This document proposes developing a miRNA-based live attenuated vaccine for SARS-CoV-2. It involves genetically engineering the virus to contain a miRNA responsive element, like for miRNA-21, in its genome. This would attenuate viral replication in vivo by degrading or suppressing viral transcripts. The vaccine could provoke a strong protective immune response without harming host cells. It may confer long-lasting immunity after one or two doses through mucosal administration like intranasal spray. Some challenges include the time needed for development and potential for secondary mutations.

1. SARS-CoV-2
Antibody produce
in response to use
a vaccine
A rational design on Covid-19 vaccine development
Presented by:
Shotabdy Roy
Mehedi Hasan
Mahidol University, Thailand
Department Of Biochemistry
Masters(MSc)

2. Background
• Enveloped, positive-sense, single-stranded RNA virus
Adapted from Kumar et.al (2020)
Morphology of SARS-CoV-2
 Coronavirus Disease 2019 (COVID-19) is a global life-
threatening disease
 Severe acute respiratory syndrome coronavirus 2
(SARS-Cov2)

3. • Beta coronavirus, Coronaviridae family
• SARS-CoV-2 belongs some of the largest genomes
among RNA viruses.(26 to 32 kb)
• Nested set of polyadenylated mRNA transcripts
• Non-structural proteins, four structural proteins and accessory
proteins
Genome organization of SARS-CoV-2
Adapted from Kumar et.al (2020)

4. Source: Microbe notes
Replication of SARS-CoV-2

5. • Non-coding ssRNA (17-24nt)
• ~60% of gene expression
• Maintenance of the
epithelial cell barrier in the
respiratory tract
• Regulation of anti-viral
host defence
• Altered miRNA expression
regulates viral replication,
immune responses, and
infection outcome
Adapted from: Richardson, et.al (2016)
Roles of microRNAs in viral acute respiratory infections

6. Objectives of the proposal
 To develop a vaccine against SARS-CoV-2 virus by conducting a potential candidate of
miRNA which will decay or translationally suppress the viral replication and enhance
innate immune response.
miRNA will be genetically engineered into positive sense RNA virus(SARS-CoV-2) as
a platform to improve live attenuated vaccine.
The proposed vaccine will also act on tissue specific and tissue non-specific
mechanism.
This vaccine could be elicited strong protective immunity without disturbing host cells.
This recombinant vaccine should have significance role on developing immunological
memory cells(B cell) which will protect against other flu-like virus.

7. Hypothesis of this proposal
• In vitro genetically engineered
SARS-CoV-2 virus containing
miRNA responsive element on
its viral genome which will
attenuate viral replication in
vivo. This can be designed a
novel miRNA-based live-
attenuated vaccine.

8. A data on SARS-CoV-2 vaccine development(2020)
 More than 165 vaccines against SARS-Cov-2 virus are now in the stage of
developing around the world by researchers of different institutes.
 Among them only 31 vaccines are now processing for clinical trials at
various phases.
Preclinical
(135+)
Phase I (19)
Phase II (11)Phase III (8)
Approved (2)
Collected from ”The new York times”

9. Some categories of vaccines(2020)
1. Whole-virus vaccines: use of an inactivated version of the coronavirus to
stimulate immune response.
2. Protein-based vaccines: use spike protein and enveloped protein and
fragment proteins of the coronavirus to stimulate immune response.
3. Viral-vector vaccines: use a virus to deliver coronavirus genes into cells and
provoke an immune response(Can Sino BIO developed a vaccine using AD5)
4. Repurposed vaccines: vaccines that already use in other diseases which also
protect against coronavirus(Ex: Bacillus Calmette-Guerin vaccine)
5. Genetic vaccines: vaccines that use one or more of the coronavirus’s own
genes to provoke an immune response. It could be RNA-based and DNA-
based vaccine.
A novel design on microRNA-based live attenuated vaccine that may lead a
role on prevention against viral pathogenicity including safety and more efficacy
purpose.

10. Why choose the miRNA-based live attenuated vaccine
• There are four main classes of vaccine-killed, subunit, mRNA and live
attenuated.
• Several advantages-more robust, increase long-lasting immunity
compared to inactivated vaccines.
• Several different mechanisms- altering the temperature of the optimal
polymerase function and deleting or mutating viral immune
antagonists.
• In developing live attenuating vaccine for both positive and negative
RNA viruses by exploiting host endogenous miRNAs.
• Generating miRNA-targeted viruses which provides a known
mechanism for attenuation, can be applied across a diverse range of
viruses.

11. Analysis through bioinformatics, 80 SARS-CoV-2 encoded miRNA
that can target the virus genome.
2 miRNA targets at the 3’UTR, 2 at the S gene
SARS-CoV-2
28 human miRNA have been predicted to have 30 targets on the virus
genome
Human
Source:
https://www.ncbi.nlm.nih.gov/genbank/
Potential Candidate selection
Proposed Methodology
General information

12. By using bioinformatics tools, screening the appropriate miRNA candidate like
(miRNA-21)
miRNA-21 expression is highly conserved in mammalian and avian species
across most of the cells, making it an excellent candidate for conducting the
research
Proposed Research Design
SARS-CoV-2 Virus
Structure of Viral genetic material (RNA)
Why choose miRNA-21??

13. Designing of miR-21-MRE
Homology sequence of miR-21
1. Au rich
2. No RBP motif
3. Low secondary structure
complexity
4. High MRE accessibility at
3’UTR end.
Engineered miRNA responsive element
Inserted into specific site
of SARS-CoV-2 genome
miR-21-MRE
Recombinant SARS-CoV-2 genome structure

14. Tissue specific mechanism
Viral RNA with miRNA target sequence No virus produce
Tissue Non-specific mechanism
Virus will be retained the ability of replication
without producing disease and promote strong
protective immunity

15. miRNA in the antiviral innate immune response
Adapted from: Stephen A.et.al, microRNAs in viral acute respiratory infections: immune regulation, biomarkers, therapy,
and vaccines(2019)

16. Advantages
• Easy to administration via intranasal spray, So get rid of needle.
• This proposed vaccine are like the natural infection that they
prevent.
• Create a long-lasting and strong innate immune response against
SARS-CoV-2 virus.
• Only 1 or 2 dose will enough create to lifetime protection against
this virus.
Disadvantages
• Take long time to commercialize the proposed vaccine in market.
• Secondary mutation can take place during producing this kind of
vaccine and lead to reversion or gain of virulence properties like
before.
• Requirement for control condition .