Arn Messager

Advanced Drug Delivery Reviews 179 (2021) 114000
Contents lists available at ScienceDirect
Advanced Drug Delivery Reviews

journal homepage: www.elsevier.com/locate/adr
Messenger RNA-based vaccines: Past, present, and future directions

in the context of the COVID-19 pandemic
Samagra Jain a, Abhijeet Venkataraman b, Marissa E. Wechsler c, Nicholas A. Peppas a,b,d,e,f,⇑
a
Department of Chemical Engineering, The University of Texas at Austin, Austin, TX, USA
b
Department of Biomedical Engineering, The University of Texas at Austin, Austin, TX, USA
c
Department of Biomedical Engineering and Chemical Engineering, The University of Texas at San Antonio, San Antonio, TX, USA
d
Institute for Biomaterials, Drug Delivery, and Regenerative Medicine, The University of Texas at Austin, Austin, TX, USA
e
Division of Molecular Pharmaceutics and Drug Delivery, College of Pharmacy, The University of Texas at Austin, Austin, TX, USA
f
Department of Surgery and Perioperative Care, Dell Medical School, The University of Texas at Austin, Austin, TX, USA
a r t i c l e i n f o a b s t r a c t
Article history: mRNA vaccines have received major attention in the fight against COVID-19. Formulations from compa-
Received 24 May 2021 nies such as Moderna and BioNTech/Pfizer have allowed us to slowly ease the social distancing measures,
Revised 27 September 2021 mask requirements, and lockdowns that have been prevalent since early 2020. This past year’s focused
Accepted 6 October 2021
work on mRNA vaccines has catapulted this technology to the forefront of public awareness and addi-
Available online 9 October 2021
tional research pursuits, thus leading to new potential for bionanotechnology principles to help drive fur-
ther innovation using mRNA. In addition to alleviating the burden of COVID-19, mRNA vaccines could
Keywords:
potentially provide long-term solutions all over the world for diseases ranging from influenza to AIDS.
mRNA
Vaccines
Herein, we provide a brief commentary based on the history and development of mRNA vaccines in
Nanotechnology the context of the COVID-19 pandemic. Furthermore, we address current research using the technology
Immunology and future directions of mRNA vaccine research.
SARS-CoV2 Ó 2021 Elsevier B.V. All rights reserved.
Drug delivery
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2. General overview of vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
3. Vaccine types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
3.1. Live-attenuated vaccines. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
3.2. Inactivated vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
3.3. Subunit/recombinant vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
3.4. Other vaccines (DNA/Toxoid) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
3.5. mRNA vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
3.5.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
3.5.2. Unique Mechanism of action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
3.5.3. Advantages over current standards of vaccination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
3.6. mRNA vaccine applications in prophylactic immunity and disease treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
3.6.1. SARS-CoV-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
3.6.2. Influenza virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
3.6.3. Zika virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
3.6.4. HIV/AIDS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
3.6.5. Dengue fever. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
3.6.6. Rabies virus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
⇑ Corresponding author.
E-mail address: [email protected] (N.A. Peppas).
https://doi.org/10.1016/j.addr.2021.114000
0169-409X/Ó 2021 Elsevier B.V. All rights reserved.
S. Jain, A. Venkataraman, M.E. Wechsler et al. Advanced Drug Delivery Reviews 179 (2021) 114000
4. Future directions and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Declaration of Competing Interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
1. Introduction deliver mRNA to the body’s tissue and harness the technique to
produce a next-generation subunit vaccine, and many leading
When the ongoing COVID-19 pandemic brought the world to a pharmaceutical companies were eager to try and do so. As of today,
standstill in the spring of 2020, the medical and scientific commu- companies such as Moderna and Pfizer/BioNTech have been suc-
nities, alongside political leaders and national governments, cessful in producing COVID-19 mRNA vaccines. Their formulations
rapidly arrived at a consensus that wide-scale vaccination against successfully passed in Phase 3 clinical trials in the fall of 2020 and
the SARS-CoV2 virus would be the most effective strategy to con- received a rare emergency use authorization from the United
trol the impact of the disease and allow for the quickest return States Food and Drug Administration (FDA). Both Moderna and Pfi-
to normalcy. This steadfast belief in the public health impact of zer/BioNTech utilized an mRNA approach requiring 2 shots, while
vaccines highlights the transformative role that they have played Johnson Johnson a viral vector approach requiring just a single
in ensuring human health. After several difficult months, billions shot. The latter approach utilized a different virus in order to
of dollars in investment, and the near single-minded focus of the genetically encode instructions intended to fight off COVID-19
scientific community, multiple novel vaccines were produced infections. Pfizer/BioNTech was the first to get approval on Decem-
against the new disease. Today, these vaccines are being adminis- ber 11, 2020, with Moderna getting approval a week later. Johnson
tered at an unprecedented rate and slowly allowing ‘‘normal” life & Johson’s vaccine was approved on February 27, 2021. All of the
to resume for societies around the world. This pandemic is not vaccines are currently approved for use in adults, while the Pfizer
yet over - many countries, particularly developing ones with low vaccine has been approved for adolescents as well. Additionally,
vaccine access and/or poor population compliance, are still being all three of these vaccines were given emergency use authorization
ravaged by the virus, new variants are frequently emerging which in various countries. On August 23, 2021, the US Food and Drug
continue to create new outbreaks and challenge the effectiveness Administration (FDA) officially approved the Pfizer/BioNTech vac-
of these vaccines, and individuals are still reeling from the eco- cine. Widespread administration of these vaccines began around
nomic and health impacts caused by the virus, but experts can con- the same time as their debut and continues today; though there
fidently state that the worst of the pandemic is behind us, and this was certainly initial hesitancy in the eyes of the general public
paradigm shift can be significantly attributed to newly developed (some of which still persists well into 2021), extensive trials and
mRNA vaccines. studies have confirmed the safety and efficacy of these vaccines.
Historically, live, killed, and subunit vaccines have been critical Over 44% of the global population [1] has now been partially vac-
in controlling the spread of similar diseases such as smallpox and cinated against COVID-19 as of September 2021, and world stabil-
hepatitis, but these diseases have been limited in their interna- ity is contingent upon the sustained success of vaccination efforts.
tional presence, infectivity and case fatalities. COVID-19 presented The breakneck pace of the development of COVID-19 mRNA
several novel challenges in this regard; the ease of spread of the vaccines highlights the benefits and utility of mRNA delivery as a
disease and the high observed fatality rate made it impossible to vaccination strategy, and it is worth exploring how this technology
ignore or downplay the impact of the virus for the general popula- can be refined and improved upon to provide prophylactic and
tion. Scientists maintained that lockdowns, social distancing/phys- therapeutic treatment solutions for a wide range of diseases. Look-
ical isolation, mask-wearing, and other stopgap measures would ing beyond COVID-19, several infectious and tropical diseases con-
continue to be necessary for public health until a vaccine for tinue to harm populations in developing countries with limited
COVID-19 became widely available, but such conditions are unsus- avenues for prevention. The foundational principles behind mRNA
tainable for extended periods of time, as evidenced by the disrup- vaccines could potentially be applied to create effective solutions
tion of economies and livelihoods around the world and the for these diseases. We examine the progression and current state
increase in suicide rates and mental health issues caused by isola- of mRNA vaccines and their modern utility, their benefits/limita-
tion. For these reasons, scientists and pharmaceutical companies tions and applications in treating and generating immunity against
faced a unique pressure from citizens and governments to create, different diseases, and how principles of bionanotechnology can be
manufacture, and widely distribute a vaccine immediately, as used to further improve mRNA vaccine applications in the future.
opposed to after the several years of development, testing, and
quality control this process normally requires.
In the spring and summer of 2020, academic research groups 2. General overview of vaccines
and pharmaceutical companies turned their attention to messen-
ger RNA (mRNA) vaccination, a different vaccination strategy Vaccines have played a pivotal role in revolutionizing human
which utilizes the host’s cellular machinery to synthesize a viral health and society over the past two centuries; their advent in
protein product and establish protective immunity within the host. the late 18000 s converged years of seminal research in germ theory
mRNA therapeutic delivery previously had limited applications in and immunology to significantly reduce the disease burden of
humans due to the instability of mRNA in vivo and was primarily many deadly infectious diseases. Vaccines are based on the funda-
being explored as an experimental strategy for cancer therapy, mental premise of protective immunity at both the individual and
but the urgency of the pandemic required rapid development of population levels. By exposing a recipient to a noninfectious ele-
a vaccine with a high safety profile, ease of scale up/production, ment of a disease-causing pathogen, the vaccine can stimulate
and strong therapeutic efficacy, which a mRNA vaccination could the host’s adaptive immune system to generate immunological
provide. Recent advances in nanotechnology, and particularly memory against the pathogen, significantly reducing the chance
lipid-based nanoparticles, offered an avenue for researchers to of infection when exposed to the pathogen in the future. Simulta-
2
neously, widespread vaccination can protect the most vulnerable
DNA: Zika virus,[12] Phase 1 clinical trial for COVID

Preclinical exploration of vaccination strategies for
members of a population through a process known as herd immu-
Future Directions in Disease Treatment/Delivery
COVID[8], orotransmucal delivery using artificial
vaccineToxoid: Exploration of PLGA-chitosan as

nity -- with enough vaccinated members in a group, the virus will
Hydrogel modulation for influenza subunit co-

have little opportunity for community spread. To date, there are
vaccines available in the United States for nearly 30 different dis-
eases ranging from influenza to Ebola, and many more worldwide
herpes[6] and COVID[7] (intranasal)

[2]. Additionally, hundreds of vaccines are currently being
researched and developed for preventative applications in treating
delivery,[10] oral delivery[11]

other diseases -- most notably SARS-CoV2 -- and vaccines and their
associated delivery strategies are also being explored for prophy-
lactic treatment against diseases such as cancer.
delivery vector[13]
Vaccination has reduced the burden of infectious disease, sec-
intelligence[9]
ond only to clean drinking water in reducing mortality worldwide
[3]. However, infectious diseases remain the second leading cause
of death worldwide, disproportionately affecting children under
the age of 5 and people in low-income countries. In fact, five of
the top ten leading causes of death in low income countries are
caused by infectious agents: lower respiratory infections (e.g.,
pneumonia), HIV/AIDS, diarrheal disease, malaria and tuberculosis
Edward Jenner, smallpox vaccine
Anthrax (1970 s) and hepatitis B
virus) - work in progressToxoid:

Typhoid, plague, cholera (1896),
[4]. While some of these killers lack a current vaccine for disease
(1796) - virtually eradicated in
DNA: 1983 (smallpox vaccine

experimentation), 2016 (Zika
control, many deaths result from vaccine-preventable disease,
indicating substantial room for improvement in vaccine technol-
ogy and administration.
Seminal Research
influenza (1940 s)
Diphtheria (1923)
humans now
3. Vaccine types
(1980 s)
The innate immune system, consisting of natural barriers such
as skin, mucous membranes, nonspecific macrophages, and
enzymes serves well as a first line of defense against many
disease-causing antigens, but fails to respond robustly and specif-
ically to antigens, possibly resulting in damage to the host through
mumps, rubella (MMR)
hepatitis A, hepatitis B
Diphtheria, influenza,
Influenza, meningitis,
excessive inflammation and delaying the response against patho-
Cholera, pertussis,
C. difficile, tetanus
gens. The human body’s adaptive immune system bridges these
typhus, measles,
Influenza, polio,
shortcomings by generating a pathogen-specific immune response

Modern Uses
and retaining memory of the pathogen’s key features. By doing so, (exploratory)
COVID-19
the system can direct cellular and humoral elements to rapidly

neutralize a pathogen when it re-enters the body, and generating
this immunological memory is the key focus of most vaccination
strategies [5].
Vaccine technology has advanced significantly from the initial
attempts in the 18th and 19th centuries to induce smallpox immu-
Only the antigenic component of the pathogen
host immune system and generates immunity
Weakened/non-virulent pathogen detected by
DNA: Insertion of DNA into host cell to create
antigenic product to host - similar to subunit

immune system, generating immunity while
is introduced into the host and presented to
nity through lesion transfusion - modern vaccines are highly pre-

antigenic productsToxoid: Introduction of
cise and carefully engineered formulations that utilize a variety

Fully killed pathogen is exposed to host
but focuses on product, not pathogen
of antigen properties to stimulate adaptive immunity. A summary

Mechanism of Action/Key Features
of the various common vaccine types and their properties are pre-
sented below in Table 1.
against future exposure
A summary of vaccine types and their properties used today.
3.1. Live-attenuated vaccines

avoiding virility
immune cells
Live-attenuated vaccines are one of the most common and

effective vaccine types, with several formulations existing for dis-
eases such as influenza, polio, and typhus, to name a few. Such vac-
cines rely on a living strain of a pathogen which has been
attenuated to be non-infectious via growth in non-human tissue;
this harmless pathogen strain is then inserted into a human and
is recognized/killed by the host immune system. Effector B and T
cells and pathogen specific antibodies are created in response to
Subunit/ Recombinant
the pathogen and will be present to neutralize future infectious

Other approaches
(DNA/Toxoid)
strains. A notable example of a live attenuated vaccine is the yearly

Live-attenuated
Vaccine Type
influenza (flu) vaccine, which is commonly developed by culturing

Inactivated
infectious influenza strains in cell cultures or fertilized chicken

embryos to produce a live attenuated vaccine administered via
Table 1
nasal spray. The CDC estimates that over 169 million influenza vac-
cines were administered in 2019 alone and that vaccination
3
reduces an individual’s chances for influenza infection by 40–60% were several benefits to pursuing research in inactivated vaccines
[14]. - namely, a stronger clinical response and more global accessibility
Live-attenuated vaccines have several benefits; they are rela- due to existing supply chains [21]. The most promising advantage
tively simple to design, generate a strong immune response, and of inactivated vaccines, however, is the generation of a more gen-
retain their bioavailability for longer periods of time, ensuring con- eral immune response. As new COVID-19 variants continue to be
sistent generation of immunity. However, they can be dangerous to discovered, mutations in the spike protein structure may render
manufacture, can cause strong adverse immune reactions in mRNA vaccines ineffective (though this is a fear yet to be realized
immunocompromised patients, and possess the potential for as current vaccines have demonstrated immunity against variants
reverse mutations to an infectious strain, such as in the infamous as well). Inactivated vaccines provide multiple antigenic features
case of the oral poliovirus vaccine inducing poliomyelitis in to the host immune system, resulting in more robust immunity
patients. These issues can largely be addressed and mitigated against spike protein-mutated variants. Though mRNA vaccines
through modern genetic engineering approaches, and in fact, sev- were more readily adopted in the West, Chinese vaccine manufac-
eral ongoing clinical trials with live-attenuated COVID-19 vaccine turers Sinovac and Sinopharm, and India’s Bharat Biotech have pro-
candidates are proceeding to determine efficacy in producing anti- duced inactivated vaccines authorized by the WHO for emergency
bodies against the virus’ characteristic spike protein [15]. However, use. These vaccines - made with an aluminum hydroxide adjuvant
a significant challenge in utilizing live-attenuated vaccines to com- - are currently being deployed in many countries around the world
bat the COVID-19 pandemic lies in their stringent transport and have demonstrated strong production of neutralizing antibod-
requirements. Vaccine manufacturers are required to ensure a ies. Additionally, the French company Valneva hopes to bring inac-
‘‘cold chain” from the point of manufacture to the point of use to tivated vaccines that are ‘‘variant-proof” to the United Kingdom,
ensure vaccine stability and effectiveness, and the high costs of but their formulation is still in early-stage clinical trials [22].
transporting live vaccines in a temperature-controlled environ-
ment and low feasibility of cold chain maintenance over long dis- 3.3. Subunit/recombinant vaccines
tances and with a large number of doses make live attenuated
vaccines an insufficient short-term solution to the pandemic. This Subunit vaccines utilize the antigenic components of the tar-
issue is seen with certain formulations of mRNA vaccines as well, geted pathogen, with the purpose of using these sections to induce
and those face the same issues in the form of limited reach [16]. a protective immune response. Generally, the components are a
specific set of proteins and/or glycoproteins. After the administra-
3.2. Inactivated vaccines tion of the vaccine, adaptive immunity can help ensure that further
exposure to that specific component will result in an immune
Inactivated vaccines include the original Salk vaccine for response. In general, subunit vaccines are used when the inacti-
poliomyelitis (polio) and the cholera vaccine [17,18]. Similar to vated vaccine proves to be ineffective in preventing complications
live-attenuated vaccines, inactivated vaccines are generally grown or is too immunogenic for the patient’s immune system. The first
in culture in non-human tissue. However, the pathogen itself is subunit vaccine was developed for inoculation against pertussis
killed to reduce the virility and prevent infection from the vaccine. (whooping cough). Initially, inactivated Bordetella pertussis bacteria
Common methods to kill the pathogen include heat and/or were used, but this caused developments of adverse reactions. This
formaldehyde. More recently, hydrogen peroxide has been led to the development of vaccines based on components of the
explored as a killing agent [19]. While the pathogen has lost viril- bacteria rather than the full inactivated bacteria. Currently, the
ity, the guiding principle behind inactivated vaccines is that the most common use of subunit vaccines is with immunization for
body is still able to produce specific antibodies that bind to the var- Hepatitis B. In addition, there are vaccines in development for
ious fragments of the killed pathogen. This action then gives the tuberculosis and cholera [23,24].
host immunity, as the body has antibodies that will be able to rec- Due to their higher safety profile, subunit vaccines are primarily
ognize part of the live pathogen. developed for use with elderly patients for applications such as
While inactivated vaccines were incredibly common in the mid- inoculation against shingles [25]. Also, many mRNA vaccines are
20th century, they produce slightly weaker immune responses in being compared to their subunit counterparts, due to subunit vac-
comparison to live-attenuated vaccines [20]. This creates a major cines’ great ability in instilling lasting immunity [26,27]. In addi-
drawback in that often several doses of inactivated vaccines are tion, subunit vaccines are being used in studies on oral delivery
needed to be effective as the body loses its immune memory over of vaccines. Since most subunit vaccines are protein-based, they
time in the absence of reproducing viruses. Thus, inactivated vac- provide an avenue to test the protection against the acidic condi-
cines are often reserved for people with weaker immune systems, tions of the gastrointestinal tract [28]. Finally, in the early stages
such as those with immunodeficiencies and the elderly, although of the COVID-19 pandemic, one vaccine approach was a subunit
utilizing an adjuvant such as aluminum hydroxide can increase vaccine. The researchers in that study focused on creating a vaccine
immunogenicity. For the case of the Salk polio vaccine, it was that targeted multiple amino acid motifs, thereby increasing the
replaced within a decade of its discovery by a Sabin oral live vac- effectiveness of the vaccine compared to the current mRNA vac-
cine due to it requiring multiple doses to be effective and the slight cines. Using a computational approach, they were able to assess
increase in polio cases as a result of the need for multiple doses the effectiveness of their designed vaccine and concluded that this
[17]. vaccine was viable for general public use [29].
As development continued on mRNA vaccines in response to
COVID-19, researchers explored more traditional strategies, such 3.4. Other vaccines (DNA/Toxoid)
as inactivated vaccines, to provide a second type of defense against
the new disease. Much of the belief in the efficacy of mRNA vacci- The most common vaccines in use today utilize the techniques
nes was predicated on the assumption that just the spike protein described above, but other vaccination strategies have demon-
was enough to elicit a robust immune response; however, certain strated potential for human use and are being explored in research
individuals and communities around the world exhibited immune settings. For example, toxoid vaccines are centered on the delivery
senescence and would require more immunostimulatory material, of only the immunostimulatory product of a disease-causing agent
such as a whole virus, to establish protective immunity. Though to generate protective immunity. Similar to subunit vaccines (but
the development using inactivated vaccines took longer, there using an antigen product as opposed to the antigen itself), these
4
vaccines have shown high efficiency in protecting against diphthe- 160 million cases and 3 million deaths in over 218 countries as of
ria, tetanus, pertussis, and C. difficile infections. Of note, a promis- May 2021 [34] .International commerce and travel were ground to
ing 2012 study by Foglia et al. succeeded in generating an a halt as cases continued to spread exponentially and fatalities
intramuscularly administered toxoid vaccine using the enterotox- steadily rose around the world. Vaccines utilizing live or killed
ins and cytotoxins produced by C. difficile. Phase I and II clinical viruses, though capable of strong and broad immunogenic
trial data indicated promising immunogenicity and safety profile responses, required years to develop and a complex manufacturing
[30]. Though toxoid vaccine applications for C. difficile are still at process to properly scale, produce, package, and deliver, which
the clinical trial stage, the popular DTaP vaccine (immunity against crippled economies and weary populations around the world could
diphtheria, tetanus, and pertussis) utilizes toxoid proteins for the not afford to wait for.
diphtheria and pertussis components. As research groups and pharmaceutical companies worked to
In response to the low immunogenicity and high safety risks of identify a feasible vaccination strategy against the novel coron-
certain modern vaccines, a novel approach to vaccination is being avirus in the spring of 2020, a unique approach unexpectedly
explored that centers on the delivery of a recombinant bacterial emerged from the field of immuno-oncology. Cancer immunother-
plasmid. DNA vaccines, which are highly similar to mRNA vaccines, apy has long been an area of interest for researchers seeking to
aim to stimulate humoral and cellular immunity by synthesizing improve upon the current standard of chemotherapy; by altering
an antigen or antigen product in vivo from an injected DNA the body’s immune response to be able to more effectively detect
sequence which will be transfected into the host genome. In labo- and eliminate tumors. Immunotherapy holds the potential to offer
ratory settings, these vaccines have demonstrated antigen produc- an effective and safe form of cancer treatment. One specific
tion against several diseases such as influenza, but translation to approach to re-engineering the body’s immune cells is the delivery
primate and human applications remains unrealized [31]. Despite of messenger RNA encoding tumor antigens to immune cells,
their intended goals, DNA vaccines remain unable to safely gener- allowing the immune system to recognize and create antibodies
ate significant immune protection in most trials. With DNA vacci- against tumors. The now-famous 2005 studies by Kariko and
nes, integration and disruption of the host genome is a significant Weissman were the first to identify mRNA’s immunostimulatory
concern, and researchers have yet to demonstrate a dose-limited properties via activation of Toll-like receptors (TLRs), as well as
administration strategy to avoid such issues. Additionally, the ben- demonstration of the fact that slight nucleotide modification could
efits of DNA vaccines over conventional vaccination methods is still allow mRNA to be engineered as a human therapeutic by avoiding
a subject of consideration; as of 2020, no DNA vaccines have been the innate immune system and producing a large amount of pro-
approved for human use. tein with a relatively high safety profile [35]. In 2007, Mockey
Of note, the Oxford-AstraZENECA vaccine emerged in late 2020 et al. explored a strategy to halt melanoma progression in mice
as an effective vaccine against COVID-19 utilizing the DNA of the models through administration of mRNA encoding melanoma-
SARS-CoV2 spike protein. This is not technically considered a associated antigen, MART1 [36]. This approach involved delivering
DNA vaccine - rather than incorporating into the host genome, the mRNA to the cytosol of dendritic cells in vitro, which would
the genetic material is delivered to the cell via a chimpanzee ade- then produce the antigenic protein and present it to CD8 + cytotoxic
novirus vector and migrates to the nucleus, where it is transcribed T lymphocytes, activating them and enabling them to lyse tumor
independently of the host genome and then follows a similar path cells. The use of mRNA was favored for several reasons over other
as mRNA vaccines. This vaccine has a much lower efficacy than the gene-editing approaches: mRNA’s quick, universal, and transient
mRNA vaccines and is further reduced against novel variants, but it translation allowed for easy and safe administration, and engi-
remains a promising option for tropical and lower-income coun- neered mRNA avoided the challenge of a premature immune
tries due to its low price per dose and less stringent storage response before presentation to cytotoxic T lymphocytes (CTLs)
requirements [32]. It is yet unclear whether there is a significant as would be observed with protein/subunit delivery. Administra-
advantage of integrating into the host genome in this particular tion of the mRNA, delivered through polyethylene glycol PEGylated
disease, but the potential drawbacks of off-target effects relegate and histidylated liposomes, resulted in a significant and specific
this as a consideration for the distant future. decrease in the rate of tumor growth, which was directly corre-
Another recent vaccine that utilizes an adenovirus vector with lated with protective CTL activity [36].
non-incorporating DNA strands is the Johnson and Johnson/Janssen Despite the promising trials with mRNA based therapeutics in
COVID-19 vaccine. This vaccine is novel in that it claims to only laboratory settings, the facts remained that mRNA is highly unsta-
require one administration, as opposed to the two required by ble inside the body and prone to degradation by immune agents
other vaccines. It demonstrated about a 66% efficacy rate against and nucleases, possesses a high potential for adverse immuno-
disease prevention, which is lower than other frontrunner vacci- genicity, and initially generates weaker protective immunity than
nes, but it is still being utilized as an important tool in the fight conventional vaccines [37]. With the modern advances in lipid
against COVID-19. However, a very small number of recipients nanoparticle technology, researchers saw an avenue to effectively
developed rare and dangerous blood clots in the brain, in a condi- deliver nucleic acid vaccines and transcribe antigenic components
tion known as cerebral venous sinus thrombosis (CVST) [33]. This in vivo [38]. From 2010 onwards, pharmaceutical companies such
was observed with the AstraZeneca vaccine in Europe as well, as Moderna, Pfizer, and BioNTech began to explore the develop-
and administration of both vaccines was temporarily paused. Reg- ment of mRNA therapeutics/vaccines and raised millions of dollars
ulators eventually decided that the benefits of administration out- in funding towards this research, though it was only until recently
weighed the risks and resumed administration. that this work came into the spotlight. With the advent of the
COVID-19 pandemic in early 2020 and the unique challenges pre-
3.5. mRNA vaccines sent in ensuring a fast, scalable, and effective international vacci-
nation strategy for the novel virus, these companies were well
3.5.1. Introduction positioned to pivot their experimental explorations with mRNA
Conventional vaccination strategies have been significantly towards a vaccination solution [39]. Moderna’s vaccine was manu-
impactful for human health, COVID-19 presented a new challenge factured with smaller independent contracts, and BioNTech part-
for not only vaccine development, but also deployment and admin- nered with the pharmaceutical giant Pfizer for logistical and
istration. The world has been hard-hit by the pandemic, with over manufacturing support [40]. By early summer, mRNA vaccines
5
had exhibited the fastest development timeline along with high ments from the past couple of decades, it has become recently
generation of immunity, and the first COVID-19 vaccines to receive possible to engineer mRNA strands with slightly modified nucleo-
emergency FDA approval in December were mRNA-based [41]. tides that are capable of activating humoral and cellular immune
In the fall and winter of 2020, mRNA vaccines began concluding responses through the production and display of protein products
their Phase 3 clinical trials; exhibiting very high efficacy against but limited immunostimulatory behavior from the mRNA strand
COVID-19, Moderna and Pfizer-BioNTech’s formulations received itself. To combat the issue of low in vivo stability and susceptibility
emergency use authorization from regulatory agencies around to degradation, modern vaccine formulations can utilize lipid
the world in a historic move and began being distributed for nanoparticles (LNPs). These ionizable formulations are usually
large-scale vaccination. These vaccines built upon the simple prin- comprised of a main amino lipid along with helper lipids (choles-
ciples demonstrated by previous work in the field - an exogenously terol, PEGylated lipid, phospholipid) to facilitate cellular uptake
engineered mRNA strand is introduced to the body and used to and release. The microfluidic mixing process to form the encapsu-
produce the antigenic component of the SARS-CoV2 spike glyco- lated lipoplexes is performed at a low pH to encourage electro-
protein. These peptide fragments are then localized to the cell static complexation of the positively charged LNP and the
membrane and presented to immune cells, resulting in immunos- negatively charged mRNA strand [48]. Experimentation is under-
timulatory activity and generation of long-term immunity. Clinical way to improve the efficacy of these carriers as well - current areas
trials pointed to over 94% transmission/infection prevention effi- of interest include engineering these carriers for better organ
cacy and 100% severe infection/death efficacy for Pfizer and Mod- specificity and targeting, drug release and endosomal escape, and
erna’s vaccine, and by December 2020, both had received cellular uptake [49]. Moderna Therapeutics was the first commer-
authorization with more traditional formulations by manufactur- cial organization to demonstrate in vivo stability of mRNA-LNP for-
ers such as AstraZeneca and Johnson and Johnson following closely mulations against influenza in the past, and the findings from that
behind [42]. A third company, CureVac, has an mRNA vaccine can- work were central to the development of their COVID-19 vaccine,
didate in late stage clinical trials with the promise of long-term as well as Pfizer’s formulation. Another critical step in the develop-
stability at regular refrigerated temperatures; however, its Phase ment of clinically applicable mRNA was discovering the ability to
3 efficacy results displayed disappointingly poor potential for clin- engineer mRNA constructs with modified 50 methylguanosine
ical translation. Scientists point to the lower mRNA dose and triphosphate caps and 30 -poly-adenosine tails. These post-
inflammatory nucleotide uridine (as opposed to the substitution transcriptional modifications are critical for ensuring proper ribo-
of pseudouridine in most other formulations) as potential reasons some binding, stability, and protection from nuclease mediated
for the initial failure [41]. However, CureVac is continuing to degradation, and ligating custom sequences can allow IVT mRNA
explore formulations with unmodified mRNA sequences. The small to avoid common degradation factors, promote longer bioavailabil-
US-based vaccine manufacturer Novavax has demonstrated a vac- ity, and assist in more effective translation [50].
cine candidate with 90% efficacy in Phase 2–3 clinical trials that Additionally, it is important to make a distinction between
also promises many of the transportation/storage benefits of the replicating and non-replicating mRNA. Non-replicating mRNA is a
CureVac vaccine, though it is yet to receive emergency approval much simpler construct and consists of a transcript encoding the
[43]. antigen of interest flanked by untranslated regions and the previ-
The development of these vaccines resulted in the largest global ously mentioned post-transcriptional additions. Such vaccines do
vaccination campaign in human history [41]. As of September not contain any extraneous genes encoding replication factors
2021, an estimated 6.1 billion doses have been administered and are meant to be degraded in a relatively short period of time
worldwide with roughly 31 million doses continuing to be given after generating the antigen [51]. The current COVID-19 mRNA
per day [1]. These vaccines have single- handedly altered the tra- vaccines utilize this form of technology as the transcript fits more
jectory of the COVID-19 pandemic, slowly reducing the need for easily in the lipid nanoparticles and is more economical to manu-
masks and stringent social distancing measures and allowing soci- facture. Replicating mRNA, in contrast, contains additional genes
ety to return to its normal state of functioning. On May 13th, 2021, encoding self-replication factors such as RNA-dependent RNA
the United States Center for Disease Control (CDC) updated their polymerase. These constructs produce additional transcripts,
guidelines to state that masks are not required in most indoor/out- allowing for sustained expression of the antigen and a longer
door settings for fully vaccinated individuals, pointing to ever- immune response [52]. However, they are much bulkier and not
decreasing case numbers and fatalities in the country - stemming yet feasible for clinical applications, though there have been many
in a large part from increased vaccination rates [44]. However, as promising recent studies in animal models [53]. Fig. 2 illustrates
the Delta variant began to proliferate in the USA in August of the structural differences between non-replicating and self-
2021 and cases/hospitalizations began to increase - including a replicating mRNA constructs.
small number of breakthrough infections but the vast majority in Following administration of the LNP-mRNA therapeutic, the
unvaccinated individuals - these guidelines were revised to goal then becomes to transiently produce and express a translated
encourage masking in high density settings and in regions of high protein product. Upon crossing the cell membrane and being
spread [45]. On August 13th, 2021, the CDC further recommended released from the LNP into the cytoplasm, the mRNA strand, con-
a third booster dose after eight months of the Moderna and Pfizer/ taining conventional post-translational modifications, such as an
BioNTech vaccines in immunocompromised individuals, with open reading frame and a poly-adenosine tail, is recognized by
plans to recommend booster doses for the general population the host cell’s ribosomes and is translated into a protein product
expected to follow soon [46]. mRNA vaccines are not the only vac- in the typical RNA-to-protein pathway. Though current vaccine
cines available against COVID-19, but they were the first ones to be formulations are non-replicating, a second class of mRNA vaccines
produced and authorized for human use, and they continue to be is also being explored; termed self-replicating, these mRNA strands
absolutely pivotal in the global effort to combat the disease. also encode viral replication machinery which can allow for sus-
Fig. 1 highlights the drastic impact that mass vaccination has had tained expression of the mRNA [55].
on decreasing the incidence of global COVID-19 cases in 2021. The translated antigen is then either localized to the cell mem-
brane or exported outside of the cell, where it can stimulate
3.5.2. Unique Mechanism of action humoral and cellular immune responses. Typically, dendritic cells
mRNA vaccines, as the name suggests, are built around the prin- (antigen-presenting cells) will phagocytize the antigen and
ciple of in vitro transcribed (IVT) mRNA. Using the unique develop- mature, migrating from around the body to lymph nodes and pre-
6
Fig. 1. Daily laboratory-confirmed SARS-CoV-2 infections in Israel (Nov 1, 2020, to April 3, 2021). Reproduced with permission from Haas et al. 2021 [47].
senting antigen fragments on their surfaces to developing T cells. 3.5.3. Advantages over current standards of vaccination
This results in the mounting of an immediate immune response Though other vaccination techniques have demonstrated high
by CD8 T cells, as well as the generation of humoral immunity cap- efficacy against many types of diseases, mRNA vaccination holds
able of neutralizing future infections from the same pathogen. B promise as a safe, controllable, and efficient alternative to
cells are activated either by direct antigen recognition at the B cell pathogen-based viruses. The key safety advantage offered by
receptor (BCR) or CD4 T cell assistance, and these cells then pro- mRNA vaccines is the non-integrating mechanism of action - with
duce antigen-specific antibodies. These antibodies can later recog- all activity localized to the cytosol, genomic disruption and off-
nize the same antigen present on the actual pathogen and rapidly target effects are not a concern as they are with DNA-based vac-
mount an immune response before proliferation and infection. This cines. Secondly, mRNA can be easily modified at the nucleic acid
humoral protection is the basis of long-term vaccine-mediated level to further reduce unwanted immunogenicity, increase effec-
immunity [56]. A general overview of the intracellular process is tive half-life, and improve safety; modifications at the untranslated
shown in Fig. 3 and an expansion on intercellular immune genera- regions (UTRs) of the molecule can also promote ribosome binding
tion is presented in Fig. 4. and protein product translation. mRNA molecules are easily pro-
Fig. 2. mRNA and saRNA protein production in antigen presenting cells. Reproduced without changes from Sandbrink and Shattock, 2020 [54]. GOI: Gene of interest; UTR:
Untranslated regions; nsPs: non-structural proteins; CTL: cytotoxic T lymphocyte.
7
duced in bioreactors, safety requirements in the manufacturing sidized by many governments around the world and is expected to
process are much less stringent than with live/inactivated vaccines decrease as manufacturing capabilities increase.
due to the absence of live virulent agents, and nanoparticle tech-
nology has improved significantly in the last decade, enabling 3.6. mRNA vaccine applications in prophylactic immunity and disease
mRNA based therapeutics to have high in vivo viability, cellular treatment
uptake, and gene expression. Clinical and laboratory trials have
shown that antigen presenting cells (APCs) are able to exhibit 3.6.1. SARS-CoV-2
mRNA protein products to a similar degree as vaccination by more We have discussed much about the history and background of
conventional methods [41]. the COVID-19 pandemic elsewhere in this paper, but it is impor-
Production of mRNA vaccines is relatively straightforward, with tant to understand the biological underpinnings of this disease as
a plasmid containing the isolated gene of interest serving as a tem- well. Coronaviruses are a general family of enveloped ssRNA
plate for the mRNA strand. This process is easily scalable and does viruses characterized by the crown shaped proteins embedded
not require the use of dangerous live viruses or carefully monitored on their outer surface. Other notable coronaviruses include SARS-
cell cultures, resulting in a high safety profile at the production CoV and MERS-CoV, which have caused smaller, more contained
stage. Subsequent purification and encapsulation into nanoparti- pandemics in recent history (Sudden Respiratory Arrest Syndrome
cles results in the final product. This manufacturing process is and Middle Eastern Respiratory Syndrome, respectively). The novel
quite rapid - Moderna was able to isolate the COVID-19 spike pro- coronavirus originated around the end of 2019 with initial out-
tein genes within 4 days of receiving the genome and had their first breaks in various countries and rapidly spread around the world
formulations ready for in vivo animal trials in 2 months [58]. How- in the spring of 2020 [60]. SARS-CoV-2 infection can present radi-
ever, although the naked mRNA is easier to produce and store com- cally differently in individuals, with most younger people with
pared to other types of vaccine elements, the final therapeutic healthy immune systems exhibiting mild to no symptoms while
requires a stringent cold chain - in the case of the Pfizer/BioNTech older people/people with co-morbidities can develop symptoms
vaccine, approximately 70 °C. This significantly affects the trans- such as a fever, pneumonia, and severe respiratory distress. Cases
portation and storage of these vaccines in tropical and developing are characterized by a significant immune response that causes tis-
countries, and these areas have been relying more on inactivated sue and organ inflammation, and in some cases, the damage from
formulations as a result. However, this technology is still new, this response can persist long after the infection [61]. Overall, the
and if the more thermostable formulations of companies such as mortality rate of this virus has been estimated to be around 4%,
CureVac pass clinical trials, then the expansion of mRNA vaccines with a significant skew towards older populations. Several variants
to these areas will become more feasible. Moderna’s formulation have been identified in many countries around the world, includ-
is stable at commercial refrigeration temperatures but distribution ing the United Kingdom, South Africa, and India. These variants
was initially lagging due to limited manufacturing capabilities. The tend to have mutations in the spike proteins that can result in an
price per dose for mRNA vaccines currently ranges from $20-$40 increased lethality rate and a decreased response from the immune
depending on the manufacturer, which is significantly more system and current vaccines/therapeutics. However, more research
expensive than more traditional formulations (which are closer is needed to confirm the unique properties of these variants as well
to $2-$10 per dose) [59]. However, this cost is currently being sub- as their response to current vaccines.
Fig. 3. Mechanism by which mRNA vaccines elicit immunity. The mRNA encoding the viral protein enters the cell where it is translated into protein by the ribosome. The
resulting protein is broken down into peptides by the proteasome or transported by the Golgi apparatus to the outside of the cell. The remaining fragments in the cell are
presented as a complex. Additionally, protein outside of the cell can be taken up by various immune cells and fragmented into smaller pieces by the endosome. Figure created
using BioRender.com.
8
Fig. 4. Cellular mechanism of immune activation. Reproduced from Ghaffari et al., 2020. [57] (1) The SARS-CoV-2 virus enters the host cell via interaction between viral
spike and host angiotensin-converting enzyme 2 (ACE2) proteins. (2,3) Following replication and release from the host cells, a subset of viruses will be engulfed and digested
by antigen-presenting cells (APCs) like macrophages or dendritic cells. (4) Fragmented SARS-CoV-2 antigen(s) will be presented to T helper cells, which in turn will interact
and activate B cells. (5) Activated B cells will proliferate and differentiate into plasma or memory B cells with high-affinity binding receptors for the original SARS-CoV-2
antigen. Plasma cells secrete their SARS-CoV-2-specific receptors in the form of IgM, IgG, or IgA antibodies. (6) Antibody-mediated neutralization occurs when SARS-CoV-2-
specific antibodies bind to viral antigen(s) and prevent virus interaction and entry into host cells.
A significant challenge with COVID-19 is its ability to spread mRNA influenza vaccines has not yet reached clinical trials, but
latently - symptom presentation in a newly infected individual there are promising results in animal models. In one study, an
can take up to 14 days, if it occurs at all, and they are able to trans- mRNA vaccine targeting the hemagglutinin of a type A influenza
mit it to others through close contact with respiratory fluids during virus was developed and injected into mice, both young and old
that time [61]. Apart from prophylactic immunity through vaccina- [63]. The vaccine was able to elicit both B- and T-cell protection.
tions, treatment options are limited; due to the rapid spread of the In addition, the vaccine was able to withstand thermal stress, as
virus, longitudinal efficacy studies have been difficult. Several ther- well as give some protection against other antigens, most notably
apeutic options have been put forth, such as a combination of the highly conserved viral nucleoprotein. The latter result indicates
treatment with hydroxychloroquine and azithromycin, nucleotide that this particular mRNA vaccine could have use for immunity
analogs such as remdisivir, and convalescent plasma. However, beyond just influenza. A similar study focused on the same target
studies utilizing these treatment options have produced conflicting but utilized a different method, oil-in-water cationic emulsion, to
results, and in most mild cases, isolated rest at home is recom- formulate the mRNA vaccine [64]. Finally, a study in 2017 used
mended for the course of the disease. In more severe cases requir- lipid nanoparticle-modified mRNA vaccines to generate rapid and
ing hospitalization, supplemental oxygen, along with fluids and increased immune responses in mice, ferrets and nonhuman pri-
possibly the aforementioned treatment options. For patients mates [65]. The results were robust enough that the study elicited
exhibiting lung failure, a mechanical ventilator can also be used the start of some Phase I clinical trials.
to assist in breathing until the body can mount a sufficient immune
response [62]. The current standard of prophylactic treatment is
3.6.3. Zika virus
vaccination; a summary of the various vaccine types is provided
The Zika virus (ZIKV) is a mosquito-borne virus which was
in Table 2.
responsible for a brief global epidemic in 2015. The virus, an envel-
oped, ssRNA flavivirus that has a disease progression similar to
3.6.2. Influenza virus dengue fever, often presents little to no symptoms in healthy adult
The influenza virus, commonly known as the ‘‘flu”, is one of the humans, but complications arise when pregnant women or young
most common viral infections today. There are four major types of children are infected; in such cases, ZIKV has been linked to severe
influenza viruses, with three types - type A, B and C - known to developmental defects, particularly in neurocognitive abilities and
infect humans. Type A is the most virulent of them, responsible brain growth. Case reports have indicated linkages between Zika
for some of the global flu pandemics. In general, most influenza virus infection and Guillain–Barré syndrome in adults and congen-
virus infections result in high fevers, runny nose, joint and muscle ital malformation in infants. Prior to the 21st century, the Zika
pain, coughing and feelings of exhaustion. Given that the infections virus demonstrated low infection rates in humans and was local-
are so common, there are yearly vaccines developed to combat the ized to regions in Asia and Africa. However, due to intercontinental
evolving viral strains. travel, several outbreaks were observed around the world with
Much like with other vaccines, mRNA vaccines are being varying levels of severity. The 2015 outbreak was severe enough
explored as an alternative approach. The current research on to warrant worldwide travel advisories and several countries
9
Table 2
A summary of the various mRNA vaccine candidates for the prophylactic treatment of SARS-CoV-2
Manufacturing Vaccine Type Vaccine Mechanism of Action Current Regulatory/Approval Status

Company
Moderna mRNA LNP-encapsulated modified mRNA encoding for COVID-19 Emergency use authorization in several countries
spike protein delivered intramuscularly over two doses with around the world
a potential third dose booster
Pfizer/BioNTech mRNA LNP-encapsulated modified mRNA encoding for COVID-19 Emergency use authorization in several countries
spike protein delivered intramuscularly over two doses with around the world
a potential third dose booster
Johnson and Johnson/ Viral vector Single dose, antigen-encoding genes delivered in a single dose Emergency use authorization in several countries
Janssen utilizing a chimpanzee adenovirus vector around the world
University of Oxford/ Viral vector Antigen-encoding genes delivered in a single dose utilizing a Lower-cost alternative to other formulations;
AstraZeneca chimpanzee adenovirus vector worldwide emergency use authorization with
focus in developing countries
Gamaleya (Sputnik-V) Viral vector Two-dose intramuscular injection of adenovirus-enclosed Limited emergency use authorization around the
antigen genes world, highest use in Eastern Hemisphere
Sinovac (CornoaVac) Inactivated virus Two-dose intramuscular injection of chemically inactivated Limited emergency authorization use around the
COVID-19 virus world, highest use in Asia and Africa
Sinopharm Inactivated vaccine Two-dose intramuscular injection of chemically inactivated Full authorization in China with emergency use
COVID-19 virus authorization in several developing countries
CanSino (Convidecia) Viral vector Single shot adenovirus vector vaccine delivered Full authorization in China with very limited
intramuscularly emergency use authorizations in developing
countries
Bharat Biotech Inactivated virus Vero cell-grown COVID-19 virus, chemically inactivated and Very limited emergency use authorizations in
(Covaxin) various tropical and developing countries
Valneva Inactivated virus Single-dose chemically inactivated virus, promises strong Phase III clinical trials
performance against variants
Novavax Subunit Thermostable formulation of COVID-19 spike protein Phase II/III clinical trials
delivered using LNPs
CureVac mRNA Unmodified mRNA encoding COVID-19 spike protein Preclinical/early clinical trials after poor Phase III
encapsulated in LNPs results in initial formulation
advised couples to delay pregnancies until viral transmission was parts of the world with limited testing capabilities. As of 2020,
under control. However, this pandemic was short-lived and most around 38 million people globally are infected with HIV, with
cases either died out on their own or were easily combated with about 20% of that number unable to receive a confirmatory test
interferon treatment. Despite the relatively low threat of the Zika (HIV.gov). AIDS was classified as an epidemic in the 20th century
virus, there is scientific interest in vaccine development as a due to the high death counts and potential for uncontrolled trans-
stepping-stone towards deadlier diseases and designing effective mission, but several therapeutic options have since become avail-
vaccine strategies for pregnant women. Several Zika virus vaccines able for HIV-exposed individuals. Pre-exposure prophylaxis and
are currently in clinical trials, and Moderna is currently in Phase 1 antiretroviral therapy can control the HIV levels for these individ-
and 2 clinical trials with a potential mRNA vaccine candidate [66]. uals, preventing the clinical presentation of AIDS, but these medi-
In a 2017 effort by Richner et al., the authors succeeded in pro- cations cannot fully rid the body of HIV and must be taken long-
ducing a lipid nanoparticle (LNP) encapsulated modified mRNA term. Additionally, they can be extremely expensive and are not
vaccine against ZIKV and observed in vivo generation of sterilizing widely available in developing countries or lower-income commu-
immunity in mice models [67]. The delivered mRNA strand, which nities, resulting in limited access to care and an inequitable disease
encoded for ZIKV structural proteins, was modified at the 50 and 30 skew towards such groups [68].
untranslated regions and had base substitutions to ease transla- mRNA vaccination has been explored as a strategy to provide
tion. Lipid nanoparticles were selected as delivery vectors due to prophylactic immunity to HIV. Vaccine development has not gone
their strong performance in previous trials with siRNA and were well historically for this disease; the high mutation rate and the
delivered to mice via intramuscular inoculation. Mice receiving challenge of generating long-term and broadly-neutralizing anti-
2 lg or 10 lg of the vaccine demonstrated high levels of induced bodies have made development difficult, and no HIV vaccines are
neutralizing antibodies against ZIKV and significantly improved currently available for use. However, with the advances in mRNA
performance over DNA plasmid/inactivated vaccines. The study technology and its recent resurgence as a potential therapeutic,
also explored safety of administration to pregnant mice, and it there may be potential avenues for the development of an effective
was observed that while safety to mother and fetus with the mRNA mRNA vaccine. In 2018, Leal et al. demonstrated that naked mRNA
vaccine was not a concern, the fetus had a significantly lower neu- could activate human dendritic cells in HIV positive patients in a
tralizing antibody titer. phase 1 clinical trial; this was observed as good tolerability to
the therapeutic and increased HIV-specific T-cell production [69].
3.6.4. HIV/AIDS However, in 2019 an error was published stating that the mRNA
Human immunodeficiency virus, or HIV, and its associated dis- sequence used contained two start codons which may have
ease, acquired immunodeficiency syndrome (AIDS), are character- impacted protein expression [70]. More recently, Saunders et al.
ized as a significant global health threat. The virus is a type of developed a full mRNA-LNP vaccine and demonstrated its antibody
human-infecting retrovirus (enveloped, ssRNA) that causes critical generation capability in primate models. Vaccination with the for-
failure of the immune system - the decreased T cell count makes mulation resulted in high titers of serum HIV-binding antibodies
the body incredibly susceptible to even simple infections like the with good binding specificity. The authors pointed to the ease of
common cold. HIV is a sexually transmitted virus and can be production and scaleup, but also the challenges that the require-
passed perinatally as well, making it particularly troublesome for ment of a cold chain could cause. The first in-human clinical trial
10
of an mRNA vaccine began in the spring of 2021 - led by Moderna 4. Future directions and perspectives
in partnership with the International AIDS Vaccine Initiative (IAVI)
and Scripps Research, the trial demonstrated that 97% of recipients It was not long ago that mRNA vaccines were on the fringes of
developed the capability to produce broadly neutralizing antibod- the scientific community with a bleak outlook for translation to
ies, with the caveats that it was only responsive against 30% of HIV human therapeutics. The COVID-19 pandemic, as devastating and
strains in the trial regions and that more research is needed to dangerous as it is, has managed to spur innovation in this novel
observe antibody maturation [71]. However, this trial was a type of vaccination, and the ongoing and future research in this
promising sign for the eventual translation of mRNA vaccines to area potentially holds huge implications for human health. Dis-
human therapeutics for HIV. eases that plague tropical populations, for example, could eventu-
ally see a reduction in mortality and case counts as work in mRNA
vaccination against Zika, Ebola virus, and HIV comes to fruition.
3.6.5. Dengue fever
The influenza virus requires a yearly vaccine reformulation due
The Dengue fever is a tropical disease brought about also by a
to its constantly evolving nature; Freyn et al. demonstrated in
mosquito-borne virus, the dengue virus (DENV). Symptoms
2020 that it was possible to design a universal mRNA vaccine
include a high fever, headache, vomiting, muscle and joint pains
against various serotypes of the influenza virus that may soon
and the characteristic skin rash. Early in the infection, the skin
reduce the burden these viruses place on society every year [78].
rashes blanch when pressed and later become a large red rash with
A subsequent phase 1 clinical trial of the same formulation in
smaller white areas. These symptoms generally take 3–14 days to
humans demonstrated that vaccination was safe and an immune
develop [72]. Currently, dengue fever is common in more than 120
response was observed against the conserved viral hemagglutinin
countries, including those in south/southeast Asia and South Amer-
protein [79].
ica [73]. After seeing an uptick in infections after World War II,
Though mRNA vaccines have come a long way in such a short
increased effort was made to understand the nature of the virus,
time, their technology is not perfected yet, as evidenced by their
including the disease transmission and progression.
slightly less-than-complete conferment of immunity against
The virus is a part of the flavivirus family, small spherical
COVID-19 and the increasingly alarming number of COVID-19
viruses with a lipid envelope, just like the Zika virus [72]. The virus
Delta variant breakthrough infections post vaccination. The current
also induces a strong immune response, making it difficult to iden-
formulations struggle with thermostability, potential for harsh side
tify the specific components of the virus that the immune system is
effects due to the impurity of the lipid nanoparticles, and may
able to recognize and target. This ability to hide from the immune
eventually become completely ineffective against new variants as
system is heightened by the virus targeting Langerhans cells, den-
the virus continues to evolve [80]. One observed issue was a small
dritic cells that present pathogens to the rest of the immune sys-
number of allergic reactions to polyethylene glycol used in stabiliz-
tem. In addition, the dengue virus is notorious for having
ing the lipid nanoparticles of the Pfizer-BioNTech vaccine [81].
multiple strains, with each strain warranting its own developed
Replacing PEG with other biocompatible stabilizing polymers, such
immune response. Both of these factors have made conventional
as poly(N-vinylpyrrolidone) (PVP) and poly(N-(2-Hydroxypropyl)
vaccines ineffective. Instead, mRNA vaccines for the dengue virus
methacrylamide) (PHPMA) may decrease these adverse reactions,
are in the works. These vaccines are designed to target some of
although much more preclinical and clinical testing is required to
the non-specific protein epitopes to increase CD8 T-cell activation
confirm the immunogenic profiles of these polymers [82]. Another
and maturation of other immune cells to confer immunity memory
potential strategy for improving the efficacy of mRNA vaccines
[74].
would be to increase their targeting of dendritic cells via surface
conjugation of specific ligands. Grafting mannose or
3.6.6. Rabies virus hydrophobic-interaction-inducing lipids to the surface of lipid
The Rabies virus is a zoonotic infectious disease that targets the nanocarriers would enable them to more effectively target effector
central nervous system. It is contracted from salivary contact from cells and generate immunity [83]. Several past and ongoing studies
an infected animal and is extremely dangerous. Early on, symp- have demonstrated the improved targeting and immunogenic abil-
toms include fever and tingling at the site of the bite/scratch and ities of nanocarriers with targeting moieties attached to the surface
can progress to violent, uncontrolled movements, loss of motion, [84]. Future strategies for improving the efficacy of intramuscu-
confusion and loss of consciousness. Another key symptom is larly administered mRNA vaccines could certainly benefit from
hydrophobia, fear of water, which is often used as the key symp- incorporating these various aspects of nanotechnology.
tom in the diagnosis of rabies. The progression time depends on One promising future direction for mRNA vaccines could be oral
how far the virus has to travel from the peripheral nervous system delivery applications. Many diseases in areas of poor hygiene in
to the central nervous system. For many, once stronger symptoms developing countries are caused by enterotoxins or enteropatho-
start showing, it is too late for treatment and the virus is generally gens such as Vibrio cholera, Escherichia coli, Salmonella, and Shi-
fatal in these situations. As of 2016, only 14 people have survived gella. These pathogens cause infection upon crossing mucosal
rabies once they started displaying symptoms [75]. barriers, and inducing the formation of an immunologically strong
However, vaccination can help mitigate this issue, particularly mucosal barrier would prevent infection. The current standard of
in developed countries. In developing countries, the current vacci- subcutaneous or intramuscular injection of vaccines elicits strong
nes are unable to keep up with the rapidly-mutating virus. Thus, a humoral and cellular immunity, but does not generate similar
new mRNA-based vaccine approach is currently being investigated immunity at mucosal surfaces. However, orally administered vac-
that targets some of less rapidly-changing parts of the virus [76]. cines must pass through the digestive tract and can confer immu-
Much like the dengue virus mRNA vaccine, the developing mRNA nity at these surfaces, allowing for prophylactic treatment against
Rabies vaccines look to create antibodies that target specific glyco- these particular toxins. Oral delivery has successfully been demon-
proteins on the virus shell. In animal studies, research has demonstrated and utilized for live-attenuated vaccines against the human
strated an increase in immune activation and maturation in rotavirus, and oral delivery of mRNA vaccines could utilize similar
response to the vaccine, as well as some preliminary clinical trials mechanisms [85]. There are several other advantages to oral
that indicate similar results, with a reasonably low amount of side delivery, including less stringent purification requirements, higher
effects [76,77]. patient compliance due to the elimination of needles, and
11
Fig. 5. Advantages of oral vaccine delivery. Advantages include the lack of a need for painful intramuscular injections and decreased generation of biohazardous plastic
material by utilizing the body’s existing gastroenteric mechanisms. Figure created using BioRender.com.
significantly decreased amount of biohazardous and plastic waste routes) [91]. Though these approaches may provide for greater
(Fig. 5) [86]. Additionally, mRNA vaccines could take advantage mucosal access and increased therapeutic adsorption than lower
of developments in nanotechnology, such as the 2014 study by GI entry, fluid disturbance from saliva and enzymatic activity create
Duran-Lobato et al., which demonstrated the in vitro targeting abil- new problems. Despite these challenges, the potential for expansion
ities of surface-modulated nanocarriers [87]. of mRNA vaccines into the parenteral delivery space remains huge
Oral delivery of mRNA vaccines, though promising, does come and the renewed interest in mRNA research promises interesting
with several associated challenges. There are significant barriers developments in the near future. Technological devices, such as
to oral delivery in general, which are compounded by mRNA’s microneedle arrays [93] and high pressure liquid jets,[94] have suc-
low stability in vivo. The digestive system is a harsh physical and cessfully demonstrated mucosal penetration capabilities with vac-
chemical environment with drastic changes in pH, and even at cine formulations, and research in nanofiber-based mucosal
the mucosal layer, mucosa-associated lymphoid tissue can cause patches for sustained vaccine release have also been explored [95].
problems of immune avoidance and epithelial layer transport An evident disparity in the COVID-19 pandemic is the difference
through tight junctions in vaccination rates between developed and developing countries;
and mucous layers may result in a lower bioavailability. mRNA according to the World Bank, of the 4 billion plus vaccines admin-
and/or associated delivery proteins would rapidly become targets istered worldwide, low-income countries have received approxi-
for proteases or exonucleases as well [88]. Previous studies in mately 1.1% of them, with the balance being concentrated in
increasing stability and bioavailability have explored utilizing pro- wealthy countries [96]. These differences can largely be attributed
tective carriers derived from chitosan/alginate [89] and freeze- to the ability of wealthy countries to pay for more doses, but also to
dried lettuce cells[90] due to their structural rigidity. Nanoparticles the stringent transportation and cold chain requirements of the
(notably polylactic acid and poly(lactic-co-glycolic) acid) in partic- current formulations, which can make delivery of doses to war-
ular allow for targeting of immune cells, improved diffusion rate mer/tropical regions challenging. As discussed earlier, ther-
across tissue/mucosal layers, and sufficient protection against bio- mostable variations of these formulations are currently under
chemical barriers [95]. Structures such as liposomes, synthetic and development and local manufacturers in the Eastern Hemisphere
natural polymeric nanocarriers, and dendrimers have all been have ramped up production of their domestic COVID-19 vaccines.
explored for oral vaccine delivery applications. Bioavailability Looking beyond COVID-19, many of the other potential applica-
and immune activation have been shown to depend on a variety tions of mRNA vaccines (HIV, Dengue fever, etc.) are most applica-
of nanoparticle parameters, including nanoparticle size/shape, sur- ble to low and middle income tropical countries which are
face charge, and surface conjugation of targeting moieties [92]. disproportionately ravaged by these diseases. Research is also
Other strategies include utilizing specific entry points in the gas- being conducted on developing mRNA vaccinations against para-
trointestinal system, including the thinner and more penetrable sites, such as the malaria parasite, which may transform the way
epithelium present in the oral cavity (sublingual and/or buccal that malaria prevention is approached in several African countries
12
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Advanced Drug Delivery Reviews 179 (2021) 114000

Contents lists available at ScienceDirect

Advanced Drug Delivery Reviews

Messenger RNA-based vaccines: Past, present, and future directions

4. Future directions and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

neously, widespread vaccination can protect the most vulnerable

DNA: Zika virus,[12] Phase 1 clinical trial for COVID

Future Directions in Disease Treatment/Delivery

COVID[8], orotransmucal delivery using artificial

vaccineToxoid: Exploration of PLGA-chitosan as

Hydrogel modulation for influenza subunit co-

herpes[6] and COVID[7] (intranasal)

delivery,[10] oral delivery[11]

Edward Jenner, smallpox vaccine

Anthrax (1970 s) and hepatitis B

virus) - work in progressToxoid:

(1796) - virtually eradicated in

DNA: 1983 (smallpox vaccine

shortcomings by generating a pathogen-specific immune response

the system can direct cellular and humoral elements to rapidly

DNA: Insertion of DNA into host cell to create

antigenic product to host - similar to subunit

is introduced into the host and presented to

nity through lesion transfusion - modern vaccines are highly pre-

cise and carefully engineered formulations that utilize a variety

but focuses on product, not pathogen

of antigen properties to stimulate adaptive immunity. A summary

3.1. Live-attenuated vaccines

Live-attenuated vaccines are one of the most common and

the pathogen and will be present to neutralize future infectious

strains. A notable example of a live attenuated vaccine is the yearly

influenza (flu) vaccine, which is commonly developed by culturing

infectious influenza strains in cell cultures or fertilized chicken

Manufacturing Vaccine Type Vaccine Mechanism of Action Current Regulatory/Approval Status

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