2. Introduction :
• Vaccines are a clinical product that is
composed of live or dead material from an
infectious agent – bacterium, virus, fungus or
parasite – that elicit protective immunity
against the pathogen when administered.
These substances are used to prevent the
spread of infectious diseases.
4. Types of vaccines
Inactivated or killed
vaccines
Live
attenuated vaccines
Subunit,
recombinant or
protein vaccines
Toxoid
Conjugate Viral vector vaccine mRNA vaccines DNA vaccines
5. Limitations of conventional vaccines:
Production
time
Mutations
and variants
Potential
allergenicity
Safety
concerns
7. Messenger RNA :
• Messenger RNA (abbreviated
mRNA) is a type of single-stranded
RNA involved in protein synthesis.
• mRNA is made from a DNA
template during the process of
transcription.
• The role of mRNA is to carry protein
information from the DNA in a cell’s
nucleus to the cell’s cytoplasm (watery
interior), where the protein-making
machinery reads the mRNA sequence
and translates each three-base codon
into its corresponding amino acid in a
growing protein chain.
8. Guanosine with a
methyl group on the
7th position.
Translational efficiency
is regulated by their
length, structures and
regulatory elements.
Coding sequence:
modification of
sequence such as
codon optimization
have contributed to
improved expression.
Properties such as
length are
important for
translation and
protection of the
mRNA molecule.
Structure of mRNA
9. mRNA vaccines
• A novel vaccine
technology which delivers
the mRNA that encoding the
antigen protein of pathogen
to the cell, expresses the
antigen protein, and then
stimulates the immune
response of the body.
11. Mechanism of action:
• The innate immune system consists of pattern-recognition receptors (PRRs), which
function to detect pathogens (pathogen-associated molecular patterns, PAMPs).
• Interaction among PRRs and PAMPs triggers the inflammatory response, the link between
the innate immunity and the adaptive immunity
• .Most traditional vaccines consisting of subunit antigens fail to activate PRRs, consequently
requiring the addition of adjuvants to provide innate immune stimulation and induce
effector responses.
• RNA, on the other hand, can directly engage some PRRs and stimulate innate immune
responses.
• Internalization of mRNA vaccines occurs primarily by non- immune cells at injection site.
12. CONTD...
RNA vaccines seem to have
‘self-adjuvanting properties by
activating host sensing
machinery. However, the innate
immune system may also
establish an antiviral response
and thereby creating a
potentially unfavorable
environment for translation of
mRNA vaccines that could
reduce vaccine effectiveness.
18. Non replicating mRNA
.
- Non-replicating mRNA vaccines encode specific antigenic proteins.
- They lack self-amplification components.
- Host cells produce the antigenic protein.
- Immune response is triggered by the displayed protein.
- Safe as they do not replicate or integrate into DNA.
- Easily customizable for targeting different diseases.
- Demonstrated success with COVID-19 mRNA vaccines like Pfizer-BioNTech and
Moderna.
20. Self amplifying mRNA
.
- Self-amplifying mRNA vaccines contain components for mRNA replication.- They encode for the target
antigen and a replicase complex.
- Simultaneous replication and antigen production occur within host cells.
- This leads to a stronger and sustained immune response.
- Lower mRNA doses are required for effectiveness.
- Potential for targeting various diseases, including viruses and cancers.
- More complex to develop than non-replicating mRNA vaccines.
- -have stringent storage and production requirements.
21. - 3' UTR is at the 3' end of mRNA.
- Contains instability factors like AREs and GREs.
- Design avoids AREs and GREs to prevent
mRNA destabilization.
- Stabilizing elements may be added to increase
mRNA stability.
- 3' UTR design impacts mRNA stability and
translation efficiency.
- Nucleoside analogs modify DNA and RNA
building blocks.
- They enhance mRNA properties when introduced.
- Examples like pseudo uridine and methylated
nucleosides make mRNA less immune recognizable.
- Improves mRNA stability and lowers immune
response risks.
- Crucial for optimizing mRNA vaccines.
- 3' poly(A) tail is at the mRNA's 3' end in eukaryotes.
- Length impacts mRNA translation and stability.
- Usually consists of dozens to hundreds of adenine bases.
- Regulated in the nucleus and cytoplasm.
- Collaborates with the 5' cap and other elements to control mRNA
function.
- 5' cap is a crucial structure at mRNA's beginning.
- Composed of N7-methylguanosine and 5',5'-triphosphate
bridge.
- Initiates translation by signaling ribosome binding.
- Protects mRNA from RNase degradation.
- Various cap types can impact mRNA stability and
translation efficiency.
- 5' UTR is crucial for translation initiation and
efficiency.
- Its secondary structure impacts translation.
- Designed to avoid upstream open reading
frames (uORFs).
- Short and less structured 5' UTR preferred for
efficiency.
- Characteristics influence mRNA translation and
are customized for vaccine design.
26. Peptide-Based mRNA Delivery
• Peptides, when positively charged, are used as carriers for mRNA vaccines and can interact
with nucleic acids through electrostatic interactions.
• Protamine as a Carrier: Protamine is a cationic peptide known for protecting mRNA from
degradation and has adjuvant activity, making it useful in early mRNA vaccine studies.
• Cationic Cell-Penetrating Peptides (CPPs): Certain cationic CPPs, like RALA and LAH4-L1, are
used to condense mRNA into nanocomplexes and enhance endosomal escape for improved
delivery.
• Anionic Peptides and Polymer Scaffolds: Anionic peptides, which are negatively charged, are
conjugated to positively charged polymers to serve as scaffolds for RNA encapsulation,
improving cell uptake and cytosolic release.
28. Cationic Nano emulsion
1. Cationic Nanoemulsion (CNE) combines
nanoemulsion with cationic lipids for RNA
delivery.
2. Nanoemulsion employs hydrophobic and
hydrophilic surfactants to create stable oil-in-
water particles.
3. MF59, an FDA-approved oil-in-water
nanoemulsion, is used with the influenza vaccine
for the elderly.
4. CNE, incorporating cationic lipids, can protect
and deliver mRNA effectively, similar to MF59.
5. CNE has shown potential in preclinical studies for
delivering mRNA vaccines, and clinical trials will
determine its efficacy in humans.
29. 1.VRPs are viral particles that deliver antigen-encoding self-amplifying mRNA into
the cytoplasm, allowing efficient antigen expression.
2. Structural Proteins from Helper Cells: The viral structural proteins required for
VRP formation are expressed in packaging (helper) cell lines to package self-
amplifying mRNA.
3. Replication Competence: VRPs can be replication-competent but attenuated or
replication-incompetent, depending on the absence of genetic regions for viral
infection.
4. Efficient Cytoplasmic Delivery: VRPs efficiently deliver RNA payloads to the
cytoplasm, leveraging the efficient internalization and genome release pathways
evolved by viruses.
Virus-Like Self-Amplifying mRNA
Particles
30. Naked mRna Vaccine delivery
method
Naked mRNA Injection: Administering the vaccine as a
buffer solution, often used in intradermal injections.
While it triggers an immune response, it's relatively
weak, and mRNA is prone to rapid degradation.
34. APPLICATIONS
• Role of mRNA vaccine in COVID-19
Pfizer COVID-19 vaccine is the first mRNA product to achieve full FDA approval.
doi: 10.3389/fimmu.2019.00594
35. • They both got nobel prize for their discoveries concerning nucleoside base modifications that
enabled the development of effective mRNA vaccine against COVID-19.
36. • Role of mRNA
vaccine in Cancer
(under Trial)
Over 50 clinical
trials are listed
on
clinicaltrials.gov
for RNA vaccine
in a number of
cancer including
blood cancers,
melanoma, brain
cancer &
prostate cancer.
https://doi.org/10.1016/j.jconrel.2022.03.032
37. There are some mRNA vaccines which are under clinical trials.
Pathgen name Companies name Current status
Influenza (flu) Sanofi
Pfizer
Moderna
NIAID
Phase 2 clinical trial
Phase 3 clinical trial
Phase 3 clinical trial
Phase 1 clinical trial
Zika virus Moderna Phase 2 clinical trial
Respiratory syncytial virus
(RSV)
Moderna Phase 3 clinical trial
Cytomegalovirus (CMV) Moderna Phase 3 clinical trial
Human immunodeficiency
virus (HIV)
Moderna
NIAID
Phase 2 clinical trial
Phase 1 clinical trial
(Source- clinicaltrials.gov)
38. ADVANTAGES
• Non-Infectious in nature :
As this type of vaccines are not constructed from
an active pathogen or even an inactivated
pathogen like in many traditional vaccines are
produced, so they are non-infectious in nature.
• Decrease the risk of localized outbreaks of
Pathogen :
In contrast traditional vaccines require the
production of pathogens, which if done at high
volume could increase the risk of localized
outbreaks of the virus at the production facility.
• Stimulating both cellular and humoral immunity :
Since the antigens are produced inside the cell,
they stimulate cellular immunity as well as
humoral immunity.
• Design Swiftly :
Like Moderna designed their mRNA-1273
vaccine for COVID-19 in 2 days.
39. ADVANTAGES
• Economically friendly:
They can be manufactured faster more cheaply & in a more standardized
fashion with fewer error rates in production .
• Risk of integration into the host genome can be averted:
As we know mRNA is translated in the cytosol, so there is no need for the RNA
to enter the cell nucleus & the risk of being integrated into the host genome is
averted.
• Optimization of open reading frame(ORF) & untranslated region (UTR) can be
possible:
Through enriching the guanine-cytosine content OR choosing UTRs known to
increase translation. This process is called sequence engineering of mRNA.
40. Advantages & disadvantages of different types of vaccine platforms
Tregoning, J.S., Brown, E.S.,
Cheeseman, H.M., Flight, K.E.,
Higham, S.L., Lemm, N.-M., Pierce,
B.F., Stirling, D.C., Wang, Z. and
Pollock, K.M. (2020), Vaccines for
COVID-19. Clin. Exp. Immunol.,
202: 162-192.
41. DISADVANTAGES
Storage
• It is one of the major issue
because mRNA is fragile, so they
must be kept at very low
temperature to avoid degrading.
• For example pfizer-BioNTech’s
BNT162b2 mRNA vaccine has to
be stored between -80ᵒC &
60ᵒC, whereas moderna mRNA-
1273 vaccine has to be stored
between -25ᵒC & -15ᵒC, reason
for this temperature difference
is that it is possible that
differences in mRNA secondary
structure could account for this
thermostability differences.
https://doi.org/10.1016/j.jconrel.2022.03.032
42. DISADVANTAGES
Recently discovered technology
Before 2020 no mRNA technology based vaccine had been authorized for use in
humans, so there is a risk of unknown effects.
The novel nature of mRNA technology has led to hesitancy & misinformation in
some populations, Addressing public concerns & providing accurate information
is crucial for building trust
Side Effects
Reactogenicity is similar to that of conventional non-RNA vaccines, there are
many side effects such as allergy, renal failure, heart failure etc.
Degradation
mRNA may also be degraded quickly after administration, this is a substantial
challenge for mRNA delivery, However appropriate carrier can avoid
degradation & enhance immune responses.
Antigenic Drift
mRNA vaccines are designed based on the genetic sequences of a specific virus
strain, if the virus undergoes significant genetic changes (antigenic drift), the
vaccine effectiveness may be reduced potentially requiring the development of
updated formulations.
43. CONCLUSION & RECENT
ADVANCEMENT
• Progress in mRNA technologies and lipid
nanoparticle based delivery systems has
allowed the development of mRNA
COVID-19 vaccines at unprecedented
speed, demonstrating the clinical
potential of lipid nanoparticle-mRNA
formulations & providing a powerful
tool against the pandemic.
• Recent improvement in mRNA vaccines
act to increase protein translation,
modulate innate & adaptive
immunogenicity & improve delivery.
https://doi.org/10.1016/j.jconrel.2022.03.032
44. • In recent years mRNA vaccines are used in animal models of influenza virus, zika
virus, rabies virus, HIV virus etc. using lipid encapsulated or naked form.
• Diverse approaches to mRNA cancer vaccines and various types of directly
injectable mRNA , have been employed in numerous cancer trials, with some
promising results showing antigen-specific T cell responses and prolonged disease
free survival in some cases.
• Highly efficient and non-toxic RNA carrier have been developed that in some cases
allow prolonged antigen expression in vivo.
• Some vaccine formulations contain novel adjuvants, while others elicit potent
responses in the absence of known adjuvants.
https://doi.org/10.1016/j.jconrel.2022.03.032
Editor's Notes
GOOD MORNING EVERYONE
TODAY I, ANUSHKA ALONG WITH SHREYA AND YOGESH WE ARE GOING TO PRESENT ON THE TOPIC MRNA VACIINES WHERE IN WE WILL BE DISCUSSING HOW AND WHY MRNA VACCINES WERE DEVELOPED, THEIR MECHANISM OF ACTION HOW THEY ARE DELIVERED INTO THE BODY THEIR TYPES AND THEIR APPLICATIONS, ADVANTAGES AND DISADVANTAGES.
Vaccination is a simple, safe, and effective way of protecting you against harmful diseases, before you come into contact with them. It uses your body’s natural defenses to build resistance to specific infections and makes your immune system stronger.
Vaccines are effective in preventing numerous infectious diseases, reducing the risk of illness, hospitalization, and death
Herd Immunity: Widespread vaccination creates herd immunity, which protects those who cannot be vaccinated, such as individuals with certain medical conditions or compromised immune systems.
As a result of widespread vaccine use, the smallpox virus has been completely eradicated and the incidence of polio, measles and other childhood diseases has been drastically reduced around the world
Vaccination programs are cost-effective, as they prevent the need for expensive medical treatments and reduce healthcare costs.
Vaccines can be categorized into several different types, each designed to trigger an immune response against specific pathogens. Here are some common types of vaccines:
Inactivated or Killed Vaccines: These vaccines contain pathogens that have been killed or inactivated, so they cannot cause the disease. Examples include the polio vaccine (IPV) and the hepatitis A vaccine.
Live Attenuated Vaccines: These vaccines contain weakened forms of the live pathogen. They mimic a natural infection, leading to a robust and long-lasting immune response. Examples include the measles, mumps, and rubella (MMR) vaccine and the oral polio vaccine (OPV).
Subunit, Recombinant, or Protein Vaccines: These vaccines use purified pieces of the pathogen, such as proteins or protein subunits. They do not contain the whole pathogen. Examples include the hepatitis B vaccine and the human papillomavirus (HPV) vaccine.
Toxoid Vaccines: These vaccines are based on the inactivated toxins produced by certain bacteria. They are used to protect against diseases caused by bacterial toxins, such as diphtheria and tetanus.
Conjugate Vaccines: These vaccines are used to protect against bacterial diseases that have a sugar-based outer coat (capsule). They combine the bacterial capsule with a protein to enhance the immune response. Examples include the Haemophilus influenzae type b (Hib) vaccine and certain types of pneumococcal vaccines.
Viral Vector Vaccines: These vaccines use a harmless virus (not the pathogen causing the disease) to deliver a piece of the pathogen to the body, triggering an immune response. The COVID-19 vaccines developed by AstraZeneca and Johnson & Johnson are examples of viral vector vaccines.
Messenger RNA (mRNA) Vaccines: mRNA vaccines, like the Pfizer-BioNTech and Moderna COVID-19 vaccines, use a small piece of genetic material from the pathogen to instruct cells to produce a harmless piece of the pathogen. This triggers an immune response.
DNA Vaccines: DNA vaccines work similarly to mRNA vaccines, but they use a small piece of the pathogen's DNA to instruct the body's cells to produce a harmless piece of the pathogen..
The majority of currently available vaccines are predominantly based on either inactivated (killed) or live attenuated approaches. Although these traditional vaccines have been used effectively against various infectious diseases, some of these have several limitations, which include their lower potential to induce a stronger immune response and poor efficacy
Apart from that these vaccines typically require longer development and production times
Conventional vaccines may need adjustments when dealing with new pathogen strains or variants, which can be time-consuming.
vaccines may contain additional ingredients or adjuvants that can lead to allergic reactions in a small number of recipients
Safety concerns :Live-attenuated vaccines use weakened forms of pathogens, which can pose safety concerns for immunocompromised individuals
Because of these reasons there was a need to introduce better vaccination approaches and that’s when mrna vaccines come to play
The 5’ capping and the 3’ poly adenylation ensures mrna stability and prevents it from degradation during its transport from nucleus into the cytoplasm
In 1961 mrna was discovered by brenner and then Malone introduced cationic lipid mediated delivery of mrna invitro
Mrna vaccines was discovere in 1990 and then from 1990 to 2019 various mrna based vaccines were developed but none of them received the fda approval and were not marketed however recently in 2020 two mrna vaccines for covid were successfully marketed
So the mrna gets gelivered into the body inside a lipid coat so as to protect it from degradation. mrna enters the cell and it goes to the ribosome machinery of the cell where it gets translated into spike protein now these spike proteins can be processed and presented to the t lymphocytes by either through cytosolic psthway or the endocytic pathway throught cytosolic pathway
SARS COV2 BINDS TO ACE2 WHICH CAUSES FUSSION OF THE VIRUS WITH THE CELL MEMBERANE AND ENDOCYTOSIS OF THE VIRUS .THE ENDOSOME HAS CATHEPSIN PROTEIN WHICH REMOVES THE VIRAL COAT AND THE MRNA IS RELEASED INTO THE CYTOPLASM WHERE IT GOES TO THE HOST RIBOSOME WHERE IT GETS TRANSLATED INTO PP1A AND PP1AB WHICH ARE THE ACTED UPON BY THE VIRAL PROTEASES TO YEILD 16 NON STRUCTURE PROTEINS WHICH FURTHER Causes FORMATION OF REPLICASE TRANSCRIPTASE COMPLEX
RTC CONVERTS THE NEGATIVE STRAND OF RNA INTO THE POSITIVE STRAND WHICH GETS TRANSLATED INTO PROTEINS
THE PROTEINS ARE THEN ASSEMBLES INTO NUCLEOCAPSID IN THE ER GOLGI INTERMEDIATE COMPARTMENT AND IS TAKEN TO THE CELL MEMBERANE WHERE IT IS RECOGNISED BY T LYMPHOCYTES TO FURTHE ELICIT IMMUNE RESPONSE
Both of them however acts as antigens for immune stimulation and inturn it teaches the body to protecht it self against a microbe
mRNA vaccines come in two main types, each with unique characteristics namely non-replicating mRNA and self-amplifying mRNA.
These two categories represent different approaches to harnessing the power of mRNA for vaccine development.
They are called "non-replicating" because they do not include components necessary for self-amplification. They only contain the instructions for a particular antigen.
mRNA includes essential elements: the cap at the 5'-end, untranslated regions (UTRs), and the poly-A sequence (poly(A)-tail) at the 3'-end.
These elements are crucial for effective translation of the mRNA, providing protection against exonucleases and ensuring proper splicing of the transcript.
they are generated through in vitro transcription, using a plasmid DNA encoding the target immunogene as a template When administered, non-replicating mRNA vaccines work by delivering the genetic instructions to the host cell's cytoplasm.
The host cell then translates these instructions into the antigenic protein, which is usually a piece of the pathogen, not the entire pathogen itself.
This protein is displayed on the cell's surface, and the immune system recognizes it as foreign, triggering an immune response.
They also have the advantage of safety, as they cannot replicate or integrate into the host's DNA.
They can also be easily tailored to target different diseases by changing the mRNA sequence to code for different antigens.
The success of COVID-19 mRNA vaccines, like the Pfizer-BioNTech and Moderna vaccines, demonstrates the potential of non-replicating mRNA vaccine technology.
Self-amplifying mRNA vaccines are a more advanced type of mRNA vaccine.
Unlike non-replicating mRNA vaccines, self-amplifying mRNA contains not only the instructions for the target antigen but also components needed for the mRNA to replicate and produce more copies within the host cells.
They are often derived from single-stranded positive-sense alphaviruses like Venezuelan equine encephalitis virus, Sindbis virus, and Semliki forest virus.
As the self-amplifying mRNA is translated into the antigenic protein, it simultaneously replicates itself, leading to higher levels of antigen production within the host cell.
This amplification process results in a stronger and more sustained immune response with a lower mRNA dose, making self-amplifying mRNA vaccines particularly attractive.
Challenges with viral vectors include the potential immunogenicity of the vector itself, which may trigger unwanted immune responses and interfere with subsequent vaccine booster injections.
Replication-capable alphaviral vectors may carry a risk of viral reactivation, similar to live attenuated vaccines.
Self-amplifying mRNA technology holds the potential to target a wide range of diseases, including viruses and cancers, and can potentially overcome some of the limitations of non-replicating mRNA vaccines.
However, developing self-amplifying mRNA vaccines is more complex than non-replicating mRNA vaccines, and they may have more stringent storage and production requirements.
Molecular design is a critical aspect of creating effective mRNA vaccines.
The 3' UTR (Untranslated Region) is located at the 3' end of the mRNA molecule.
It is a concentrated area of mRNA instability factors, including A + U-rich elements (AREs) and GU-rich elements (GREs).
Designing the 3' UTR involves avoiding AREs and GREs, which can destabilize mRNA.
Additionally, stabilizing elements may be introduced to enhance mRNA stability and prolong its half-life.
The specific 3' UTR design can influence mRNA stability and translation efficiency.
The 3' poly(A) tail is located at the 3' end of eukaryotic mRNA.
Its length can affect mRNA translation and stability.
Typically, eukaryotic mRNA has poly(A) tails composed of dozens to hundreds of adenine bases.
The length of the poly(A) tail is regulated in the nucleus and cytoplasm and plays a role in mRNA stability and translation efficiency.
It cooperates with the 5' cap and other elements to regulate mRNA function.
The 5' cap is a critical structural element at the beginning of an mRNA molecule.
It consists of a positively charged base, N7-methylguanosine, and a negatively charged 5',5'-triphosphate bridge.
The 5' cap is essential for initiating mRNA translation, as it signals ribosomes to bind and start protein synthesis.
It also protects the mRNA from degradation by RNases.
Different types of 5' caps exist, and they can influence mRNA stability and translation efficiency.
The 5' UTR (Untranslated Region) is a part of the mRNA molecule that plays a role in translation initiation and efficiency.
The secondary structure of the 5' UTR can affect translation initiation.
5' UTRs are usually designed to avoid upstream open reading frames (uORFs) to prevent erroneous translation.
A short and less structured 5' UTR is often preferred for efficient translation initiation.
The characteristics of the 5' UTR can influence mRNA translation efficiency, and they are tailored for specific vaccine design.
Nucleoside analogs are modified versions of nucleosides, the building blocks of DNA and RNA.
Modified nucleoside analogs can be introduced into mRNA to improve its properties.
For example, certain analogs, like pseudouridine and methylated nucleosides, can make the mRNA less recognizable by the immune system.
These modifications help increase mRNA stability and reduce the risk of inducing unwanted immune responses.
Nucleoside analog design is a critical aspect of optimizing mRNA vaccines.
Molecular design is a critical aspect of creating effective mRNA vaccines.
mRNA molecules for vaccines consist of several key elements:
5' cap: A structure at the beginning of the mRNA molecule important for stability and translation initiation.
5' UTR (Untranslated Region): This region influences the mRNA's translation efficiency.
Open Reading Frame (ORF): This section contains the genetic instructions for the target antigen.
3' UTR: The 3' untranslated region is important for mRNA stability.
Poly(A) tail: The polyadenine tail at the 3' end is crucial for stability and translation.
The combination of these elements can be precisely engineered to optimize mRNA stability and translation efficiency for vaccine development.
Efficient delivery is critical for ensuring mRNA reaches the host cell's cytoplasm to stimulate the production of specific antigens.
Overcoming these challenges is vital for the success of mRNA vaccines in preventing or treating diseases.
The first challenge beingbarrier being the size mRNA molecules are relatively large, making it difficult for them to cross the cell membrane via simple diffusion. The second being
Negative Charge: The negative charge of mRNA molecules can cause electrostatic repulsion with the negatively charged cell membrane. And the last is
Vulnerability to Degradation: mRNA is highly susceptible to degradation by RNAases in the skin and blood
So Researchers have investigated many methods to deliver mRNA vaccines to overcome the challeges discussed in the previous slide
For example, delivery carriers, such as lipid-derived and polymer-derived materials, dramatically increased cellular uptake of RNAs, thus receiving tremendous attention in recent years mRNA vaccines were also delivered as free mRNA). Additionally, dendritic cells were loaded with mRNA vaccines ex vivo and transferred to the hosts
In this section, I am going to focus on the technologies for formulating and delivering mRNA vaccines in carrier-mediated, naked, and DC-based forms.
LNPs play a crucial role in delivering mRNA vaccines, including those used for COVID-19. They provide a protective and efficient means of delivering genetic material into target cells, ultimately triggering an immune response and enabling the production of specific proteins for disease protection and treatment. The specifics of LNP-mediated mRNA release and their interactions with cells continue to be subjects of ongoing research and investigation.
LNPs can encapsulate RNA molecules, shielding them from enzymatic degradation. Typically, the encapsulation efficiency of mRNA by LNPs is high, ensuring the protection of mRNA molecules. They typically consist of four key components:
Ionizable Cationic Lipid: This lipid aids in the self-assembly of LNP particles and is essential for the release of mRNA into the cytoplasm after endocytosis.
Lipid-Conjugated Polyethylene Glycol (PEG): PEG helps stabilize the nanoparticles during preparation and provides a hydrophilic outer layer, prolonging circulation time after administration.
- Cholesterol: Cholesterol serves as a stabilizing agent.
-Phospholipids: These maintain the two-layer lipid structure of LNPs.
- Cationic peptides with positively charged amino groups, such as lysine and arginine residues, are used to deliver mRNA through electrostatic interactions.
- Protamine is a cationic peptide that can protect mRNA from RNase degradation and has adjuvant properties.
- Protamine can form complexes with mRNA and protect it from degradation, improving stability and efficacy.
- Protamine-mRNA complexes have adjuvant activity through activation of TLR7.
- Cationic cell-penetrating peptides (CPPs) like RALA and LAH4-L1 facilitate the delivery of mRNA and enhance endosomal escape.
- Some CPPs are amphipathic, which helps disrupt endosome membranes at low pH and improve mRNA release.
- Anionic peptides, while unable to complex with RNA due to their negative charges, can be conjugated to positively charged polymers for RNA encapsulation.
- Clinical trials involving protamine-mRNA complexes and naked mRNA delivered via ID or IM routes showed well-tolerated vaccines but did not induce sufficient immune responses against vaccine targets.
- More research is needed to enhance the efficacy of mRNA vaccines using peptide carriers.
Polymeric materials serve as effective carriers for mRNA vaccines, protecting the RNA from degradation and facilitating intracellular delivery. To enhance stability and safety, structural modifications, such as incorporating lipid chains and biodegradable components, have been explored.
Cationic polymers like polyethylenimine (PEI), polyamidoamine (PAMAM) dendrimers, and polysaccharides effectively condense and deliver negatively charged RNA molecules. For example, PEI formulations have successfully delivered mRNA vaccines against HIV, influenza, and other diseases. PAMAM dendrimers protected against Ebola, H1N1 influenza, and Toxoplasma gondii challenges. The microfluidic mixing method was employed for mRNA vaccine formulation.
Anionic polymers, like PLGA, have been used in combination with cationic lipids to create lipid-polymer hybrid formulations. These hybrids have shown efficacy in delivering mRNA vaccines, delaying tumor growth in mice.
Polymer-based mRNA vaccines have demonstrated therapeutic effects in preclinical studies. Future research aims to develop biodegradable and highly efficient polymers for clinical translation.
Cationic nanoemulsion (CNE) is an innovative approach to delivering mRNA vaccines effectively. It combines the benefits of nanoemulsion, a stable oil-in-water particle system, with cationic lipids that can absorb negatively charged RNA molecules. One example of a nanoemulsion-based adjuvant is MF59, which is FDA-approved for use with influenza vaccines in older adults.
CNE enhances vaccine efficacy through the release of cytokines and recruitment of immune cells without triggering specific immune receptors. By incorporating cationic lipids, such as DOTAP, into the formulation, CNE can protect mRNA from degradation and efficiently deliver it to target cells.
In preclinical studies, CNE has successfully delivered self-amplifying mRNA vaccines against various viral and bacterial infections, including respiratory syncytial virus, cytomegalovirus, and HIV. These vaccines induced high levels of antigen-specific antibodies and effective immune responses in animal models, demonstrating the promise of CNE as a vaccine delivery system.
While CNE has shown great potential in preclinical studies, its effectiveness in humans is yet to be fully evaluated through clinical trials. This technology holds promise for the future of mRNA vaccine development and delivery, offering a new avenue to combat infectious diseases.
Virus-like self-amplifying mRNA particles (VRPs) offer a unique approach to mRNA vaccine delivery. These particles utilize viral structural proteins, expressed from helper cell lines, to package self-amplifying mRNA. The advantage of VRPs lies in their efficient cytoplasmic delivery of the RNA payload, resembling how viruses naturally enter and release their genetic material into cells.
VRPs have been derived from various viruses, including alphaviruses, flaviviruses, and rhabdoviruses. They've been used to target a wide range of infections and cancers, showing promise in preclinical studies. For instance, VRPs based on Venezuelan equine encephalitis virus (VEEV) successfully expressed dengue virus antigens, protecting non-human primates from viral challenge. Kunjin virus-derived VRPs expressing GM-CSF led to the elimination of primary tumors and reduced lung metastases in mouse models.
Despite their potential, there are challenges associated with VRP-based mRNA vaccines. Scaling up production can be complex, requiring specialized manufacturing processes. Additionally, there's a risk of the immune system producing antibodies against the viral vectors, which has been observed in clinical trials. Future development in this field aims to enhance vaccine efficacy, streamline production, and minimize the impact of anti-vector immune responses.
Naked mRNA injection means that the delivery of the vaccine is only done in a buffer solution.[63] This mode of mRNA uptake has been known since the 1990s.[21] The first worldwide clinical studies used intradermal injections of naked mRNA for vaccination.[64][65] A variety of methods have been used to deliver naked mRNA, such as subcutaneous, intravenous, and intratumoral injections. Although naked mRNA delivery causes an immune response, the effect is relatively weak, and after injection the mRNA is often rapidly degraded.[58]
various methods of administering mRNA vaccines have been explored, each with its own advantages and disadvantages.
1. **Intravenous Injections (IV):**
- Administered directly into the bloodstream, providing a large volume for vaccine delivery.
- Offers direct access to immune cells and lymphoid organs, increasing vaccine efficacy.
- Disadvantages include hindrance by plasma proteins, enzymes, and mechanical forces, as well as potential systemic side effects like spleen injury and lymphocyte depletion.
2. **Subcutaneous Injections (SC):**
- Administered under the skin's epidermis and dermis in the subcutis layer.
- Allows for a larger injection volume, reducing pressure and pain at the site.
- Disadvantages include lower absorption rates and potential mRNA degradation.
3. **Intramuscular Injections (IM):**
- Administered into the muscles, where there is a network of blood vessels and immune cells.
- IM-administered mRNA vaccines remain at the injection site and draining lymph nodes for an extended period.
- Proven efficacy for certain mRNA vaccines, such as those against SARS-CoV-2.
4. **Intradermal Injections:**
- Administered in the dermis layer of the skin, which contains vascular and lymphatic vessels.
- Allows for the transport of mRNA vaccines and antigen-presenting cells (APCs) to draining lymph nodes for B and T cell activation.
5. **Intranodal Injections:**
- Deliver mRNA vaccines to peripheral lymphoid organs where APCs and immune cells interact.
- The efficacy for mRNA vaccines is still being explored, and this method may require ultrasound guidance for administration.
Good morning all of you! As earlier part about mRNA vaccine already discussed by anushka & shreya , Now I will discuss about Application advantages & disadvantages of mRNA vaccine.
As slide showing there was huge amount of disappointation till 2020 among the researchers who were working on mRNA vaccine , & after a decades of years & period of failure Finally a time of success came in 2020 & mRNA vaccine formed against COVID-19 & successfully tested, approved & distributed in the society for there well being.
But between this journey from continuous failure to success a major Question which arrised among research community was , what is the reason behind this failure, many hypothesis came & lastly it was concluded that N1-methyl-pseudouridine modified mRNA is responsible for the increasing in efficacy of mRNA vaccine.
& further it was also proved by comparing mRNA based vaccine CureVac(CvnCov) & vaccines of pfizer-BoiNTech & Moderna, & Found that after delivering with same stretegy, there was difference in efficacy between CureVac & vaccines of pfizer – BioNtech & moderna , & it occurs due to N-1-methyl-pseudouridine modified mRNA.
Till now there is only disease (COVID-19) for which mRNA vaccine is available in market, & pfizer COVID-19 vaccine is the first mRNA product to achieve full FDA approval.
Generally RNA vaccine for COVID-19 can be based on two different types of RNA, namely mRNA vaccine & saRNA vaccine, As the structure of mRNA & saRNA are similar but a key diiference is saRNA contain code for viral relpicase enzyme & helps in making multiple copies of the viral RNA, once it’s in a cell so this make it cost effective.
Recently two scientist, Ms. Katalin kariko & Mr. Drew weisman got the nobel prize in physiology or medicine 2023, for their discoveries concerning nucleoside base modification that enabled the development of effective mRNA vaccine against COVID-19. & they create a great achievement in this new era of vaccinology.
Currently over 50 clinical trials are listed on clinicaltrials.gov for RNA baesd vaccine in a number of cancer including blood cancers, melanoma, brain cancer, & prostate cancer.
This technolgy has a scope for personalized cancer vaccine, where the RNA sequence in the vaccine is designed to code for cancer-specific antigens.
There are also some mRNA vaccines which are under clinical trials is listed here, like vaccine for influenza virus, pfizer & moderna are working on it under phase 3 clinical trial, as currently flu vaccine which are available in market have low effectiveness about 40-60% so there is need for vaccine with high effectiveness. Vaccine for zika virus, which is under phase 2 clinical trial , vaccine for respiratory syncytial virus which is also under phase 3 clinical trial, vaccine for cytomegalovirus which is also under phase 3 clinical trial, & vaccine for human immunodeficiency virus, which is also under phase 2 and phase 1 clinical trial respectively.
As safety should be first priority in any prevention control so in the case of mRNA based vaccine active pathogens are not used in the construction of vaccine like in many traditional vaccines are produced.
As discussed in 1st point there is no active pathogen is involved so this lead to decrease in the risk of localized outbreaks of pathogen
Due to production of antigen inside the cell it will lead to stimulation of both cellular as well as humoral immunity.
As designing of this mRNA based vaccine can be done very fast as compare to traditional vaccine
These mRNA based vaccine are more cheaper than other traditional vaccine
As the translation of mRNA is occur in cytoplasm so there is no need for the RNA to enter the nucleus, & this will lead to decrease in the risk of being integration into the host genome
This can be done by enriching the guanine-cytosine (GC content) OR by choosing UTR known to increase translation & this process is known as sequence engineering of mRNA
This slide showing the Advantages & disadvantages of different types of vaccine platform in comparision to mRNA based vaccine
As Storage is one of the major issue, because mRNA is very fragile so they must be kept at very low temperature to avoid degradation. For example BNT162b2 mRNA vaccine has to be stored between -80c & -60c whereas mRNA-1273 vaccine has to be stored between -25c & -15c & reason for this temperature difference is reported by Nature in nov. 2020 that “it is possible that difference in mRNA secondary structure could account for the thermostability differences.
As before 2020 there was no mRNA based vaccine had been authorized for use in humans, so there is a risk of unknown effect. & there is a need of providing accurate information about mRNA technology in public for building trust.
As reactogenicity is similar to that of conventional non-RNA vaccine, there are also many side effects such as allergy, renal failure, heart failure etc.
mRNA may also be degraded quickly after administration & this is a substantial challenge for mRNA delivery, however appropriate carrier can avoid degradation & enhance immune response
