory infectious diseases: the delivery perspectives

Dendritic cells targeted chitosan nanoparticles for nasal DNA immunization against SARS CoV nucleocapsid protein

Maltose-Binding Protein (MBP), a Secretion-Enhancing Tag for Mammalian Protein Expression Systems

A 193-amino acid fragment of the SARS coronavirus S protein efficiently binds angiotensin-converting enzyme 2

Airway M cells arise in the lower airway due to RANKL signaling and reside in the bronchiolar epithelium associated with iBALT in murine models of respiratory disease

DNA vaccine that targets hemagglutinin to MHC class II molecules rapidly induces antibody-mediated protection against influenza

A Dose Effects of Recombinant Adenovirus Immunization in Rodents

Mucosal vaccination via the respiratory tract

Chitosan-DNA nanoparticles enhanced the immunogenicity of multivalent DNA vaccination on mice against Trueperella pyogenes infection

Development of a DNA Vaccine for melanoma metastasis by inhalation based on an analysis of transgene expression characteristics of naked pDNA and a ternary complex in mouse lung tissues

Thank-you Dr. Jordan B. Peterson.

There are no conflicts of interest to report.

This article is protected by copyright. All rights reserved

This article is protected by copyright. All rights reserved