Vaccine Development Morgan Brisse

Emerging Concepts
and Technologies in
Vaccine Development
Morgan Brisse1,2, Sophia M. Vrba2, Natalie Kirk 2,3, Yuying Liang2 and Hinh Ly2*
1 Biochemistry, Molecular Biology, and Biophysics Graduate Program, University of Minnesota Twin Cities, St. Paul, MN,
United States, 2 Department of Veterinary & Biomedical Sciences, University of Minnesota Twin Cities, St. Paul, MN, United
States, 3 Comparative Molecular Biosciences Graduate Program, University of Minnesota Twin Cities, St. Paul, MN, United States
Despite the success of vaccination to greatly mitigate or eliminate threat of diseases
caused by pathogens, there are still known diseases and emerging pathogens for which
the development of successful vaccines against them is inherently difficult. In addition,
vaccine development for people with compromised immunity and other pre-existing
medical conditions has remained a major challenge. Besides the traditional inactivated or
live attenuated, virus-vectored and subunit vaccines, emerging non-viral vaccine
technologies, such as viral-like particle and nanoparticle vaccines, DNA/RNA vaccines,
and rational vaccine design, offer innovative approaches to address existing challenges of
vaccine development. They have also significantly advanced our understanding of vaccine
immunology and can guide future vaccine development for many diseases, including
rapidly emerging infectious diseases, such as COVID-19, and diseases that have not
traditionally been addressed by vaccination, such as cancers and substance abuse. This
review provides an integrative discussion of new non-viral vaccine development
technologies and their use to address the most fundamental and ongoing challenges of
vaccine development.
Keywords: non-viral DNA-RNA vaccines, nanoparticle vaccines, virus-like particle vaccines, cancer vaccines,
substance abuse, noncommunicable disease, infectious disease, COVID19
Beginning with the discovery of cowpox inoculation that can protect humans against smallpox
infection by Edward Jenner in the late 18th century, vaccination has become an important means to
prevent disease. Despite the success of vaccination in the eradication or control of some major
pathogens, several challenges remain in vaccine development and administration. Several
widespread infectious diseases such as HIV, tuberculosis, and influenza continue to pose great
challenges for fully protective vaccination. Emerging and reemerging pathogens present a pressing
need for expediting vaccine development and approval as a rapid response to epidemics, such as the
current COVID-19 global pandemic caused by the SARS-CoV-2 virus. The advantages and
disadvantages of the various vaccine platforms can make the choice for preferred platform(s) to
use for vaccine development during a pandemic complicated. The traditional methods to produce a
vaccine, such as live attenuated and inactivated vaccines or protein subunit vaccines have their
Frontiers in Immunology | 1 September 2020 | Volume 11 | Article 583077
Edited by:
Katie Ewer,
University of Oxford, United Kingdom
Reviewed by:
Arun Kumar,
Coalition for Epidemic Preparedness
Innovations (CEPI), Norway
Hannah Sharpe,
University of Oxford, United Kingdom
Hinh Ly
[email protected]
Specialty section:
This article was submitted to
Vaccines and Molecular
a section of the journal
Frontiers in Immunology
Received: 14 July 2020
Accepted: 14 September 2020
Published: 30 September 2020
Brisse M, Vrba SM, Kirk N, Liang Y and
Ly H (2020) Emerging Concepts and
Technologies in Vaccine Development.
Front. Immunol. 11:583077.
doi: 10.3389/fimmu.2020.583077
published: 30 September 2020
doi: 10.3389/fimmu.2020.583077
advantages and disadvantages, which have been extensively
reviewed elsewhere (1–3). Briefly, live attenuated vaccines
present the risk of reversion to a highly pathogenic form while
inactivated vaccines may not be sufficiently immunogenic or in
some cases can lead to an enhanced disease pathology (3).
Additionally, most pandemic vaccines have to be clinically
tested during an active outbreak in order to obtain sufficient
safety and efficacy data, thereby limiting the number of vaccine
candidates that can be deployed to save life during an
emergency situation.
Less conventional approaches to vaccinology include the nonviral
vaccine technologies that are the topic of this review, as well as
viral vector platforms. Viral vector vaccines rely on antigen
delivered on an unrelated, non-pathogenic viral backbone. This
technology was developed almost forty years ago using a vaccinia
virus vector expressing the hepatitis B surface antigen (HBsAg),
which provided protective immunity to chimpanzees exposed to
hepatitis B (4, 5). Since then, viral vectors have been used
successfully in many veterinary species (6–12), although only a
single viral vector has been licensed for human vaccination (rVSVZEBOV
for Ebola virus) (13). A number of viruses have been
developed as vectors for vaccine development, including poxviruses,
adenoviruses, herpesviruses, arenaviruses, retroviruses,
paramyxoviruses, and flaviviruses, among others (14–16). The
main advantage of viral vectors over traditional vaccines is their
ability to evoke a robust adaptive immune response in the absence
of an adjuvant (17). However, the tradeoff for enhanced
immunogenicity is the concern for potential reversion of the
attenuated viral vector to virulence, especially when using a
replication-competent vector (18). Replication-defective and
single-cycle viral vectors are attractive alternatives that have an
increased safety profile and, in some cases, are still able to elicit a
strong immune response (19, 20). More details about the known
viral vectors and their recent advances in vaccine development will
be discussed in our forthcoming review article (Vrba, S.M., et al.,
in preparation).
Other fundamental challenges toward successful vaccination
include the ever-changing and highly divergent nature of some
viruses that allow for the potential to escape vaccine coverage, preexisting
immunity of the vaccinated populations, and pre-existing
medical conditions that can prevent vaccines from being fully
effective and safe. Vaccination could also provide an enticing
alternative therapy against diseases such as cancers and substance
abuse. However, the efficacy of these vaccines is limited by the
disease complexity and the lack of a more complete understanding
of protective immunity in these medical conditions. The relative
contribution and balance of the different arms of host immunity,
i.e., antibodies and cell-mediated responses, toward protection
without adverse effects remains a challenging issue that needs to be
addressed for individual disease (21). Furthermore, the immune
response to vaccination can be influenced by numerous factors
such as gender, age, co-existing medical conditions, genetic
variations, and lifestyle (22). While vaccines have traditionally
been delivered as inactivated or attenuated preparations, recent
developments of non-viral vaccine systems offer potential
additional solutions to meet the new challenges of vaccine
development, especially during epidemic or pandemic situations.
This article focuses on new non-viral vaccine development
technologies and their implications for combating on-going and
emerging communicable and non-communicable diseases.
Virus-Like Particle and Nanoparticle
Subunit Vaccines
Subunit vaccines deliver antigens as purified proteins, which confer
the advantage of enhanced safety and scalability compared to
whole-pathogen vaccines due to the lack of the requirement for
the expression of all viral components and the ability to express and
purify any particular antigens of interest in large quantity. A
disadvantage of subunit vaccines is that they are generally less
immunogenic in nature and therefore require adjuvants and
repeated vaccination doses (2). Several approaches have been
used to increase the immunogenicity and stability of subunit
vaccines, such as virus-like particle (VLP) vaccines and
nanoparticle (NP) vaccines.
VLP vaccines use platforms capable of producing particles
that mimic the structure of authentic viruses. VLP vaccines can
be produced by expressing antigenic proteins in a eukaryotic or
prokaryotic system, resulting in the formation of particles with
an inherent ability of the antigenic proteins to self-assemble
(23) (Figure 1). Alternatively, VLP vaccines can also be made
by producing blank VLP templates and then chemically linking
antigenic peptides onto the pre-formed particles (23). Because
these VLPs do not contain a viral genome, they are unable to
replicate in cells and therefore have an improved safety profile
compared to live viral vaccines (24). Yet, VLP vaccines can
often fully activate immune systems of the vaccinated
individual. VLPs are taken up by dendritic cells, where they
are processed and presented onMHC class I and II molecules to
activate the adaptive immune response. Subsequent stimulation
of CD8+ T cells and CD4+ T helper cells leads to activation of
cell mediated responses and B cells (and antibody production),
respectively (23, 25–29). As a result, VLP vaccines are
considered highly immunogenic and can stimulate robust
cellular and humoral immune responses due to their highly
repetitive display of antigenic epitopes (30, 31). A number of
VLP vaccine candidates are now clinically applicable with some
notable examples including the hepatitis B vaccine (HBV)
Engerix (32), the human papillomavirus vaccine (HPV)
Cervarix (33) from GlaxoSmithKline (GSK), the HBV vaccine
Recombivax® (34), and the human papillomavirus (HPV)
vaccine Gardasil® (35) from Merck & Co, Inc. VLP vaccines
that are currently in clinical trials include vaccines for malaria
(36, 37), influenza (38), rotavirus (39, 40), tuberculosis (41),
Zika virus (42), and HIV (43, 44). Efforts to further increase the
immunogenicity of VLP vaccines include optimizing antigen
design and production platforms (of primarily bacterial
origin) (45).
Brisse et al. Non-Viral Vaccine Technologies
Frontiers in Immunology | 2 September 2020 | Volume 11 | Article 583077
NP vaccines are produced by chemically crosslinking protein
antigens and carrier molecules to increase immunogenicity and
decrease degradation of the antigens (45). These carriers can be
organic (primarily lipid-based) or in-organic (primarily
polymeric or metal based) (Figure 2) (46–49). More recently,
self-assembling protein NPs, which consist of oligomers of
monomeric protein, have been found in some cases to also
provide the benefit normally afforded by a carrier (47, 50). NPs
have similarly high rates of stability as VLPs, but they do not
stimulate the innate immune response to the same extent as
VLPs. However, NPs are simpler in design than VLPs by lacking
the multiple protein components of VLPs, which further
decrease their cost of production and increase their
reproducibility and safety. The challenges associated with
decreased immunogenicity of NP vaccines as compared to VLP
vaccines can be partly addressed by adjusting the carrier to the
desired antigen, based on factors such as size, surface charge,
shape and hydrophobicity (46, 47, 50). Additionally, carriers can
be used to directly target NPs to immune cells and to increase
cross-presentation by antigen-presenting cells (APCs) (46, 47).
DNA and RNA Vaccines
Another promising area of vaccine development includes
vaccines that are based on nucleic acids: DNA or RNA
vaccines. These vaccines have gained popularity due to their
cost-effectiveness, ease of design and production, attractive
biosafety profile, and, in the case of DNA, stability. Nucleic
acid vaccines have gained particular attention for their potential
to rapidly produce vaccines against emerging infectious diseases
such as those currently being tested against SARS-CoV-2, the
causative agent of COVID-19, which will be discussed in some
detail below.
The immunogenic and protective efficacy of DNA vaccines
have been demonstrated repeatedly in vitro and in small animal
models, and a limited number of DNA vaccines have been
approved for veterinary use (51). However, DNA vaccines tend
FIGURE 1 | Schematic of VLP vaccine production. The methodology to produce VLP vaccines is summarized in this cartoon. In brief, VLP vaccines are produced
by transfecting eukaryotic cells or transforming bacterial cells with a DNA plasmid encoding an antigenic peptide attached to a viral capsid and/or other protein that
is sufficient to form VLPs. The antigenic peptide is present on the outside of the VLP which becomes available for interaction with the immune system. Antigens
conjugated with a chemical crosslinker can also be attached to VLPs containing external proteins conjugated to a complementary chemical crosslinker, which will
result in antigens being linked to the VLP and being presented on the outside edges. Figure created using BioRender software.
Brisse et al. Non-Viral Vaccine Technologies
Frontiers in Immunology | 3 September 2020 | Volume 11 | Article 583077
to induce poor immune responses in humans and other large
animal models (52). One possible explanation may be that
intramuscular injection, which has been the most studied route
of DNA vaccine administration in humans, tends to elicit mostly
cell-mediated immune responses (53), which is likely due to
significantly lower APC populations residing in muscles and
antigen presentation dominated by MHC I (51). Alternatively,
DNA vaccines can be coated with gold NPs and administered
intradermally by a gene gun (Figure 3). While preliminary data
suggest that this method may increase humoral responses to
DNA-based vaccines (51), it is limited by its low dose per
administration (54). In vivo electroporation (permeabilization
of the skin by an electric current to allow plasmid DNA uptake)
has thus far been shown to have the highest immunogenicity in
multiple small animal models (51, 54) and has been tested in two
phase I clinical trials for HIV vaccination with some promising
results. In the first clinical trial, the immune system was primed
with a DNA vaccine encoding the IL12 gene followed by a boost
dose with the recombinant VSV-based HIV vaccine (55). The
second trial evaluated the cellular immunity induced by HIV
DNA vaccines through intramuscular injection administered by
electroporation (56). Other efforts are being undertaken to
increase the immunogenicity of DNA vaccines such as codon
optimization, optimal promoter usage and epigenetic design,
generating nanocarrier plasmids to increase stability and
plasmids fused to proteins that specifically target APCs,
adjuvant use (which will be discussed in some detail below)
and short hairpin RNA (shRNA) targeting of host cells that
decrease immunogenicity to DNA vaccines. These approaches
have been extensively reviewed elsewhere (51, 54).
A recent development involves the successful use ofmRNA as a
protective vaccine. While mRNA was originally found to be viable
for in vivo gene transfer in the early 1990’s, the development of
mRNA vaccines was initiated much later due to the inherent
instability of mRNA compared to DNA (57). The efficacy of
mRNA vaccines can be increased by several factors, such as
ensuring mRNA purity, adding 5’ Kozak and cap sequences, 3’
poly-A sequences and modified nucleosides to increase mRNA
stability and decrease detection by the receptors of innate immune
cells, codon optimization, introduction by intramuscular, and
intradermal injection to reduce RNA degradation, and by
generating thermostable mRNA (57–59) (Figures 4, 5). Methods
to encapsulate RNA have also been explored to increase the
stability and immunogenicity of RNA vaccines, as has been used
with exosome encapsulated RNA (60) and RNA-transfected
dendritic cells (61, 62). When fully optimized, RNA vaccines
may have an immunogenic advantage over DNA vaccines due
to the presence of multiple cellular pathways that activate innate
immunity in response to foreign RNA such as the toll-like
receptors (TLRs) and RIG-I-like receptors (RLRs) (63, 64).
In addition to the aforementioned non-replicating RNA,
RNA vaccines can include self-replicating or self-amplifying
RNA molecules that are normally based on positive-strand RNA
viruses of which the structural genes are replaced by antigens (57,
58) (Figure 4). One study comparing the efficacy of conventional
mRNA versus self-amplifying RNA found that both were effective
FIGURE 2 | Schematic of NP vaccine production. The methodology to produce NP vaccines is summarized in this cartoon. In brief, NP vaccines are produced by
assembling a complex of antigens, a linker molecule, and a carrier molecule by chemical conjugation. Figure created using BioRender software.
Brisse et al. Non-Viral Vaccine Technologies
Frontiers in Immunology | 4 September 2020 | Volume 11 | Article 583077
in protecting mice against influenza infection, but that selfamplifying
RNA elicited protection at a much lower RNA dose
and induced a delayed yet longer-lasting antigen expression (65).
Self-replicating RNA transfected into dendritic cells (66) has
been shown to induce an immune response in vivo (67). RNA
vaccines have been used in a number of studies in animal models
(68) and have recently completed phase 1 clinical trials for rabies
(57, 69) and influenza (68, 70). Both trials had similar safety
profiles with most patients experiencing mild to moderate
reactions to administration and a few patients experiencing
more severe reactions. Both vaccines also demonstrated
immunogenicity through neutralizing antibody levels, though
antibody levels with the rabies vaccine were more highly
dependent on dose and route, with needle-free intradermal
dose able to sustain neutralizing antibody levels in half of the
number of vaccinated individuals one year after injection but not
with those receiving intramuscular or intradermal injections (57,
69). Additionally, phase 1 clinical trials are currently underway
to test the self-replicating RNA vaccines for HIV and Zika
virus (68).
Several challenges face the development of DNA/RNA
vaccines. First, while DNA and RNA vaccines may avoid the
safety concerns due to microorganism-based vaccine
formulations, they have safety concerns of their own. While an
early study suggested that DNA vaccination might result in some
instances of random chromosomal integration, it was
determined that this occurred with a significantly lower
frequency than random genetic mutations (71). However, a
subsequent study did not observe chromosomal integration to
occur following DNA vaccination (51). The possibility of
introducing unwanted bacterial DNA elements (such as
antibiotic resistance genes to the gut microbiome) has been
raised as a safety concern for DNA vaccination, but as of yet it
has not been proven (51). As such, regulatory guidelines have
been put in place for new DNA vaccine clinical trials in the
United States and Europe (72). Vaccine formulation based on
mRNA has the advantage of being produced in cell-free systems
that can eliminate the concern of bacterial contamination and
also lack the potential for chromosomal integration and longlasting
expression (57). While the World Health Organization
(WHO) has recently classified mRNA vaccines as its own
therapeutic class (73), similar regulations have not yet been
developed due to the more limited testing of mRNA vaccines
in humans.
It has been found that DNA vaccination primarily induces
antigen expression at the site of administration with significantly
lower levels being observed elsewhere (51), which may partly
explain its poor immunogenicity. While less is known about the
levels of on- and/or off-target expressions seen with RNA
vaccination, they are presumed to be generally lower than DNA
vaccine due to the decreased stability of RNA. However, off-target
antigen expressionmay be relativelyminor in shaping the immune
response, as the route and mode of DNA/RNA vaccine delivery
can markedly alter vaccine immunity, but the mechanism is yet to
be fully understood. Generally, intramuscular or intradermal
injection is used in animal models and human volunteers to
elicit protection against infectious disease to maximize delivery
to APCs, while intraperitoneal or intravenous injection has been
used in selected animal models to induce systemic expression in
therapeutic models, such as cancer vaccination (57). These
findings implicating localized dosage routes as most effective for
eliciting immunity from nucleic acid vaccines may help explain
why gene gun and electroporation have been found to be the most
effective routes for DNA vaccine administration. The most
effective dosage routes may also be similar for RNA vaccines, as
intradermal and intramuscular injection have repeatedly been
found to be the most effective delivery routes for RNA vaccines,
and needle-free delivery systems may also be more effective than
injections (57). Interestingly, immunity can still result from
injection of naked RNA in certain models, but it has been found
FIGURE 3 | Methods of improving DNA vaccines. The various methods that have been developed to improve the stability and immunogenicity of DNA vaccines are
summarized in this chart. A number of design and delivery mechanisms have contributed to improving the performance of nucleic acid vaccines, such as methods of
clinical delivery, genetic engineering, and linking nucleic acid vaccines to cells or biomolecules. Figure created using BioRender software.
Brisse et al. Non-Viral Vaccine Technologies
Frontiers in Immunology | 5 September 2020 | Volume 11 | Article 583077
in particular that IV administration of naked RNA results in rapid
RNA degradation (57).
Rationally Designed Vaccines
A key aspect of non-viral vaccine development involves the
selection of antigens that can effectively elicit a protective
immune response. Whereas traditional vaccines are generally
developed through attenuation or inactivation of pathogens and
through the incorporation of few selected antigens as vaccine
components, new technologies have recently been applied
toward antigen discovery and design. For example, “reverse
vaccinology” refers to the ability to screen the complete
antigen sets based on whole-genome sequencing of pathogens
for the ability to induce protective antibody responses.
Combinations of reverse vaccinology and traditional vaccine
approaches allow for an efficient development of immunogenic
vaccine candidates (74, 75).
Bioinformatic tools have contributed greatly to vaccine design
and evaluation in recent years. Computational approaches are
continually improving in their ability to predict T and B-cell
epitopes from the complete antigen pools and to rationally design
antigens with potential long-lasting protective immunity. Such
algorithms calculate antigen-antibody interaction energies and
structures (76) that increasingly bridge modeling based on
FIGURE 4 | Methods of improving RNA vaccines. The various methods that have been developed to improve the stability and immunogenicity of RNA vaccines are
summarized in this chart. A number of design and delivery mechanisms have contributed to improving the performance of nucleic acid vaccines, such as methods of
clinical delivery, genetic engineering, and linking nucleic acid vaccines to cells or biomolecules. Figure created using BioRender software.
Brisse et al. Non-Viral Vaccine Technologies
Frontiers in Immunology | 6 September 2020 | Volume 11 | Article 583077
existing templates and free-modeling based on heterologous
structures and database consensus design (77). Deep sequencing
combined with computational analysis allows for thoroughly
characterizing the B cell repertoire in survivors of disease to
identify the protective immunity (78).
A computationally designed antigen found to be protective in
animal models was first reported for the respiratory syncytial
virus (RSV) F antigen in 2013 (79). Many challenging vaccine
targets have since been developed rationally for HIV (80–82),
hepatitis C (83, 84), herpes (85), Zika (86, 87), RSV (82), HPV
(88), as well as for bacteria (82, 89), fungi (90), and cancers (91).
Rational vaccine design has also been utilized to improve VLP
and NP vaccines by selecting a repetitive and predictive protein
backbone structure for enhanced antigen presentation (92).
Finally, rational antigen design is being explored for activation
of dendritic cells (93) such as targeting C-type lectin receptors to
activate antigen presentation in the context of the pathogens
(e.g., Ebola and HIV) (94). The first rationally-designed vaccine
to undergo human clinical trials is the anti-malarial vaccine
Mosquirix, which was approved for use by the European
Medicines Agency in 2015 (95). Human clinical trials have not
yet begun for other rationally designed vaccines, however. A key
point to note is that rationally designed vaccines require a
comprehensive knowledge of the biology and immune
response to a pathogen (95), and rational design is therefore
more difficult to implement for rapidly emerging diseases.
A major challenge to rational vaccine antigen design is the lack
of knowledge of T cell epitopes compared to B cell epitopes. Most
successful antigens are expected to elicit both B and T cell responses.
Quantitative databases have been developed more for predicting B
cell receptor (BCR) epitopes than T cell receptor (TCR) epitopes.
BCR epitopes can be predicted in part by the structural and
chemical properties of the epitopes due to BCRs recognizing
primary and tertiary antigen structures, while TCR epitopes have
to be predicted based on known TCR epitopes because they only
recognize the primary structures. Increasing capacity for
identification of TCR epitopes by machine learning from known
epitopes will likely help to mitigate this inequity (74, 75, 96).
Vaccines for Immunosuppressed
A fundamental challenge to vaccination is the limited
immunogenic response to vaccines seen in immunosuppressed
individuals, namely, young children, the elderly, and those who
are immunocompromised for medical reasons. The underlying
causes of immunosuppression in each of these populations vary,
and their underlying mechanisms should be taken into account
when creating the best vaccine approach.
Young children, in particular infants and neonates, are
considered to be immunosuppressed due to age-specific immune
FIGURE 5 | Methods of improving DNA and RNA vaccines. The various methods that have been developed to improve the stability and immunogenicity of both
DNA and RNA vaccines are summarized in this chart. A number of design and delivery mechanisms have contributed to improving the performance of nucleic acid
vaccines, such as methods of clinical delivery, genetic engineering, and linking nucleic acid vaccines to cells or biomolecules. Figure created using
BioRender software.
Brisse et al. Non-Viral Vaccine Technologies
Frontiers in Immunology | 7 September 2020 | Volume 11 | Article 583077
system developments that result in children being particularly
susceptible to infection (97). The specific mechanisms for the
immunosuppression in this population vary, but one prominent
example is the decreased expression of Th17 supporting cytokines
by TLR receptors and increased expression of anti-inflammatory
cytokines in neonates and particularly in premature newborns
(98). On the other hand, the immunosuppressive phenomenon
observed in the elderly has been referred to as immunosenescence
(99) and it is caused by a number of complex changes resulting in
impaired innate and adaptive immune responses (100–104),
degradation of lymphoid architecture (105), and increasing
proinflammatory cytokines and chemokines (106, 107). To
highlight a few important changes, dendritic cells have reduced
uptake of antigens (108, 109), macrophages are unable to
phagocytose apoptotic cells (110), the number of naïve T cells
decreases (111, 112), and B cell repertoire decreases (113). These
age-related changes in the ability of the immune systemto respond
to infection differ from the challenges to vaccination presented by
conditions or medications that result in immunosuppression. One
such example of a medication that results in immunosuppression
is steroids, which have been reviewed extensively elsewhere (114).
Steroids exert many effects on immune cells, such as the
reprogramming of dendritic cells to tolerogenic dendritic cells
(115–117). These cells induce the formation of regulatory T
cells (118).
The development of vaccines that can potently activate the
innate immune response without using live attenuated vaccines is
a central focus of vaccine development for immunocompromised
individuals. This is particularly relevant for subunit vaccines, as
they do not contain potential viral genomic elements that can act
as pattern-associated molecular patterns (PAMPs) to activate
innate immune responses. A major approach toward increasing
the immunogenic response to a subunit vaccine is through the use
of adjuvants. Originally discovered by including food products in
equine vaccines and inducing sites of localized sterile
inflammation and abscesses, the adjuvant repertoire has since
been greatly expanded (119). The so-called “first generation
adjuvants”, which remain the most common adjuvants in
clinical use, include aluminum salts (alum) and mineral oil-inwater
emulsions, which function by promoting the migration of
APCs to the sites of intramuscular injection (120). However, the
use of these adjuvants is limited greatly by the recruitment of only
a comparatively small population of immune cells that are made
up of APCs (120) and a markedly Th2 response with little to no
cellular immune response (120). This has become the aim of
current research to design new adjuvants that can increase the
breadth of the innate immune responses to the vaccine.
Much effort has been focused on enhancing the usable
adjuvant repertoire to further customize the immune response
and to avoid the Th2-dominated immune response seen with
some adjuvants (e.g., alum) and instead support a Th1 response
in certain circumstances. Specifically, a response skewed toward
Th2 response is desired for antibody production and
antiparasitic immune response, while a Th1 response is desired
for intracellular or viral pathogens. Skewing toward Th1 or Th2
is thought to occur after APCs stimulate certain cytokine gene
expression profiles (121). For example, LPS-derivative–based
AS04 is being used in hepatitis B and HPV vaccines (122) and
has been found to increase cell-mediated immune responses in
patients with end-stage renal disease (123). Other adjuvants that
have been developed to induce a Th1 immune response include
IC31® (124, 125), GLA-SE (125, 126), and CAF01 (125, 127). In
addition to the Th1 skewed immune response that these
adjuvants displayed, GLA-SE induced antibodies and CAF01
showed a Th1/Th17 response (125). Increasing the breadth of the
immune response to vaccines thus can enhance the safety and
effectiveness of vaccines for both immunosuppressed
populations as well as the general population.
There has been an increasing effort toward developing new
vaccines that may induce a safe and immunogenic response in
immunocompromised individuals. As an example, DNA
vaccines could be used to encode for antibodies that could be
safely and temporarily expressed in immunocompromised
patients, such as throughout the course of an influenza season.
Recent studies that tested the efficacy of influenza neutralizing
antibodies found that protection against lethal disease could be
conferred by plasmids expressing antibodies given intramuscularly
by electroporation (128). However, several major considerations
need to be fully addressed before these techniques can be
developed for human use. Specifically, the duration and the
stability of plasmid vaccination have not yet been fully
characterized in humans. Additionally, it has been shown that
anti-dsDNA antibodies can be produced by primary B cells
isolated from mice treated with plasmid DNA (129), which
appear similar to anti-dsDNA antibodies that have been shown
to be expressed during systemic lupus erythematosus (130, 131).
The anti-dsDNA antibodies may prefer to bind to certain CpGrich
sequences on bacterial DNA of the plasmid (129), which
might serve as a means for DNA vaccine optimization. Finally, the
purity of plasmid DNA stocks needs to be thoroughly confirmed
in order to avoid possible stimulation of unwanted
immune reactions.
Other novel adjuvant approaches include the surfactant and
emulsifier-based AS03 that are currently being used in influenzapandemic
vaccines (132). LPS-derivative–based AS04 is being
used in hepatitis B and HPV vaccines (122) and has been found
to increase cell-mediated immune responses in patients with
end-stage renal disease (123). Lipid products that form micelles
in solution and act as solid particle carriers are another form of
adjuvants that can activate innate immunity, as seen with CAF01
(133, 134) and AS01B/E formulation from GlaxoSmithKline
(135), which are used in the only currently available vaccine
for malaria (136, 137). Several other adjuvants currently in use
primarily function as TLR agonists (138–140). TLR agonists have
shown promise in aged and young mice (141) as various TLR
agonists [e.g., CpG (TLR9), poly(I:C) (TLR3), and pam3CSK4
(TLR1 and TLR2)] can induce expression of co-stimulatory
molecules on APCs (141). Another adjuvant approach taken to
overcome the immune challenges presented by young children is
the use of ß-glucan. These sugars, found in the cell walls of some
pathogens, activate dendritic cells through the CLEC71-SYKCARD9
pathway, and it was shown to provide protection against
Brisse et al. Non-Viral Vaccine Technologies
Frontiers in Immunology | 8 September 2020 | Volume 11 | Article 583077
tuberculosis infection (142). Recently, defective-interfering (DI)
viral particle vaccines have also been explored for use as
adjuvants to increase the innate immune response (143–152).
These are VLPs with aberrant and defective genomes, which have
been found in some cases to increase the innate immune
response when compared to the replicating virion. Taken
together, several innovative strategies are currently being
developed to increase the immunogenicity and safety of
vaccines for immunocompromised populations.
Vaccines With Non-Traditional Antigens
Because of their increased safety and versatility, such as the ability
to deliver a diverse range of molecules as antigens, VLP and NP
vaccines have the potential for use to provide immunity against
non-protein antigens. A prime example is the development of NP
vaccines to treat substance abuse disorders by attaching a drug
molecule to a hapten carrier (Figure 2). Vaccines against drugs of
abuse aim to elicit a humoral (antibody) response that can
neutralize the drug target before it crosses the blood-brain
barrier to induce psychotropic effects, thereby decreasing
positive associations with and hopefully dependency on the
addictive drug. Such vaccines have an advantage for long-term
therapeutic use over pharmaceuticals targeting neural receptors by
eliminating the medical complications and safety concerns of
directly modulating neural signaling network. They also differ
from other vaccines in that they are given to active users of drugs
of abuse to prevent escalation of use or relapse and do not depend
on herd immunity for effectiveness, so their efficacy is determined
by individual responses to the vaccines (153).
A hapten carrier, which is a potent B cell antigen, is used to
stimulate B cell responses and thereby also activates B cell
responses to the attached drug. Therefore, hapten and linker
design are of particular interest to ensure structural integrity and
to maximize B cell responses (153–170). The vaccine can also be
linked to a protein carrier designed to activate T cell responses
(and particularly CD4 T cell response to then activate B cells)
(153, 160, 171–181), though there has not been a clear
determination of whether an increased CD4 Th2:Th1 ratio
correlates with efficacy for vaccines against drugs of abuse
(153). Finally, an additional consideration in designing
vaccines against drugs of abuse is determining whether to
target the drug itself or its possibly more psychotropic
metabolites that can provide a greater level of protection. A
prime example of this is heroin vaccines seemingly being most
effective when they can structurally mimic the psychotropic
heroin metabolite 6-acetylmorphine (6AM) (166, 172, 182). It
should be noted that clinical trial results have only been reported
for vaccines against nicotine and cocaine addictions, with the
vaccines demonstrating efficacy only in a subset of patients that
were able to achieve high neutralizing antibody titers (153, 183–
186). The recent vaccine developments to ameliorate drug abuse
have been reviewed more extensively elsewhere (153, 160, 187).
Additionally, VLP and NP vaccines are being used in toxoid
vaccine formulations, which provide quick neutralization against
a cytotoxic molecule (primarily bacterial toxins) that cannot be
expressed in its full and activated form. The most well-known
example is the diphtheria, tetanus and pertussis (DTaP)
inactivated subunit vaccine which has been in use for decades
and can elicit effective immune responses against the toxin
produced by any of these bacteria if/when the vaccinated
individual happens to be exposed to them. The pertussis
component of DTaP has a demonstrated high level of safety
that it is one of only two known vaccines (besides influenza) that
is given to pregnant women in several countries (188). Current
clinical trials are focusing on testing potentially more effective
toxoid vaccines for pertussis (189) as well as Haemophilius
influenzae type b, polio virus, and hepatitis B virus (190). It
has also been found that using bacterial membrane or red blood
cell (RBC) membrane micelles as a carrier can significantly
increase the immunogenicity of the vaccines, and pre-clinical
testing is currently underway to use these carriers in vaccine
development against the multi-drug resistant bacteria
Staphylococcus aureus (MRSA) (191).
Therapeutic Vaccines for
Noncommunicable Diseases
As our capabilities for vaccine development and production have
expanded, a paradigm shift has recently taken place to use
vaccines for disease treatment in addition to disease
prevention. These therapeutic vaccine designs rely on the
identification of protein markers unique to a disease phenotype
which may evade the development of an immune response due
to the markers not being recognized by the APCs. For example,
cancer vaccines to elicit immune responses against cancerspecific
antigens are one of the most widely studied therapeutic
vaccines to date due to the inherent challenges of developing
effective cancer therapeutics and a need for targeted treatment.
The immunosuppressive environment present in cancers has
made developing cancer vaccines a significant challenge,
especially for vaccines that rely on viral vectors. Therefore, the
improved safety profile of non-viral vaccines offers an attractive
potential for cancer vaccine development. Non-viral vaccines
also confer an additional advantage for developing cancer
vaccines in that cancer vaccines may be most effective when an
antigen specific to the mutational profile of the individual cancer
is used (192–194), particularly in combination to overcome
immune tolerance (195). The time needed to make a vaccine
against an individual antigen or against a combination of
individual antigens is greatly reduced with non-viral vaccines
due to the ease of encoding an antigen on a nucleic acid vaccine
or purifying protein for a subunit vaccine in comparison to
incorporating a personalized antigen into a viral vaccine,
growing viral stocks and verifying its expression (196).
Nucleic acid vaccines have been a key area in recent
developments for cancer treatment. While a number of DNA
cancer vaccine candidates have entered into early phase clinical
trials (197), RNA vaccines are thought to have particularly
encouraging potential due to their increased immunogenicity
compared to DNA vaccines (68, 198). Preliminary results
indicate that intranodal injection of naked tumor antigenencoding
mRNA can control tumor growth in mouse cancer
models (199–202). Additionally, naked mRNA was found to be
Brisse et al. Non-Viral Vaccine Technologies
Frontiers in Immunology | 9 September 2020 | Volume 11 | Article 583077
immunogenic via intradermal injection in a phase I/II clinical
trial for prostate cancer (203). However, a key challenge in
developing RNA cancer vaccines has been the need to further
ensure the stability of the RNA and to increase its targeting to
APCs (204, 205) in order to overcome the immunosuppressive
environment of cancers. Developing a delivery vehicle for RNA
cancer vaccines has therefore been a central focus of research and
development in this area.
Loading RNA into liposomes is one method that has shown
some success in controlling cancer growth in mouse models
(206–208) and has demonstrated some preliminary efficacy in
early stage clinical trials for use as a delivery system for anticancer
genes (209, 210) and siRNAs (211), with different
liposome constructs targeting RNA localization to the spleen.
RNA-loaded liposomes can also be targeted directly to T cells by
using RNA that encodes for anti-CD3 along with the cancer
antigens, bypassing the need for recognition by APCs. This
concept has notably been tested in conjunction with chimeric
antigen receptor (CAR) T cell therapy (198, 212, 213). Another
method bypasses targeting RNA to APCs by directly transfecting
dendritic cells (DCs) with RNA extracted from tumors or RNA
encoding tumor antigens (214–217) (Figure 4) and then
introducing the engineered DCs into patients with a
combination of cytokines and/or checkpoint blockades (218,
219). However, DC-directed RNA vaccines are currently
limited by the restrained immune environment present in
cancers, which can limit the activity of DCs and increases the
activity of regulatory T cells (216, 220, 221). It is thought that
these challenges could be mitigated by optimizing the use of
cytokines and other factors that would act as adjuvants in
combination with cancer RNA vaccines (195, 222–225) and by
optimizing DC isolation and culturing conditions (226). A few
DC-directed RNA vaccine candidates are currently in clinical
trials, including those in phase III (196, 221).
Subunit vaccines have also been developed for use as cancer
vaccines (227), which are being tested with many of the same
delivery systems as nucleic acid-based cancer vaccines to
maximize vaccine targeting to immune cells (228). NP-based
vaccines in particular have been developed and tested for use as
cancer treatments (228–235), the most notable of which are
several HPV vaccines for prevention of cervical cancer (236).
While less development has been done on VLP-based cancer
vaccines, one notable target that has been used is the widely
expressed cancer antigen human epidermal growth factor
receptor-2/neu (HER2), which has shown to be immunogenic
in mouse models (237–244) and in early clinical testing in
humans (245) and dogs (246), but as a whole these vaccines
have had to undergo additional design in order to overcome B
cell tolerance (227, 247) and to fully characterize their antitumor
Because subunit vaccines require antigen presentation in
order to elicit an immune response, a primary challenge in
VLP- and NP-based cancer vaccine developments has been
optimizing their uptake by APCs (228). Vaccine uptake by
APCs can be optimized by engineering VLP- and NP-based
cancer vaccines to resemble the structure of viral particles as
closely as possible, such as by using certain types of carriers
(liposomes, polymers and ferritin cages), sizes (20–45 nm) and a
spherical shape (Figure 4). Subsequently, these vaccines may be
most successful when combined with checkpoint blockade
treatment by encouraging clonal expansion of lymphocytes
(248). VLPs and NPs can also be used as immuno-enhancers,
e.g., to deliver cytokines and TLR agonists to target sites,
which has been found to boost localized immune responses
while avoiding immunopathogenic and possibly systemic
inflammation (46).
Vaccines for Rapidly Emerging Viral
Emerging and reemerging pathogens, such as West Nile virus,
pandemic influenza virus, Ebola virus, dengue virus, Zika virus,
and the on-going global pandemic SARS-CoV-2 pose great
challenges to the public health system. Rapid development and
deployment of vaccines are critical to quickly build up resistance
against these and other disease “X”, which is a term used by the
WHO to refer to future unknown disease pandemics (249). The
ideal vaccine platform in a pandemic situation must be costeffective
and can be rapidly developed and produced on a large
scale to meet global demands. Temperature sensitivity is also a
consideration, as cold chain storage can be particularly difficult
to maintain in developing countries. Development of heat stable
vaccines like the oral bovine rotavirus pentavalent vaccine (BRVPV,
Rotasil® by the Serum Institute of India), which was
prequalified by the WHO in 2018, can provide protection
against serious diseases in regions where transportation and
refrigeration are unreliable (250). In comparison, the only FDA
approved Ebola virus vaccine (rVSV-ZEBOV, ERVEBO® by
Merck and Co., Inc.) must be stored at −80°C or −60°C (251),
which presents a major obstacle for affected countries. Rapid
production of low cost, scalable, and temperature stable vaccines
is an ongoing challenge in the face of emerged and emerging
global disease pandemics.
Currently, rapid development of vaccines is greatly limited by
the resources and regulatory policies needed to bring a vaccine
from its conceptualization stage to the clinic, which has been
estimated to cost between $200 and $500 million dollars and to
take 5–18 years (252). Vaccines also tend to be manufactured in
countries with larger economic and technical prowess and more
robust disease surveillance systems than developing countries and
therefore can unfairly influence the equity of vaccine distribution
and usage. This was seen in the 2009–2010 influenza pandemic,
where 80% of the vaccines were manufactured and used in seven
industrialized regions (United States, Canada, Australia, western
Europe, Russia, China, and Japan), while the majority of
developing regions in the world did not receive any pandemic
influenza vaccines until January 2010, 9 months after the WHO
declared the influenza pandemic (253). In addition, as mentioned
previously, most pandemic vaccines have to be clinically tested
during an active outbreak in order to obtain sufficient safety and
efficacy data, thereby limiting the number of vaccine candidates
that can be deployed to save lives. This was seen during the Ebola
outbreak of 2013–2015, when two vaccines were fully developed in
Brisse et al. Non-Viral Vaccine Technologies
Frontiers in Immunology | 10 September 2020 | Volume 11 | Article 583077
advance of clinical trials but only one (the simian adenovirusbased
Ebola vaccine ChAd3-EBO-Z) was tested early enough in
the outbreak to obtain sufficient clinical data (254). Similar
challenges are also seen in selecting vaccine candidates for the
large sample sizes needed for phase III clinical trials for HIV
vaccine candidates. Statistical ranking systems to prioritize
candidates are being developed to aid in this selection process
(255). Zoonotic diseases present additional considerations, as it is
economical to vaccinate the multiple species that may act as
reservoirs of the pathogen(s) in order to control the spread of
the disease. The first vaccine to provide protection in multiple
species is the simian adenovirus-based vaccine candidate
ChAdOx1 RVF, which has been shown to provide effective
protection against Rift Valley Fever virus in sheep, goats, and
cattle and is currently undergoing testing in larger livestock field
trials and in humans (254, 256).
Other measures have been undertaken to expedite the process
of vaccine development and reduce the cost of vaccine
production. International institutions allow for collaborative
groups to rapidly co-operate on vaccine development and
shorten the vaccine manufacturing process. The Coalition for
Epidemic Preparedness and Innovations (CEPI) provides funds
for clinical trial and stockpiling of vaccines that would not have
market incentive in a traditional funding mechanism of vaccine
development and manufacturing (257). Such international
collaborations will help to bridge the differing vaccine
development policies and investitures across countries and use
these combined resources to develop vaccines to primarily
benefit those living in either underdeveloped or developing
nations (258). In a recent example of this, CEPI, Gavi, and the
WHO have come together to form COVAX, the vaccines pillar of
the Access to COVID-19 Tools (ACT) Accelerator, with the
mission to expedite the production of a COVID-19 vaccine to be
equitably distributed throughout the world (259).
Technical challenges in vaccine production process can be an
impediment. For example, the use of fertilized chicken eggs in
vaccine production can pose challenges such as the restricted
capacity of egg production, egg allergies, and the emergence of
viruses with egg-culture-adapted mutations that can reduce vaccine
efficacy (260). The use of animal cells for certain vaccines can also
present significant challenges of cost, slow production rates, and
potential high risk of contaminations. Other vaccine production
systems, such as VLP vaccines produced in yeasts, insect cells and
bacterial systems, as well as DNA/RNA vaccines, can benefit from
increased robustness of antigen production, decreased risk of
contaminations, and quicker time of response (252). This may
especially be the case for DNA vaccines, where the increasing
capacity of next-generation DNA sequencing, for example, has
lowered the time for development of a DNA vaccine from 20
months following the 2003 SARS outbreak to 3.25months following
the 2016 Zika outbreak (261).
COVID-19 Pandemic as a Case Study to
Rapidly Develop Non-Viral Vaccines
The ongoing COVID-19 pandemic has presented unique
opportunities as well as challenges for vaccine development.
Unlike the influenza vaccines, no coronavirus vaccines existed
prior to the COVID-19 pandemic. Such a rapid and widespread
need for a completely novel vaccine for COVID-19 has resulted in a
drive to significantly reduce the length of time required to produce a
new vaccine. It has also highlighted the necessity to use non-viral
vaccine platforms with overlapping stages of vaccine development,
including preclinical and clinical testing and manufacturing that
would otherwise be required to happen in a stepwise process for a
traditional vaccine development effort (262–264). However, a rapid
progression of clinical testing will need to be balanced by the need
for obtaining quality data on vaccine safety and efficacy (265),
especially considering previous reports of pathological antibodydependent
enhancement responses in some patients immunized
with the 2003 SARS vaccine candidates (266, 267). As with previous
pandemics, widespread global availability and resource
management will be another key consideration for vaccine
selection, especially considering the near ubiquitous presence of
COVID-19 around the globe and its disproportionate impact on
populations of low socio-economic status (268, 269). It is also likely
that the approval of multiple vaccine candidates will be most
optimal to controlling and ending the pandemic should more
than one vaccine prove to be effective in preventing COVID-19
disease. Multiple COVID-19 vaccines would allow for more clinical
and regulatory choices to accommodate differences in patient
responses (particularly in more vulnerable patient populations)
and manufacturing and distribution capabilities (270, 271).
Perhaps, with these considerations in mind, non-viral COVID-19
vaccine platforms (e.g., DNA and mRNA) have been selected
among the first candidates to enter clinical testing, partly for their
aforementioned reasons of safety profiles and relative ease of
manufacturing (Table 1). Some of the RNA-based COVID-19
vaccines (all of which are currently in various stages of clinical
trials) include but are not necessarily limited to:
1. The mRNA-1273 vaccine developed by the U.S. biotech
company Moderna (272).
2. The mRNA CVnCoV vaccine developed by the German
company CureVac (273).
3. A group of 4 RNA vaccines under the name BNT162
developed by the German company Biontech that consists
of two nucleoside-modified mRNAs, a uridine-containing
mRNA and a self-amplifying mRNA (274), which in an early
phase I/II trial, the nucleoside-modified mRNA BNT1621b
has been shown to elicit neutralizing antibodies (275) and is
better tolerated particularly in older adults than BNT1621a
(276, 277).
4. The self-amplifying mRNA LNP-nCoVsaRNA (COVAC1)
vaccine from the Imperial College London (278).
5. The mRNA vaccine LUNAR-COV19 (ARCT-021) from US
company Arcturus Therapeutics (279).
6. An unnamed mRNA vaccine candidate from Chinese
company Yunnan Walvax Biotechnology (280).
Some of the COVID-19 DNA vaccines (all of which are also
in various stages of clinical trials) include but are not necessarily
limited to:
Brisse et al. Non-Viral Vaccine Technologies
Frontiers in Immunology | 11 September 2020 | Volume 11 | Article 583077
1. The INO-4800 vaccine developed by the U.S. pharmaceutical
company Inovio (281) with preliminary phase I data
suggesting that 94% of participants might have developed an
immune response against it following vaccine administration
by electroporation (282) and that vaccination in rhesus
macaques elicited neutralizing antibodies against both the
D614 and G614 SARS-CoV-2 strains (283).
2. The GX-19 vaccine developed by the South Korean company
Genexine (284).
3. The AG0301-COVID19 vaccine developed by the Japanese
company AnGes, Inc. (285, 286).
4. The ZyCoV-D vaccine developed by the Indian company
Cadila Healthcare Ltd (287).
5. The live bacteria-mediated plasmid delivery system bacTRLSpike
developed by the Canadian company Symvivo (288).
Genetically engineered APCs are also being pursued as
potential COVID-19 vaccine candidates, with DCs transfected
with lentiviral vectors expressing COVID-19 antigens currently
being tested in China (289, 290) and in the United States (291).
Meanwhile, COVID-19 VLP- and NP-based vaccines have also
advanced into clinical trials, including the NP NVX-CoV2373
TABLE 1 | Non-viral vaccines currently in development for SARS-CoV-2*.
Vaccine Name Vaccine type Company and Country Preliminary results
mRNA-1273 mRNA Moderna, USA • Self-reported preliminary data indicating all patients developed neutralizing antibody
response. Patients developed moderate side effects with highest dose (250 ug)
were eliminated from future study.
• Entered phase III clinical trials in July 2020 with targeted enrollment of 30,000
CVnCoV mRNA CureVac, Germany • Entered phase II clinical trials in August 2020
BNT162 mRNA (4 candidates) Biontech, Germany • Early phase I/II trial data showed that patients who received nucleoside-modified
mRNA BNT1621b produced neutralizing antibodies.
• Further phase I/II clinical trial data showed that BNT1621a and BNT1621b produced
similar neutralizing antibody titers but that BNT1621b was associated with less
systemic responses particularly in older adults.
• BNT162b was selected to continue in phase II/III clinical trials.
mRNA (self-amplifying) Imperial College London, UK • Entered phase I/II clinical trials in June 2020
• Transitioned to phase II clinical trials in July 2020
mRNA Arcturus Therapeutics, USA • Entered phase I/II clinical trials in July 2020
Unnamed mRNA
mRNA Yunnan Walvax
Biotechnology co, China
INO-4800 DNA Inovio, USA • Preliminary phase I data suggest that 94% of participants developed an immune
response against the vaccine.
• Preprint suggests that a single dose seroconverted vaccinated rhesus macaques.
Neutralizing antibodies were produced against the D614 and G614 strains and
memory responses lasted at least 4 months after vaccination.
GX-19 DNA Genexine, South Korea • Entered phase I/II clinical trials in June 2020
AG0301-COVID19 DNA AnGes Inc, Japan • Entered phase I/II clinical trials in July 2020
ZyCoV-D DNA Cadila Healthcare Ltd, India • Entered phase I/II clinical trials in July 2020
bacTRL-Spike DNA (live bacteria
Symvivo, Canada
LV-SMENP-DC APC (lentiviral) Shenzhen Geno-Immune
Medical Institute, China
Covid-19/aAPC APC (lentiviral) Shenzhen Geno-Immune
Medical Institute, China
AV-COVID-19 APC (antigen-loaded) Aivita Biomedical, USA • Entered phase I/II clinical trials in May 2020
NVX-CoV2373 NP Novavax, USA • Entered phase I/II clinical trials in May 2020
• Self-reported data from phase I indicate that the vaccine was well tolerated and
induced neutralizing antibody responses in all patients after two doses.
SCB-2019 NP Clover Biopharmaceuticals,
• Entered phase I/II clinical trials in May 2020
COVAX-19 NP GeneCure Biotechnologies,
• Entered phase I clinical trials in June 2020
MVC-COV1901 NP Medigen Vaccine Biologics
corp, Taiwan
• Entered phase I clinical trials in July 2020
AdmirSC-2f NP Adimmune corp, Taiwan • Entered phase I clinical trials in August 2020
Unnamed spike
protein vaccine
NP University of Queensland,
• Entered phase I clinical trials in June 2020
Unnamed VLP
VLP Medicago, Canada • Entered phase I clinical trials in June 2020
*COVID19 vaccine data compiled with the aid of the BioRender COVID-19 Vaccine and Drug tracker:
This chart summarizes the name, type of vaccine, company and country of origin and preliminary data on existing non-viral vaccines for SARS-CoV-2 that are currently undergoing clinical testing.
Brisse et al. Non-Viral Vaccine Technologies
Frontiers in Immunology | 12 September 2020 | Volume 11 | Article 583077
vaccine from the U.S. company Novavax (292), the NP SCB-2019
vaccine from the Chinese company Clover Biopharmaceuticals
(293), the NP COVAX-19 vaccine from the U.S. company
GeneCure Biotechnologies (294), the NP vaccine from the
Taiwanese company Medigen Vaccine Biologics (295), the NP
vaccine AdmirSC-2f from Taiwanese company Adimmune corp
(296), an unnamed NP vaccine from the University of Queensland
(297, 298) and an unnamed VLP vaccine from the Canadian
company Medicago (299). CEPI has collaborated in the
development and testing of a selected number of these vaccine
candidates (273, 281, 298).
It should also be noted that several viral vectored vaccine
candidates for COVID-19 have also entered in clinical testing
(Table 2). Several adenovirus vectored vaccines are currently
the furthest along in clinical testing. One example is the vaccine
candidate AZD1222 (formerly known as ChAdOx1 nCov-19), a
replication-defective chimpanzee adenovirus developed by
Oxford University which entered phase III clinical trials in
August 2020. This viral vector was chosen due to its previous
application as a vaccine vector for Middle East respiratory
syndrome coronavirus (MERS-CoV). The ChAdOx1 vector
encoding the spike (S) protein provided protection against six
different strains of MERS-CoV in rhesus macaques (25),
demonstrating its ability to be an effective vaccine for
coronaviruses. Specific to COVID-19, AZ1222 was found to
induce humoral and cell mediated immune repsonses in phase
I/II cliical trial and did not result in any instances of severe side
effects (300). It has recently found that AZD1222 could induce
a robust humoral, CD8 and Th1 dominant CD4 response in
mice and rhesus macaques and that both a prime and a primeboost
regimen protected rhesus macaques against COVID-19
related pneumonia. However, it should be noted that there was
no difference in the amount of nasal virus shedding in
vaccinated vs unvaccinated animals challenged with SARSCoV-
2 (301).
Two other replication-incompetent adenoviral vectored
vaccines for COVID-19 have also entered clinical trials. The
Ad5-nCoV candidate from Chinese company CanSino biologics
was shown in early clinical trial data to induce significant
antibody and T cell responses after a single dose and to have
only rare instances of severe side effects that were more prevalent
among the higher dose groups (302, 303). The Chinese
government has recently approved the vaccine for use among
its members of the armed forces (304). Additionally, the
Ad26.COV2.S from Johnson & Johnson induced antibody and
T cell responses in rhesus macaques after a single dose, and
antibody titers negatively correlated with viral titers during viral
challenge (305). Finally, the Gam-COVID-Vac candidate from
the Gamaleya Research Institute of Epidemiology and
Microbiology in Russia is another adenovirus-based vaccine
that is the first COVID-19 vaccine to gain government
approval for widespread use after a phase I trial. Phase III
trials for this vaccine began in August 2020 (306).
Finally, two COVID-19 vaccine candidates based on liveattenuated
measles platforms have also entered into early clinical
trials. The TMV-083 candidate from the Institut Pasteur and
with collaboration with CEPI is a measles vectored vaccine
expressing a modified SARS-CoV-2 surface glycoprotein that
entered phase I clinical trials in August 2020 (307), while the
V591 candidate from Merck also entered phase I clinical trials in
August 2020 (308). Many other viral vectored vaccine candidates
for COVID-19 are also in preclinical stages of development.
TABLE 2 | Viral vaccines currently in development for SARS-CoV-2*.
Vaccine Name Vaccine
Company and Country Preliminary results
AZD1222 (ChAdOx1
Adenovirus Oxford University, UK • Vector was shown to protect rhesus macaques against six strains of MERS-CoV.
• Early phase I/II clinical trial data show that vaccine was well tolerated and induced humoral
and cell-mediated responses.
• Vaccine was found to induce robust humoral, CD8 and Th1 dominated CD4 responses in
mice and rhesus macaques, and that both a prime and prime-boost regimen protected
rhesus macaques against COVID-19 related pneumonia.
• Entered phase III clinical trials in August 2020
Ad5-nCoV Adenovirus CanSino biologics, China • Early phase I/II clinical trial data show that vaccine induced antibody and cell-mediated
responses after a single dose and was well tolerated.
• Entered phase III clinical trials in August 2020
• Approved by the Chinese government for use by its members of the armed forces
Ad26.COV2.S Adenovirus Johnson and Johnson, USA • Vaccine was found to induce antibody and T cell responses in rhesus macaques after a single
dose, and antibody titers negatively correlated with viral titers during viral challenge.
• Entered phase III clinical trials in August 2020
Gam-COVID-Vac Adenovirus Gamaleya Research Institute
of Epidemiology and
Microbiology, Russia
• Approved for widespread use by the Russian government before the release of clinical trial
• Entered phase III clinical trials in August 2020
TMV-083 Measles Institut Pasteur, France • Entered phase I clinical trials in August 2020
V591 Measles Merck, USA • Entered phase I clinical trials in August 2020
*COVID19 vaccine data compiled with the aid of the BioRender COVID-19 Vaccine and Drug tracker:
This chart summarizes the name, viral vector used, company and country of origin, and preliminary data on existing viral vaccines for SARS-CoV-2 that are currently undergoing
clinical testing.
Brisse et al. Non-Viral Vaccine Technologies
Frontiers in Immunology | 13 September 2020 | Volume 11 | Article 583077
The emergence of new non-viral vaccine technologies has
significantly advanced the scope and efficacy of traditional vaccine
formulations that are generally based on single protein subunit
vaccines or attenuated or killed vaccines. Non-viral vaccine
technologies have allowed for new applications to address
ongoing challenges of vaccination with customization in the areas
of safety, immunogenicity, breadth of protection, scalability, and
ease of production. These new technologies have also expanded the
notion of what is possible with vaccination by extending their reach
to once untenable areas, such as cancer treatment and neutralization
of drugs of abuse. It is clear that continued development and
optimization of vaccines will require multi-faceted approaches
that can only be implemented with extensive cross-field
collaboration and periodic review of the current state of
vaccinology. These challenges as well as opportunities ensure that
vaccine development will remain on the cutting edge of science for
decades to come to combat new and emerging pathogens as well as
other noncommunicable diseases.
MB, SV, NK, and HL contributed to the literature review and
writing of the manuscript. MB prepared all figures and tables. All
authors contributed to the article and approved the
submitted version.
This work was supported in parts by NIH NIAID grant R01
AI131586, USDA-NIFA-Capacity Funds (Hatch and Animal
Health), and the University of Minnesota School of Medicine
Academic Investment Research Program (AIRP) and COVID-19
Rapid Response Funds to HL and YL, USDA-NIFA AFRI grant
#2019-05384 and Minnesota Agricultural Experiment Station Rapid
Agricultural Response Fund to HL, and by a pre-doctoral NIH
fellowship T32 DA007097 to MB. NIH T32 training grant in
ComparativeMedicine and Pathology (5T32 OD010993-17) for NK.
1. Liu MA. Immunologic basis of vaccine vectors. Immunity (2010) 33:504–15.
doi: 10.1016/j.immuni.2010.10.004
2. Plotkin S. History of vaccination. Proc Natl Acad Sci U S A (2014)
111:12283–7. doi: 10.1073/pnas.1400472111
3. Rauch S, Jasny E, Schmidt KE, Petsch B. New vaccine technologies to
combat outbreaksituations. Front Immunol (2018) 9(1963):1–24.
doi: 10.3389/fimmu.2018.01963
4. Smith GL, MacKett M, Moss B. Infectious vaccinia virus recombinants that
express hepatitis B virus surface antigen. Nature (1983) 302:490–5.
doi: 10.1038/302490a0
5. Moss B, Smith GL, Gerin JL, Purcell RH. Live recombinant vaccinia virus
protects chimpanzees against hepatitis B. Nature (1984) 311:67–9.
doi: 10.1038/311067a0
6. Tartaglia J, Jarrett O, Neil JC, Desmettre P, Paoletti E. Protection of cats
against feline leukemia virus by vaccination witha canarypox virus
recombinant, ALVAC-FL. J Virol(1993) 67:2370–5. doi: 10.1128/
7. Darteil R, Bublot M, Laplace E, Bouquet JF, Audonnet JC, Riviè M.
Herpesvirus of turkey recombinant viruses expressing infectious bursal
disease virus (IBDV) VP2 immunogen induce protection against an IBDV
virulent challenge in chickens. Virology (1995) 211:481–90. doi: 10.1006/
8. Pastoret PP, Brochier B. The development and use of a vaccinia-rabies
recombinant oral vaccine for the control of wildlife rabies; A link between
Jenner and Pasteur. Epidemiol Infect (1996) 116:235–40. doi: 10.1017/
9. Welter J, Taylor J, Tartaglia J, Paoletti E, Stephensen CB. Vaccination
against Canine Distemper Virus Infection in Infant Ferrets with and without
Maternal Antibody Protection, Using Recombinant Attenuated Poxvirus
Vaccines. J Virol (2000) 74:6358–67. doi: 10.1128/jvi.74.14.6358-6367.2000
10. Pardo MC, Tanner P, Bauman J, Silver K, Fischer L. Immunization of
Puppies in the Presence of Maternally Derived Antibodies Against Canine
Distemper Virus. J Comp Pathol (2007) 137:S72. doi: 10.1016/
11. Jas D, Coupier C, Toulemonde CE, Guigal PM, Poulet H. Three-year
duration of immunity in cats vaccinated with a canarypox-vectored
recombinant rabies virus vaccine. Vaccine (2012) 30:6991–6. doi: 10.1016/
12. Esaki M, Godoy A, Rosenberger JK, Rosenberger SC, Gardin Y, Yasuda A,
et al. Protection and antibody response caused by turkey herpesvirus vector
newcastle disease vaccine. Avian Dis (2013) 57:750–5. doi: 10.1637/10540-
13. First FDA-approved vaccine for the prevention of Ebola virus disease, marking
a critical milestone in public health preparedness and response | FDA .
Available at:
public-health (Accessed September 1, 2020).
14. Draper SJ, Heeney JL. Viruses as vaccine vectors for infectious diseases and
cancer. Nat Rev Microbiol (2010) 8:62–73. doi: 10.1038/nrmicro2240
15. Dhanwani R, Zhou Y, Huang Q, Verma V, Dileepan M, Ly H, et al. A Novel
Live Pichinde Virus-Based Vaccine Vector Induces Enhanced Humoral and
Cellular Immunity after a Booster Dose. J Virol (2015) 90:2551–60.
doi: 10.1128/JVI.02705-15
16. Dhanwani R, Ly H, Liang Y. Recombinant Tri-Segmented Pichinde Virus as
a Novel Live Viral Vaccine Platform. In: NJ Clifton, editor. Methods in
molecular biology. New York, NY: Humana Press (2017). p. 169–79.
doi: 10.1007/978-1-4939-6869-5_10
17. Peng B, Wang LR, Gomez-Roman VR, Davis-Warren A, Montefiori DC,
Kalyanaraman VS, et al. Replicating Rather than Nonreplicating Adenovirus-
HumanImmunodeficiency Virus Recombinant Vaccines Are Better at Eliciting
Potent Cellular Immunity andPriming High-Titer Antibodies. J Virol (2017)
79:10200–9. doi: 10.1128/jvi.79.16.10200-10209.2005
18. Condit RC, Williamson AL, Sheets R, Seligman SJ, Monath TP, Excler JL,
et al. Unique safety issues associated with virus-vectored vaccines: Potential
for and theoretical consequences of recombination with wild type virus
strains. Vaccine (2016) 34:6610–6. doi: 10.1016/j.vaccine.2016.04.060
19. Dudek T, Knipe DM. Replication-defective viruses as vaccines and vaccine
vectors. Virology (2006) 344:230–9. doi: 10.1016/j.virol.2005.09.020
20. Barry M. Single-cycle adenovirus vectors in the current vaccine landscape.
Expert Rev Vaccines (2018) 17:163–73. doi: 10.1080/14760584.2018.1419067
21. Clem AS. Fundamentals of vaccine immunology. J GlobalInfect Dis (2011)
3:373–8. doi: 10.4103/0974-777X.77299
22. Zimmermann P, Curtis N. Factors that influence the immune response
tovaccination. Clin Microbiol Rev (2019) 32:e00084–18. doi: 10.1128/
23. Kushnir N, Streatfield SJ, Yusibov V. Virus-like particles as a highly efficient
vaccine platform: Diversity of targets and production systems and advances
in clinical development. Vaccine (2012) 31:58–83. doi: 10.1016/
24. Mohsen MO, Zha L, Cabral-Miranda G, Bachmann MF. Major findings and
recent advances in virus–like particle (VLP)-based vaccines. Semin Immunol
(2017) 34:123–32. doi: 10.1016/j.smim.2017.08.014
Brisse et al. Non-Viral Vaccine Technologies
Frontiers in Immunology | 14 September 2020 | Volume 11 | Article 583077
25. Deml L, Speth C, Dierich MP, Wolf H, Wagner R. Recombinant HIV-1
Pr55gag virus-like particles: Potent stimulators of innate and acquired
immune responses. Mol Immunol (2005) 42:259–77. doi: 10.1016/
26. Paliard X, Liu Y, Wagner R, Wolf H, Baenziger J, Walker CM. Priming of
strong, broad, and long-lived HIV type 1 p55(gag)-specific CD8+ cytotoxic
T cells after administration of a virus-like particle vaccine in rhesus
macaques. AIDS Res Hum Retroviruses (2000) 16:273–82. doi: 10.1089/
27. Murata K, Lechmann M, Qiao M, Gunji T, Altert HJ, Liang TJ.
Immunization with hepatitis C virus-like particles protects mice from
recombinant hepatitis C virus-vaccinia infection. Proc Natl Acad Sci
U.S.A. (2003) 100:6753–8. doi: 10.1073/pnas.1131929100
28. Schirmbeck R, Böhm W, Reimann J. Virus-like Particles Induce MHC
Class I-Restricted T-Cell Responses: Lessons learned from the hepatitis
B small surface antigen. Intervirology (1996) 39:111–9. doi: 10.1159/
29. Win SJ, Ward VK, Dunbar PR, Young SL, Baird MA. Cross-presentation of
epitopes on virus-like particles via the MHC i receptor recycling pathway.
Immunol Cell Biol (2011) 89:681–8. doi: 10.1038/icb.2010.161
30. Grgacic EVL, Anderson DA. Virus-like particles: Passport to immune
recognition. Methods (2006) 40:60–5. doi: 10.1016/j.ymeth.2006.07.018
31. Chackerian B. Virus-like particles: Flexible platforms for vaccine
development. Expert Rev Vaccines (2007) 6:381–90. doi: 10.1586/
32. glaxosmithkline. ENGERIX-B [Hepatitis B Vaccine (Recombinant)] injectable
suspension, for intramuscular use. Available at:
33. glaxosmithkline. Cervarix, Suspension for Intramuscular Injection. Available
34. RECOMBIVAX HB®. Hepatitis B Vaccine (Recombinant) Suspension for
intramuscular injection. Available at:
35. Merck. GARDASIL® [Human Papillomavirus Quadrivalent (Types 6, 11, 16,
and 18) Vaccine, Recombinant] Suspension for intramuscular injection.
Available at:
36. Agnandji ST, Lell B, Fernandes JF, Abossolo BP, Methogo BGNO,
Kabwende AL, et al. A phase 3 trial of RTS,S/AS01 malaria vaccine in
African infants. N Engl J Med (2012) 367:2284–95. doi: 10.1056/
37. Agnandji ST, Lell B, Soulanoudjingar SS, Fernandes JF, Abossolo BP,
Conzelmann C, et al. First results of phase 3 trial of RTS,S/AS01 malaria
vaccine in African children. N Engl J Med (2011) 365:1863–75. doi: 10.1056/
38. Song JM, Wang BZ, Park KM, van Rooijen N, Quan FS, Kim MC, et al.
Influenza virus-like particles containing M2 induce broadly
crossprotective immunity. PloS One (2011) 6:e14538. doi: 10.1371/
39. El-Attar L, Oliver SL, Mackie A, Charpilienne A, Poncet D, Cohen J, et al.
Comparison of the efficacy of rotavirus VLP vaccines to a live homologous
rotavirus vaccine in a pig model of rotavirus disease. Vaccine (2009)
27:3201–8. doi: 10.1016/j.vaccine.2009.03.043
40. Zhou H, Guo L, Wang M, Qu J, Zhao Z, Wang J, et al. Prime immunization
with rotavirus VLP 2/6 followed by boosting withan adenovirus expressing
VP6 induces protective immunization against rotavirus inmice. Virol J
(2011) 8(3):1–8. doi: 10.1186/1743-422X-8-3
41. Dhanasooraj D, Kumar RA, Mundayoor S. Subunit protein vaccine delivery
system for tuberculosis based on hepatitis B virus core VLP (HBc-VLP)
particles. In: Methods in Molecular Biology. Totowa, NJ: Humana Press Inc.
(2016). p. 377–92. doi: 10.1007/978-1-4939-3389-1_26
42. Boigard H, Alimova A, Martin GR, Katz A, Gottlieb P, Galarza JM. Zika
virus-like particle (VLP) based vaccine. PloS Negl Trop Dis (2017) 11:
e0005608. doi: 10.1371/journal.pntd.0005608
43. Franco D, LiuW, Gardiner DF, Hahn BH, Ho DD. CD40L-containing virus-like
particle as a candidate HIV-1 vaccine targeting dendritic cells. J Acquir Immune
Defic Syndr (2011) 56:393–400. doi: 10.1097/QAI.0b013e31820b844e
44. Pillay S, Shephard EG, Meyers AE, Williamson AL, Rybicki EP. HIV-1 subtype
C chimaeric VLPs boost cellular immune responses inmice. J Immune
Based Ther Vaccines (2010) 8(7):1–6. doi: 10.1186/1476-8518-8-7
45. Frietze KM, Peabody DS, Chackerian B. Engineering virus-like particles as
vaccine platforms. Curr Opin Virol (2016) 18:44–9. doi: 10.1016/
46. Pati R, Shevtsov M, Sonawane A. Nanoparticle vaccines against infectious
diseases.Front Immunol (2018) 9(2224):1–16. doi: 10.3389/fimmu.
47. Zhao L, Seth A, Wibowo N, Zhao CX, Mitter N, Yu C, et al. Nanoparticle
vaccines. Vaccine (2014) 32:327–37. doi: 10.1016/j.vaccine.2013.11.069
48. Han J, Zhao D, Li D, Wang X, Jin Z, Zhao K. Polymer-based nanomaterials
and applications for vaccines anddrugs. Polym (Basel) (2018) 10(31):1–14.
doi: 10.3390/polym10010031
49. Li H, Li Y, Wang X, Hou Y, Hong X, Gong T, et al. Rational design of
polymeric hybrid micelles to overcome lymphaticand intracellular delivery
barriers in cancer immunotherapy. Theranostics (2017) 7:4383–98.
doi: 10.7150/thno.20745
50. Al-Halifa S, Gauthier L, Arpin D, Bourgault S, Archambault D.
Nanoparticle-Based Vaccines Against RespiratoryViruses. Front Immunol
(2019) 10:22. doi: 10.3389/fimmu.2019.00022
51. Hobernik D, Bros M. DNA vaccines—How far from clinical use? Int J Mol
(2018) 19:3605. doi: 10.3390/ijms19113605
52. Khan KH. DNA vaccines: Roles against diseases. GERMS (2013) 3:26–35.
doi: 10.11599/germs.2013.1034
53. Kennedy NJ, Spithill TW, Tennent J, Wood PR, Piedrafita D. DNA vaccines
in sheep: CTLA-4 mediated targeting and CpG motifs enhance
immunogenicity in a DNA prime/protein boost strategy. Vaccine (2006)
24:970–9. doi: 10.1016/j.vaccine.2005.08.076
54. Li L, Saade F, Petrovsky N. The future of human DNA vaccines. J Biotechnol
(2012) 162:171–82. doi: 10.1016/j.jbiotec.2012.08.012
55. Li SS, Kochar NK, Elizaga M, Hay CM, Wilson GJ, Cohen KW, et al. DNA
priming increases frequency of T-cell responses to a vesicularstomatitis virus
HIV vaccine with specific enhancement of CD8 T-cell responses by
interleukin-12plasmid DNA. Clin Vaccine Immunol (2017) 24:1–
14e00263-17. doi: 10.1128/CVI.00263-17
56. Haidari G, Cope A, Miller A, Venables S, Yan C, Ridgers H, et al. Combined
skin and muscle vaccination differentially impact thequality of effector T cell
functions: The CUTHIVAC-001 randomized trial. SciRep (2017) 7:13011.
doi: 10.1038/s41598-017-13331-1
57. Zhang C, Maruggi G, Shan H, Li J. Advances in mRNA vaccines for
infectious diseases. Front Immunol (2019) 10:594. doi: 10.3389/fimmu.
58. Lundstrom K. Latest development on RNA-based drugs and vaccines.Futur
Sci OA (2018) 4(5):FSO300. doi: 10.4155/fsoa-2017-0151
59. Uchida S, Yoshinaga N, Yanagihara K, Yuba E, Kataoka K, Itaka K.
Designing immunostimulatory double stranded messenger RNA with
maintained translational activity through hybridization with poly A
sequences for effective vaccination. Biomaterials (2018) 150:162–70.
doi: 10.1016/J.BIOMATERIALS.2017.09.033
60. Hood JL. Post isolation modification of exosomes for nanomedicine applications.
Nanomedicine (2016) 11:1745–56. doi: 10.2217/nnm-2016-0102
61. Benteyn D, Heirman C, Bonehill A, Thielemans K, Breckpot K. mRNAbased
dendritic cell vaccines. Expert Rev Vaccines (2014) 14:161–76.
doi: 10.1586/14760584.2014.957684
62. Diken M, Kreiter S, Selmi A, Britten CM, Huber C, Türeci Ö, et al. Selective
uptake of naked vaccine RNA by dendritic cells is driven by
macropinocytosis and abrogated upon DC maturation. Gene Ther (2011)
18:702–8. doi: 10.1038/gt.2011.17
63. Van den Boorn JG, Hartmann G. Turning Tumors into Vaccines: Co-opting
the Innate Immune System. Immunity (2013) 39:27–37. doi: 10.1016/
64. Elion DL, Cook RS, Elion DL, Cook RS. Harnessing RIG-I and intrinsic
immunity in the tumor microenvironment for therapeutic cancer
treatment. Oncotarget (2018) 9:29007–17. doi: 10.18632/oncotarget.
65. Vogel AB, Lambert L, Kinnear E, Busse D, Erbar S, Reuter KC, et al. Self-
Amplifying RNA Vaccines Give Equivalent Protection against Influenza to
mRNA Vaccines but at Much Lower Doses. Mol Ther (2018) 26:446–55.
doi: 10.1016/j.ymthe.2017.11.017
Brisse et al. Non-Viral Vaccine Technologies
Frontiers in Immunology | 15 September 2020 | Volume 11 | Article 583077
66. Démoulins T, Englezou PC, Milona P, Ruggli N, Tirelli N, Pichon C, et al.
Self-replicating RNA vaccine delivery to dendriticcells. Methods Mol Biol
(2017) 1499:37–75. doi: 10.1007/978-1-4939-6481-9_3
67. Englezou PC, Sapet C, Démoulins T, Milona P, Ebensen T, Schulze K, et al.
Self-Amplifying Replicon RNA Delivery to Dendritic Cells by Cationic
Lipids. Mol Ther Nucleic Acids (2018) 12:118–34. doi: 10.1016/
68. Pardi N, Hogan MJ, Porter FW, Weissman D. mRNA vaccines-a new era in
vaccinology. Nat Rev Drug Discovery (2018) 17:261–79. doi: 10.1038/
69. Alberer M, Gnad-Vogt U, Hong HS, Mehr KT, Backert L, Finak G, et al.
Safety and immunogenicity of a mRNA rabies vaccine in healthy adults: an
open-label, non-randomised, prospective, first-in-human phase 1 clinical
trial. Lancet (2017) 390:1511–20. doi: 10.1016/S0140-6736(17)31665-3
70. Bahl K, Senn JJ, Yuzhakov O, Bulychev A, Brito LA, Hassett KJ, et al.
Preclinical and Clinical Demonstration of Immunogenicity by mRNA
Vaccines against H10N8 and H7N9 Influenza Viruses. Mol Ther (2017)
25:1316–27. doi: 10.1016/j.ymthe.2017.03.035
71. Wang Z, Troilo PJ, Wang X, Griffiths TG, Pacchione SJ, Barnum AB, et al.
Detection of integration of plasmid DNA into host genomic DNA following
intramuscular injection and electroporation. Gene Ther (2004) 11:711–21.
doi: 10.1038/
72. Myhr AI. DNA Vaccines: Regulatory Considerations and Safety Aspects.
Curr Issues Mol Biol (2017) 22:79–88. doi: 10.21775/cimb.022.079
73. Kramps T, Elbers K. Introduction to RNA vaccines. Methods MolBiol (2017)
1499:1–11. doi: 10.1007/978-1-4939-6481-9_1
74. Rueckert C, Guzmán CA. Vaccines: From Empirical Development to
RationalDesign. PloS Pathog (2012) 8:e1003001. doi: 10.1371/journal.
75. Peri C, Gagni P, Combi F, Gori A, Chiari M, Longhi R, et al. Rational epitope
design for protein targeting. ACS Chem Biol (2013) 8:397–404. doi: 10.1021/
76. Yamashita T. Toward rational antibody design: Recent advancements in
molecular dynamics simulations. Int Immunol (2018) 30:133–40.
doi: 10.1093/intimm/dxx077
77. He L, Zhu J. Computational tools for epitope vaccine design and evaluation.
Curr Opin Virol (2015) 11:103–12. doi: 10.1016/J.COVIRO.2015.03.013
78. Rappuoli R, Bottomley MJ, D’Oro U, Finco O, De Gregorio E. Reverse
vaccinology 2.0: Human immunology instructs vaccine antigen design. J Exp
Med (2016) 213:469–81. doi: 10.1084/JEM.20151960
79. McLellan JS, Chen M, Joyce MG, Sastry M, Stewart-Jones GBE, Yang Y, et al.
Structure-based design of a fusion glycoprotein vaccine for respiratory
syncytial virus. Sci (80 ) (2013) 342:592–8. doi: 10.1126/science.1243283
80. Wu X, Yang ZY, Li Y, Hogerkorp CM, Schief WR, Seaman MS, et al. Rational
design of envelope identifies broadly neutralizing human monoclonal
antibodies to HIV-1. Sci (80) (2010) 329:856–61. doi: 10.1126/
81. Zhou T, Xu K. Structural features of broadly neutralizing antibodies and
rational design of vaccine. In: Advances in Experimental Medicine and Biology.
New York, NY: Springer New York LLC (2018). p. 73–95. doi: 10.1007/978-
82. Ferlenghi I, Cozzi R, Scarselli M. Structural Vaccinology: A Threedimensional
View for Vaccine Development. Curr Top Med Chem (2013)
13:2629–37. doi: 10.2174/15680266113136660187
83. Keck M-L, Wrensch F, Pierce BG, Baumert TF, Foung SKH. Mapping
Determinants of Virus Neutralization and Viral Escape forRational Design
of a Hepatitis C Virus Vaccine. Front Immunol (2018) 9:1194. doi: 10.3389/
84. He L, Tzarum N, Lin X, Shapero B, Sou C, Mann CJ, et al. Proof of concept
for rational design of hepatitis C virus E2 core nanoparticle vaccines. Sci Adv
(2020) 6:eaaz6225. doi: 10.1126/sciadv.aaz6225
85. Kaufmann JK, Flechtner JB. Evolution of rational vaccine designs for genital
herpes immunotherapy. Curr Opin Virol (2016) 17:80–6. doi: 10.1016/
86. López-Camacho C, Abbink P, Larocca RA, Dejnirattisai W, Boyd M,
Badamchi-Zadeh A, et al. Rational Zika vaccine design via the modulation
of antigen membraneanchors in chimpanzee adenoviral vectors. Nat
Commun (2018) 9(1):2441. doi: 10.1038/s41467-018-04859-5
87. Tai W, Chen J, Zhao G, Geng Q, He L, Chen Y, et al. Rational Design of Zika
Virus Subunit Vaccine with EnhancedEfficacy. J Virol (2019) 93(17):e02187-
18. doi: 10.1128/jvi.02187-18
88. Li Z, Song S, He M, Wang D, Shi J, Liu X, et al. Rational design of a tripletype
human papillomavirus vaccine bycompromising viral-type specificity.
Nat Commun (2018) 9(1):5360. doi: 10.1038/s41467-018-07199-6
89. Ong E, Wong MU, He Y. Identification of new features from known
bacterial protectivevaccine antigens enhances rational vaccine design.
Front Immunol (2017) 8:1382. doi: 10.3389/fimmu.2017.01382
90. Hurtgen BJ, Hung CY. Rational design to T lymphocyte epitope-based
vaccines against Coccidioides infection. Methods Mol Biol (2017) 1625:45–
64. doi: 10.1007/978-1-4939-7104-6_4
91. Martı́nez-Sáez N, Peregrina JM, Corzana F. Principles of mucin structure:
Implications for the rational design of cancer vaccines derived from
MUC1-glycopeptides. Chem Soc Rev (2017) 46:7154–75. doi: 10.1039/
92. Karch CP, Burkhard P. Vaccine technologies: From whole organisms to
rationally designed protein assemblies. Biochem Pharmacol (2016) 120:1–14.
doi: 10.1016/j.bcp.2016.05.001
93. Zupančič E, Curato C, Paisana M, Rodrigues C, Porat Z, Viana AS, et al.
Rational design of nanoparticles towards targeting antigen-presenting cells
and improved T cell priming. J Control Release (2017) 258:182–95.
doi: 10.1016/j.jconrel.2017.05.014
94. Porkolab V, Chabrol E, Varga N, Ordanini S, Sutkevičiute I, Thépaut M,
et al. Rational-Differential Design of Highly Specific Glycomimetic Ligands:
Targeting DC-SIGN and Excluding Langerin Recognition. ACS Chem Biol
(2018) 13:600–8. doi: 10.1021/acschembio.7b00958
95. Oyarzún P, Kobe B. Recombinant and epitope-based vaccines on the road to the
market and implications for vaccine design and production. Hum Vaccines
Immunother (2016) 12:763–7. doi: 10.1080/21645515.2015.1094595
96. Liljeroos L, Malito E, Ferlenghi I, Bottomley MJ. Structural and
Computational Biology in the Design of ImmunogenicVaccine
Antigens. J Immunol Res (2015) 2015:156241. doi: 10.1155/2015/156241
97. Zhang X, Zhivaki D, Lo-Man R. Unique aspects of the perinatal immune
system. Nat Rev Immunol (2017) 17:495–507. doi: 10.1038/nri.2017.54
98. Kollmann TR, Levy O, Montgomery RR, Goriely S. Innate Immune Function
by Toll-like Receptors: Distinct Responses in Newborns and the Elderly.
Immunity (2012) 37:771–83. doi: 10.1016/j.immuni.2012.10.014
99. Aiello A, Farzaneh F, Candore G, Caruso C, Davinelli S, Gambino CM, et al.
Immunosenescence and its hallmarks: How to oppose aging strategically? A
review of potential options for therapeutic intervention. Front Immunol
(2019) 10:2247:2247. doi: 10.3389/fimmu.2019.02247
100. Hazeldine J, Lord JM. Innate immunesenescence: underlying mechanisms
and clinical relevance. Biogerontology (2015) 16:187–201. doi: 10.1007/
101. Montgomery RR, Shaw AC. Paradoxical changes in innate immunity in
aging: recent progress and new directions. J Leukoc Biol (2015) 98:937–43.
doi: 10.1189/jlb.5mr0315-104r
102. Chinn IK, Blackburn CC, Manley NR, Sempowski GD. Changes in primary
lymphoid organs with aging. Semin Immunol (2012) 24:309–20.
doi: 10.1016/j.smim.2012.04.005
103. Kline GH, Hayden TA, Klinman NR. B cell maintenance in aged mice
reflects both increased B cell longevity and decreased B cell generation.
J Immunol (1999) 162:3342–9.
104. Stephan RP, Lill-Elghanian DA, Witte PL. Development of B cells in aged
mice: decline in the ability of pro-Bcells to respond to IL-7 but not to other
growth factors. J Immunol (1997) 158:1598–609.
105. Becklund BR, Purton JF, Ramsey C, Favre S, Vogt TK, Martin CE, et al. The
aged lymphoid tissue environment fails to support naive T cellhomeostasis.
Sci Rep (2016) 6:30842. doi: 10.1038/srep30842
106. Ferrucci L, Corsi A, Lauretani F, Bandinelli S, Bartali B, Taub DD, et al. The
origins of age-related proinflammatory state. Blood (2005) 105:2294–9.
doi: 10.1182/blood-2004-07-2599
107. De Martinis M, Franceschi C, Monti D, Ginaldi L. Inflamm-ageing and
lifelong antigenic load as major determinants of ageing rate and longevity.
FEBS Lett (2005) 579:2035–9. doi: 10.1016/j.febslet.2005.02.055
108. Cumberbatch M, Dearman RJ, Kimber I. Influence of ageing on Langerhans
cell migration in mice: Identification of a putative deficiency of epidermal
Brisse et al. Non-Viral Vaccine Technologies
Frontiers in Immunology | 16 September 2020 | Volume 11 | Article 583077
interleukin-1a. Immunology (2002) 105:466–77. doi: 10.1046/j.1365-
109. Desai A, Grolleau-Julius A, Yung R. Leukocyte function in the aging immune
system. J Leukoc Biol (2010) 87:1001–9. doi: 10.1189/jlb.0809542
110. Aprahamian T, Takemura Y, Goukassian D, Walsh K. Ageing is associated
with diminished apoptotic cell clearance in vivo. Clin Exp Immunol (2008)
152:448–55. doi: 10.1111/j.1365-2249.2008.03658.x
111. Cambier J. Immunosenescence: a problem of lymphopoiesis, homeostasis,
microenvironment, and signaling. John Cambier. Immunol Rev (2005)
205:5–6. doi: 10.1111/j.0105-2896.2005.00276.x
112. Wertheimer AM, Bennett MS, Park B, Uhrlaub JL, Martinez C, Pulko V,
et al. Aging and Cytomegalovirus Infection Differentially and Jointly Affect
Distinct Circulating T Cell Subsets in Humans. J Immunol (2014) 192:2143–
55. doi: 10.4049/jimmunol.1301721
113. Kogut I, Scholz JL, Cancro MP, Cambier JC. B cell maintenance and function
in aging. Semin Immunol (2012) 24:342–9. doi: 10.1016/j.smim.2012.04.004
114. Coutinho AE, Chapman KE. The anti-inflammatory and immunosuppressive
effects of glucocorticoids, recent developments and
mechanistic insights. Mol Cell Endocrinol (2011) 335:2–13. doi: 10.1016/
115. Rea D, Van Kooten C, Van Meijgaarden KE, Ottenhoff THM, Melief CJM,
Offringa R. Glucocorticoids transform CD40-triggering of dendritic cells into
an alternative activation pathway resulting in antigen-presenting cells that
secrete IL-10. Blood (2000) 95:3162–7. doi: 10.1182/blood.v95.10.3162
116. Rutella S, Lemoli RM. Regulatory T cells and tolerogenic dendritic cells:
From basic biology to clinical applications. Immunol Lett (2004) 94:11–26.
doi: 10.1016/j.imlet.2004.04.015
117. Luther C, Adamopoulou E, Stoeckle C, Brucklacher-Waldert V, Rosenkranz
D, Stoltze L, et al. Prednisolone Treatment Induces Tolerogenic Dendritic
Cells and a Regulatory Milieu in Myasthenia Gravis Patients. J Immunol
(2009) 183:841–8. doi: 10.4049/jimmunol.0802046
118. Chamorro S, Garcı́a-Vallejo JJ, Unger WWJ, Fernandes RJ, Bruijns SCM,
Laban S, et al. TLR Triggering on Tolerogenic Dendritic Cells Results in
TLR2 Up-Regulation and a Reduced Proinflammatory Immune Program. J
Immunol (2009) 183:2984–94. doi: 10.4049/jimmunol.0801155
119. Pasquale A, Preiss S, Silva F, Garçon N. Vaccine Adjuvants: from 1920 to
2015 and Beyond.Vaccines (2015) 3:320–43. doi: 10.3390/vaccines3020320
120. Moyer TJ, Zmolek AC, Irvine DJ. Beyond antigens and adjuvants:
Formulating futurevaccines. J Clin Invest (2016) 126:799–808. doi: 10.1172/
121. Grogan JL, Mohrs M, Harmon B, Lacy DA, Sedat JW, Locksley RM. Early
transcription and silencing of cytokine genes underlie polarization of T
helper cell subsets. Immunity (2001) 14:205–15. doi: 10.1016/S1074-7613
122. Didierlaurent AM, Morel S, Lockman L, Giannini SL, Bisteau M, Carlsen H,
et al. AS04, an Aluminum Salt- and TLR4 Agonist-Based Adjuvant System,
Induces a Transient Localized Innate Immune Response Leading to
Enhanced Adaptive Immunity. J Immunol (2009) 183:6186–97.
doi: 10.4049/jimmunol.0901474
123. Kundi M. New hepatitis B vaccine formulated with an improved adjuvant
system. Expert Rev Vaccines (2007) 6:133–40. doi: 10.1586/14760584.6.2.133
124. Olafsdottir TA, Lingnau K, Nagy E, Jonsdottir I. IC31 ® , a Two-Component
Novel Adjuvant Mixed with a Conjugate Vaccine Enhances Protective
Immunity against Pneumococcal Disease in Neonatal Mice. Scand J
Immunol (2009) 69:194–202. doi: 10.1111/j.1365-3083.2008.02225.x
125. Knudsen NPH, Olsen A, Buonsanti C, Follmann F, Zhang Y, Coler RN, et al.
Different human vaccine adjuvants promote distinct antigen-independent
immunological signatures tailored to different pathogens. Sci Rep (2016) 6:1–
13. doi: 10.1038/srep19570
126. Behzad H, Huckriede ALW, Haynes L, Gentleman B, Coyle K, Wilschut JC,
et al. GLA-SE, a synthetic toll-like receptor 4 agonist, enhances T-cell
responses to influenza vaccine in older adults. J Infect Dis (2012) 205:466–
73. doi: 10.1093/infdis/jir769
127. Agger EM, Rosenkrands I, Hansen J, Brahimi K, Vandahl BS, Aagaard C,
et al. Cationic Liposomes Formulated with Synthetic Mycobacterial
Cordfactor (CAF01): A Versatile Adjuvant for Vaccines with Different
Immunological Requirements. PloS One (2008) 3:e3116. doi: 10.1371/
128. Elliott STC, Kallewaard NL, Benjamin E, Wachter-Rosati L, McAuliffe JM,
Patel A, et al. DMAb inoculation of synthetic cross reactive antibodies
protectsagainst lethal influenza A and B infections. NPJ Vaccines (2017) 2
(18):1–18. doi: 10.1038/s41541-017-0020-x
129. Uccellini MB, Busto P, Debatis M, Marshak-Rothstein A, Viglianti GA.
Selective binding of anti-DNA antibodies to native dsDNA fragmentsof
differing sequence. Immunol Lett (2012) 143:85–91. doi: 10.1016/
130. Vallin H, Perers A, Alm GV, Ronnblom L. Anti-Double-Stranded DNA
Antibodies and Immunostimulatory Plasmid DNA in Combination Mimic the
Endogenous IFN-a Inducer in Systemic Lupus Erythematosus. J Immunol (1999)
131. Wellmann U, Letz M, Herrmann M, Angermuller S, Kalden JR, Winkler TH.
The evolution of human anti-double-stranded DNAautoantibodies. Proc
Natl Acad Sci (2005) 102:9258–63. doi: 10.1073/pnas.0500132102
132. Morel S, Didierlaurent A, Bourguignon P, Delhaye S, Baras B, Jacob V, et al.
Adjuvant System AS03 containing a-tocopherol modulates innate immune
response and leads to improved adaptive immunity. Vaccine (2011) 29:2461–
73. doi: 10.1016/j.vaccine.2011.01.011
133. Henriksen-Lacey M, Christensen D, Bramwell VW, Lindenstrøm T, Agger
EM, Andersen P, et al. Liposomal cationic charge and antigen adsorption are
important properties for the efficient deposition of antigen at the injection
site and ability of the vaccine to induce a CMI response. J Control Release
(2010) 145:102–8. doi: 10.1016/j.jconrel.2010.03.027
134. van Dissel JT, Joosten SA, Hoff ST, Soonawala D, Prins C, Hokey DA, et al. A
novel liposomal adjuvant system, CAF01, promotes long-lived
Mycobacterium tuberculosis-specific T-cell responses in human. Vaccine
(2014) 32:7098–107. doi: 10.1016/j.vaccine.2014.10.036
135. Garçon N, Chomez P, Van Mechelen M. GlaxoSmithKline Adjuvant Systems
in vaccines: Concepts, achievements and perspectives. Expert Rev Vaccines
(2007) 6:723–39. doi: 10.1586/14760584.6.5.723
136. Bejon P, Lusingu J, Olotu A, Leach A, Lievens M, Vekemans J, et al. Efficacy
of RTS,S/AS01E vaccine against malaria in children 5 to 17 months of age. N
Engl J Med (2008) 359:2521–32. doi: 10.1056/NEJMoa0807381
137. Olotu A, Lusingu J, Leach A, Lievens M, Vekemans J, Msham S, et al. Efficacy
of RTS,S/AS01E malaria vaccine and exploratory analysis on anticircumsporozoite
antibody titres and protection in children aged 5-17
months in Kenya and Tanzania: A randomised controlled trial. Lancet
Infect Dis (2011) 11:102–9. doi: 10.1016/S1473-3099(10)70262-0
138. Stanberry L, Strugnell R. Vaccines of the future. Perspect Vaccinol (2011)
1:151–99. doi: 10.1016/j.pervac.2011.05.006
139. Duthie MS, Windish HP, Fox CB, Reed SG. Use of defined TLR ligands as
adjuvants within humanvaccines. Immunol Rev (2011) 239:178–96.
doi: 10.1111/j.1600-065X.2010.00978.x
140. Gnjatic S, Sawhney NB, Bhardwaj N. Toll-like receptor agonists are they good
adjuvants? Cancer J (2010) 16:382–91. doi: 10.1097/PPO.0b013e3181eaca65
141. Jones SC, Brahmakshatriya V, Huston G, Dibble J, Swain SL. TLR-Activated
Dendritic Cells Enhance the Response of Aged Naive CD4 T Cells via an IL-
6–Dependent Mechanism. J Immunol (2010) 185:6783–94. doi: 10.4049/
142. Gill MA, Palucka AK, Barton T, Ghaffar F, Jafri H, Banchereau J, et al.
Mobilization of plasmacytoid and myeloid dendritic cells to mucosal
sites in children with respiratory syncytial virus and other viral
respiratory infections. J Infect Dis (2005) 191:1105–15. doi: 10.1086/
143. Dimmock NJ, Easton AJ. Defective Interfering Influenza Virus RNAs: Time
To Reevaluate Their Clinical Potential as Broad-Spectrum Antivirals? J Virol
(2014) 88:5217–27. doi: 10.1128/JVI.03193-13
144. Dimmock NJ, Rainsford EW, Scott PD,Marriott AC. Influenza Virus Protecting
RNA: an Effective Prophylactic and Therapeutic Antiviral. J Virol (2008)
82:8570–8. doi: 10.1128/jvi.00743-08
145. Duhaut SD, Dimmock NJ. Defective influenza A virus generated entirely
from plasmids: Its RNA is expressed in infected mouse lung and modulates
disease. J Virol Methods (2003) 108:75–82. doi: 10.1016/S0166-0934(02)
146. Scott PD, Meng B, Marriott AC, Easton AJ, Dimmock NJ. Defective
interfering virus protects elderly mice from influenza. Virol J (2011) 8:212.
doi: 10.1186/1743-422X-8-212
Brisse et al. Non-Viral Vaccine Technologies
Frontiers in Immunology | 17 September 2020 | Volume 11 | Article 583077
147. Dimmock NJ, Dove BK, Scott PD, Meng B, Taylor I, Cheung L, et al. Cloned
Defective Interfering Influenza Virus Protects Ferrets from Pandemic 2009
Influenza A Virus and Allows Protective Immunity to Be Established. PloS
One (2012) 7:49394. doi: 10.1371/journal.pone.0049394
148. Dimmock NJ, Dove BK, Meng B, Scott PD, Taylor I, Cheung L, et al.
Comparison of the protection of ferrets against pandemic 2009 influenza A
virus (H1N1) by 244 DI influenza virus and oseltamivir. Antiviral Res (2012)
96:376–85. doi: 10.1016/j.antiviral.2012.09.017
149. Scott PD, Meng B, Marriott AC, Easton AJ, Dimmock NJ. Defective
interfering influenza virus confers only short-lived protection against
influenza virus disease: Evidence for a role for adaptive immunity in DI
virus-mediated protection in vivo. Vaccine (2011) 29:6584–91. doi: 10.1016/
150. Scott PD, Meng B, Marriott AC, Easton AJ, Dimmock NJ. Defective
interfering influenza A virus protects in vivo against disease caused by a
heterologous influenza B virus. J Gen Virol (2011) 92:2122–32. doi: 10.1099/
151. Easton AJ, Scott PD, Edworthy NL, Meng B, Marriott AC, Dimmock NJ. A
novel broad-spectrum treatment for respiratory virus infections: Influenzabased
defective interfering virus provides protection against pneumovirus
infection in vivo. Vaccine (2011) 29:2777–84. doi: 10.1016/j.vaccine.
152. Yang Y, Lyu T, Zhou R, He X, Ye K, Xie Q, et al. The antiviral and antitumor
effects of defective interfering particles/genomes and their mechanisms.
Front Microbiol (2019) 10:1852:1852. doi: 10.3389/fmicb.2019.01852
153. Pravetoni M. Biologics to treat substance use disorders: Current status and
new directions. Hum Vaccines Immunother (2016) 12:3005–19. doi: 10.1080/
154. Carrera MRA, Ashley JA, Parsons LH, Wirsching P, Koob GF, Janda KD.
Suppression of psychoactive effects of cocaine by active immunization.
Nature (1995) 378:727–30. doi: 10.1038/378727a0
155. Jalah R, Torres OB, Mayorov AV, Li F, Antoline JFG, Jacobson AE, et al.
Efficacy, but Not Antibody Titer or Affinity, of a Heroin Hapten Conjugate
Vaccine Correlates with Increasing Hapten Densities on Tetanus Toxoid, but
Not on CRM197 Carriers. Bioconjug Chem (2015) 26:1041–53. doi: 10.1021/
156. McCluskie MJ, Thorn J, Mehelic PR, Kolhe P, Bhattacharya K, Finneman JI,
et al. Molecular attributes of conjugate antigen influence function of
antibodies induced by anti-nicotine vaccine in mice and non-human
primates. Int Immunopharmacol (2015) 25:518–27. doi: 10.1016/
157. Kimishima A, Wenthur CJ, Zhou B, Janda KD. An advance in prescription
opioid vaccines: Overdose mortality reduction and extraordinary alteration
of drug half-life. ACS Chem Biol (2017) 12:36–40. doi: 10.1021/
158. Bremer PT, Kimishima A, Schlosburg JE, Zhou B, Collins KC, Janda KD.
Combatting Synthetic Designer Opioids: A Conjugate Vaccine Ablates
Lethal Doses of Fentanyl Class Drugs. Angew Chemie Int Ed (2016)
55:3772–5. doi: 10.1002/anie.201511654
159. Collins KC, Schlosburg JE, Bremer PT, Janda KD. Methamphetamine
Vaccines: Improvement through Hapten Design. J Med Chem (2016)
59:3878–85. doi: 10.1021/acs.jmedchem.6b00084
160. Bremer PT, Janda KD. Conjugate Vaccine Immunotherapy for Substance
Use Disorder. Pharmacol Rev (2017) 69:298–315. doi: 10.1124/pr.117.013904
161. Sulima A, Jalah R, Antoline JFG, Torres OB, Imler GH, Deschamps JR, et al.
A Stable Heroin Analogue That Can Serve as a Vaccine Hapten to Induce
Antibodies That Block the Effects of Heroin and Its Metabolites in Rodents
and That Cross-React Immunologically with Related Drugs of Abuse. J Med
Chem (2018) 61:329–43. doi: 10.1021/acs.jmedchem.7b01427
162. Nguyen JD, Bremer PT, Ducime A, Creehan KM, Kisby BR, Taffe MA, et al.
Active vaccination attenuates the psychostimulant effects of a-PVP and
MDPV in rats. Neuropharmacology (2017) 116:1–8. doi: 10.1016/
163. Isomura S, Wirsching P, Janda KD. An immunotherapeutic program for the
treatment of nicotine addiction: Hapten design and synthesis. J Org Chem
(2001) 66:4115–21. doi: 10.1021/jo001442w
164. Peterson EC, Gunnell M, Che Y, Goforth RL, Carroll FI, Henry R, et al. Using
hapten design to discover therapeutic monoclonal antibodies for treating
methamphetamine abuse. J Pharmacol Exp Ther (2007) 322:30–9.
doi: 10.1124/jpet.106.117150
165. Moreno AY, Mayorov AV, Janda KD. Impact of distinct chemical structures
for the development of a methamphetamine vaccine. J Am Chem Soc (2011)
133:6587–95. doi: 10.1021/ja108807j
166. Stowe GN, Vendruscolo LF, Edwards S, Schlosburg JE, Misra KK, Schulteis G,
et al. A vaccine strategy that induces protective immunity against heroin. J Med
Chem (2011) 54:5195–204. doi: 10.1021/jm200461m
167. Pravetoni M, Le Naour M, Harmon TM, Tucker AM, Portoghese PS, Pentel
PR. An oxycodone conjugate vaccine elicits drug-specific antibodies that
reduce oxycodone distribution to brain and hot-plate analgesia. J Pharmacol
Exp Ther (2012) 341:225–32. doi: 10.1124/jpet.111.189506
168. Pryde DC, Jones LH, Gervais DP, Stead DR, Blakemore DC, Selby MD, et al.
Selection of a Novel Anti-Nicotine Vaccine: Influence of Antigen Design on
Antibody Function in Mice. PloS One (2013) 8:e76557. doi: 10.1371/
169. Pravetoni M, Le Naour M, Tucker AM, Harmon TM, Hawley TM,
Portoghese PS, et al. Reduced antinociception of opioids in rats and mice
by vaccination with immunogens containing oxycodone and hydrocodone
haptens. J Med Chem (2013) 56:915–23. doi: 10.1021/jm3013745
170. Collins KC, Janda KD. Investigating hapten clustering as a strategy to
enhance vaccines against drugs of abuse. Bioconjug Chem (2014) 25:593–
600. doi: 10.1021/bc500016k
171. Stevens MW, Gunnell MG, Tawney R, Owens SM. Optimization of a
methamphetamine conjugate vaccine for antibody production in mice. Int
Immunopharmacol (2016) 35:137–41. doi: 10.1016/j.intimp.2016.03.028
172. Bremer PT, Schlosburg JE, Banks ML, Steele FF, Zhou B, Poklis JL, et al.
Development of a Clinically Viable Heroin Vaccine. J Am Chem Soc (2017)
139:8601–11. doi: 10.1021/jacs.7b03334
173. Pravetoni M, Pentel PR, Potter DN, Chartoff EH, Tally L, LeSage MG. Effects
of an oxycodone conjugate vaccine on oxycodone self-administration and
oxycodone-induced brain gene expression in rats. PloS One (2014) 9:101807.
doi: 10.1371/journal.pone.0101807
174. Kosten TR, Rosen M, Bond J, Settles M, Roberts JSC, Shields J, et al. Human
therapeutic cocaine vaccine: Safety and immunogenicity. Vaccine (2002)
20:1196–204. doi: 10.1016/S0264-410X(01)00425-X
175. Martell BA, Mitchell E, Poling J, Gonsai K, Kosten TR. Vaccine
pharmacotherapy for the treatment of cocaine dependence. Biol Psychiatry
(2005) 58:158–64. doi: 10.1016/j.biopsych.2005.04.032
176. Moreno AY, Azar MR, Warren NA, Dickerson TJ, Koob GF, Janda KD.
A critical evaluation of a nicotine vaccine within a self-administration
behavioral model. Mol Pharm (2010) 7:431–41. doi: 10.1021/mp
177. Rüedi-Bettschen D, Wood SL, Gunnell MG, West CM, Pidaparthi RR,
Carroll FI, et al. Vaccination protects rats from methamphetamineinduced
impairment of behavioral responding for food. Vaccine (2013)
31:4596–602. doi: 10.1016/j.vaccine.2013.07.038
178. Cornish KE, De Villiers SHL, Pravetoni M, Pentel PR. Immunogenicity of
individual vaccine components in a bivalent nicotine vaccine differ according
to vaccine formulation and administration conditions. PloS One (2013)
8:82557. doi: 10.1371/journal.pone.0082557
179. Kosten TR, Domingo CB, Shorter D, Orson F, Green C, Somoza E, et al.
Vaccine for cocaine dependence: A randomized double-blind placebocontrolled
efficacy trial. Drug Alcohol Depend (2014) 140:42–7.
doi: 10.1016/j.drugalcdep.2014.04.003
180. Pravetoni M, Vervacke JS, Distefano MD, Tucker AM, Laudenbach M,
Pentel PR. Effect of Currently Approved Carriers and Adjuvants on the Pre-
Clinical Efficacy of a Conjugate Vaccine against Oxycodone in Mice and
Rats. PloS One (2014) 9:e96547. doi: 10.1371/journal.pone.0096547
181. McCluskie MJ, Thorn J, Gervais DP, Stead DR, Zhang N, Benoit M, et al.
Anti-nicotine vaccines: Comparison of adjuvanted CRM197 and Qb-VLP
conjugate formulations for immunogenicity and function in non-human
primates. Int Immunopharmacol (2015) 29:663–71. doi: 10.1016/
182. Schlosburg JE, Vendruscolo LF, Bremer PT, Lockner JW, Wade CL, Nunes
AAK, et al. Dynamic vaccine blocks relapse to compulsive intake of
heroin. Proc Natl Acad Sci U S A (2013) 110:9036–41. doi: 10.1073/
Brisse et al. Non-Viral Vaccine Technologies
Frontiers in Immunology | 18 September 2020 | Volume 11 | Article 583077
183. Cornuz J, Zwahlen S, Jungi WF, Osterwalder J, Klingler K, van Melle G, et al.
A vaccine against nicotine for smoking cessation: A randomizedcontrolled
trial. PloS One (2008) 3(6):e2547. doi: 10.1371/journal.pone.0002547
184. Hatsukami DK, Jorenby DE, Gonzales D, Rigotti NA, Glover ED, Oncken
CA, et al. Immunogenicity and smoking-cessation outcomes for a novel
nicotine immunotherapeutic. Clin Pharmacol Ther (2011) 89:392–9.
doi: 10.1038/clpt.2010.317
185. Fahim RE, Kessler PD, Kalnik MW. Therapeutic vaccines against tobacco
addiction. Expert Rev Vaccines (2013) 12:333–42. doi: 10.1586/erv.13.13
186. Martell BA, Orson FM, Poling J, Mitchell E, Rossen RD, Gardner T, et al.
Cocaine vaccine for the treatment of cocaine dependence in methadonemaintained
patients: A randomized, double-blind, placebo-controlled
efficacy trial. Arch Gen Psychiatry (2009) 66:1116–23. doi: 10.1001/
187. Townsend EA, Banks ML. Preclinical Evaluation of Vaccines to Treat Opioid
Use Disorders: How Close are We to a Clinically Viable Therapeutic? CNS
Drugs (2020) 34:449–61. doi: 10.1007/s40263-020-00722-8
188. Raya BA, Edward KM, Scheifele DW, Halperin SA. Pertussis and influenza
immunisation during pregnancy: a landscape review. Lancet Infect Dis
(2017) 17:209–22. doi: 10.1016/S1473-3099(17)30190-1
189. Hozbor D. New Pertussis Vaccines: A Need and aChallenge. Pertussis
Infection and Vaccines (book), New Pertussis Vaccines: A Need and a
Challenge (book section) (2019). pp. 115–26. doi: 10.1007/5584_2019_407
190. Kitchin NRE. Review of diphtheria, tetanus and pertussis vaccines in clinical
development. Expert Rev Vaccines (2011) 10:605–15. doi: 10.1586/erv.11.60
191. Angsantikul P, Fang RH, Zhang L. Toxoid Vaccination against Bacterial
Infection Using Cell Membrane-Coated Nanoparticles. Bioconjug Chem
(2018) 29:604–12. doi: 10.1021/acs.bioconjchem.7b00692
192. Li L, Goedegebuure SP, Gillanders WE. Preclinical and clinical development
of neoantigen vaccines. Ann Oncol (2017) 28:xii11–7. doi: 10.1093/annonc/
193. Aurisicchio L, Pallocca M, Ciliberto G, Palombo F. The perfect personalized
cancer therapy: Cancer vaccines against neoantigens. J Exp Clin Cancer Res
(2018) 37:86. doi: 10.1186/s13046-018-0751-1
194. Maeng HM, Berzofsky JA. Strategies for developing and optimizing
cancervaccines. F1000Research (2019) 8:654. doi: 10.12688/f1000
195. Tagliamonte M, Petrizzo A, Tornesello ML, Buonaguro FM, Buonaguro L.
Antigen-specific vaccines for cancer treatment. Hum Vaccines Immunother
(2014) 10:3332–46. doi: 10.4161/21645515.2014.973317
196. Sahin U, Türeci Ö. Personalized vaccines for cancer immunotherapy. Sci (80)
(2018) 359:1355–60. doi: 10.1126/science.aar7112
197. Lopes A, Vandermeulen G, Préat V. Cancer DNA vaccines: current
preclinical and clinical developmentsand future perspectives. J Exp Clin
Cancer Res (2019) 38:146. doi: 10.1186/s13046-019-1154-7
198. Sayour EJ, Mendez-Gomez HR, Mitchell DA. Cancer vaccine
immunotherapy with RNA-loadedliposomes. Int J Mol Sci (2018) 19:2890.
doi: 10.3390/ijms19102890
199. Granstein RD, Ding W, Ozawa H. Induction of anti-tumor immunity with
epidermal cells pulsed with tumor- derived RNA or intradermal
administration of RNA. J Invest Dermatol (2000) 114:632–6. doi: 10.1046/
200. Scheel B, Aulwurm S, Probst J, Stitz L, Hoerr I, Rammensee H-G, et al.
Therapeutic anti-tumor immunity triggered by injections of
immunostimulating single-stranded RNA. Eur J Immunol (2006) 36:2807–
16. doi: 10.1002/eji.200635910
201. Van Lint S, Goyvaerts C, Maenhout S, Goethals L, Disy A, Benteyn D, et al.
Preclinical evaluation of TriMix and antigen mRNA-based antitumor
therapy. Cancer Res (2012) 72:1661–71. doi: 10.1158/0008-5472.CAN-11-
202. Bialkowski L, Van Weijnen A, Van Der Jeught K, Renmans D, Daszkiewicz
L, Heirman C, et al. Intralymphatic mRNA vaccine induces CD8 T-cell
responses that inhibit the growth of mucosally located tumours. Sci Rep
(2016) 6:1–15. doi: 10.1038/srep22509
203. Kübler H, Scheel B, Gnad-Vogt U, Miller K, Schultze-Seemann W, vom
Dorp F, et al. Self-adjuvanted mRNA vaccination in advanced prostate
cancer patients: a first-in-man phase I/IIa study. J Immunother Cancer
(2015) 3:26. doi: 10.1186/s40425-015-0068-y
204. Audouy SAL, De Leij LFMH, Hoekstra D, Molema G. In vivo characteristics
of cationic liposomes as delivery vectors for gene therapy. Pharm Res (2002)
19:1599–605. doi: 10.1023/A:1020989709019
205. Lorenzer C, Dirin M, Winkler AM, Baumann V, Winkler J. Going beyond
the liver: Progress and challenges of targeted delivery of siRNA therapeutics.
J Control Release (2015) 203:1–15. doi: 10.1016/j.jconrel.2015.02.003
206. Hess PR, Boczkowski D, Nair SK, Snyder D, Gilboa E. Vaccination with
mRNAs encoding tumor-associated antigens and granulocyte-macrophage
colony-stimulating factor efficiently primes CTL responses, but is insufficient
to overcome tolerance to a model tumor/self antigen. Cancer Immunol
Immunother (2006) 55:672–83. doi: 10.1007/s00262-005-0064-z
207. Phua KKL, Staats HF, Leong KW, Nair SK. Intranasal mRNA nanoparticle
vaccination induces prophylactic andtherapeutic anti-tumor immunity. Sci
Rep (2014) 4:5128. doi: 10.1038/srep05128
208. Oberli MA, Reichmuth AM, Dorkin JR, Mitchell MJ, Fenton OS, Jaklenec A,
et al. Lipid Nanoparticle Assisted mRNA Delivery for Potent Cancer
Immunotherapy. Nano Lett (2017) 17:1326–35. doi: 10.1021/acs.nanolett.
209. Lu C, Stewart DJ, Lee JJ, Ji L, Ramesh R, Jayachandran G, et al. Phase I
clinical trial of systemically administeredTUSC2(FUS1)-nanoparticles
mediating functional gene transfer in humans. PloS One (2012) 7(4):
e34833. doi: 10.1371/journal.pone.0034833
210. Cho EA, Moloney FJ, Cai H, Au-Yeung A, China C, Scolyer RA, et al. Safety
and tolerability of an intratumorally injected DNAzyme, Dz13, in patients
with nodular basal-cell carcinoma: A phase 1 first-in-human trial
(DISCOVER). Lancet (2013) 381:1835–43. doi: 10.1016/S0140-6736(12)
211. Wagner MJ, Mitra R, Mcarthur MJ, Baze W, Barnhart K, Wu SY, et al.
Preclinical mammalian safety studies of EPHARNA (DOPC Nanoliposomal
EphA2-Targeted siRNA). Mol Cancer Ther (2017) 16:1114–23. doi: 10.1158/
212. Porter DL, Levine BL, Kalos M, Bagg A, June CH. Chimeric Antigen
Receptor–Modified T Cells in Chronic Lymphoid Leukemia. N Engl J Med
(2011) 365:725–33. doi: 10.1056/NEJMoa1103849
213. Stadler CR, Bähr-Mahmud H, Celik L, Hebich B, Roth AS, Roth RP, et al.
Elimination of large tumors in mice by mRNA-encoded bispecific antibodies.
Nat Med (2017) 23:815–7. doi: 10.1038/nm.4356
214. Wilgenhof S, Van Nuffel AMT, Benteyn D, Corthals J, Aerts C, Heirman C,
et al. A phase IB study on intravenous synthetic mRNA electroporated
dendritic cell immunotherapy in pretreated advanced melanoma patients.
Ann Oncol (2013) 24:2686–93. doi: 10.1093/annonc/mdt245
215. Kranz LM, Diken M, Haas H, Kreiter S, Loquai C, Reuter KC, et al. Systemic
RNA delivery to dendritic cells exploits antiviral defence for cancer
immunotherapy. Nature (2016) 534:396–401. doi: 10.1038/nature18300
216. McNamara MA, Nair SK, Holl EK. RNA-Based Vaccines in Cancer
Immunotherapy. J Immunol Res (2015) 2015:794528. doi: 10.1155/2015/
217. Sayour EJ, De Leon G, Pham C, Grippin A, Kemeny H, Chua J, et al. Systemic
activation of antigen-presenting cells via RNA-Loadednanoparticles.
Oncoimmunology (2017) 6(1):e1256527. doi: 10.1080/2162402X.
218. Wilgenhof S, Corthals J, Heirman C, Van Baren N, Lucas S, Kvistborg P, et al.
Phase II study of autologous monocyte-derived mRNA electroporated
dendritic cells (TriMixDC-MEL) plus ipilimumab in patientswith pretreated
advanced melanoma. J Clin Oncol (2016) 34:1330–8. doi: 10.1200/
219. Wang Y, Zhang L, Xu Z, Miao L, Huang L. mRNA Vaccine with Antigen-
Specific Checkpoint Blockade Induces an Enhanced Immune Response
against Established Melanoma. Mol Ther (2018) 26:420–34. doi: 10.1016/
220. Tan YS, Sansanaphongpricha K, Prince MEP, Sun D, Wolf GT, Lei
YL. Engineering Vaccines to Reprogram Immunity against Head and
Neck Cancer. J Dent Res (2018) 97:627–34. doi: 10.1177/002203451
221. Mastelic-Gavillet B, Balint K, Boudousquie C, Gannon PO, Kandalaft LE.
Personalized dendritic cell vaccines-recent breakthroughs andencouraging
clinical results. Front Immunol (2019) 10:766. doi: 10.3389/fimmu.
Brisse et al. Non-Viral Vaccine Technologies
Frontiers in Immunology | 19 September 2020 | Volume 11 | Article 583077
222. Pollard C, De Koker S, Saelens X, Vanham G, Grooten J. Challenges and
advances towards the rational design of mRNA vaccines. Trends Mol Med
(2013) 19:705–13. doi: 10.1016/j.molmed.2013.09.002
223. Becker PD, Guzmán CA. Rational design of vaccination strategies to
promote antigen entryinto the MHC class I-restricted presentation
pathway. Transfus MedHemother (2017) 77:398–411. doi: 10.1159/
224. Gatti-Mays ME, Redman JM, Collins JM, Bilusic M. Cancer vaccines: Enhanced
immunogenic modulation through therapeutic combinations. Hum Vaccines
Immunother (2017) 13:2561–74. doi: 10.1080/21645515.2017.1364322
225. Vermaelen K. Vaccine strategies to improve anticancer cellular
immuneresponses. Front Immunol (2019) 10:8. doi: 10.3389/fimmu.
226. Perez CR, De Palma M. Engineering dendritic cell vaccines to improve
cancerimmunotherapy. Nat Commun (2019) 10:5408. doi: 10.1038/s41467-
227. Sander AF, Lollini P-L. Virus-like antigen display for cancer vaccine
development, what is the potential? Expert Rev Vaccines (2018) 17:285–8.
doi: 10.1080/14760584.2018.1455505
228. Wen R, Umeano AC, Kou Y, Xu J, Farooqi AA. Nanoparticle systems for cancer
vaccine. Nanomedicine (2019) 8:627–48. doi: 10.2217/nnm-2018-0147
229. Butts C, Socinski MA, Mitchell PL, Thatcher N, Havel L, Krzakowski M, et al.
Tecemotide (L-BLP25) versus placebo after chemoradiotherapy for stage III
non-small-cell lung cancer (START): A randomised, double-blind, phase 3
trial. Lancet Oncol (2014) 15:59–68. doi: 10.1016/S1470-2045(13)70510-2
230. Rosalia RA, Cruz LJ, van Duikeren S, Tromp AT, Silva AL, Jiskoot W, et al.
CD40-targeted dendritic cell delivery of PLGA-nanoparticle vaccines induce
potent anti-tumor responses. Biomaterials (2015) 40:88–97. doi: 10.1016/
231. Schuster J, Lai RK, Recht LD, Reardon DA, Paleologos NA, Groves MD, et al.
multicenter trial of rindopepimut (CDX-110) in newly diagnosed
glioblastoma: The ACT III study. Neuro Oncol (2015) 17:854–61.
doi: 10.1093/neuonc/nou348
232. Riabov V, Tretyakova I, Alexander RB, Pushko P, Klyushnenkova EN.
Anti-tumor effect of the alphavirus-based virus-like particle vector
expressing prostate-specific antigen in a HLA-DR transgenic mouse
model of prostate cancer. Vaccine (2015) 33:5386–95. doi: 10.1016/
233. Singer C, Pfeiler G, Hubalek M, Bartsch R, Stoeger H, Pichler A, et al. Efficacy
and safety of the therapeutic cancervaccine tecemotide (L-BLP25) in early
breast cancer: Results from a prospective, randomized,neoadjuvant phase-II
study (ABCSG-34). Eur J Cancer (2020) 132:43–52. doi: doi:10.1016/
234. Varypataki EM, Benne N, Bouwstra J, Jiskoot W, Ossendorp F. Efficient
eradication of established tumors in mice with cationic liposome-based
synthetic long-peptide vaccines. Cancer Immunol Res (2017) 5:222–33.
doi: 10.1158/2326-6066.CIR-16-0283
235. Zupančič E, Curato C, Kim JS, Yeini E, Porat Z, Viana AS, et al.
Nanoparticulate vaccine inhibits tumor growth via improved T cell
recruitment into melanoma and huHER2 breast cancer. Nanomed
Nanotechnol Biol Med (2018) 14:835–47. doi: 10.1016/j.nano.2017.12.011
236. Stanley M. Tumour virus vaccines: Hepatitis B virus and humanpapillomavirus.
Philos Trans R Soc B Biol Sci (2017) 372:20160268.
doi: 10.1098/rstb.2016.0268
237. Tegerstedt K, Lindencrona JA, Curcio C, Andreasson K, Tullus C, Forni G,
et al. A single vaccination with polyomavirus VP1/VP2Her2 virus-like
particles prevents outgrowth of HER-2/neu-expressing tumors. Cancer Res
(2005) 65:5953–7. doi: 10.1158/0008-5472.CAN-05-0335
238. Tegerstedt K, Franzén A, Ramqvist T, Dalianis T. Dendritic cells loaded with
polyomavirus VP1/VP2Her2 virus-like particles efficiently prevent
outgrowth of a Her2/neu expressing tumor. Cancer Immunol Immunother
(2007) 56:1335–44. doi: 10.1007/s00262-007-0281-8
239. Andreasson K, Tegerstedt K, Eriksson M, Curcio C, Cavallo F, Forni G, et al.
Murine pneumotropic virus chimeric Her2/ neu virus-like particles as
prophylactic and therapeutic vaccines against Her2/ neu expressing
tumors. Int J Cancer (2009) 124:150–6. doi: 10.1002/ijc.23920
240. Jalali SA, Sankian M, Tavakkol-Afshari J, Jaafari MR. Induction of tumorspecific
immunity by multi-epitope rat HER2/neu-derived peptides
encapsulated in LPD Nanoparticles. Nanomed Nanotechnol Biol Med
(2012) 8:692–701. doi: 10.1016/j.nano.2011.09.010
241. Singer J, Manzano-Szalai K, Fazekas J, Thell K, Bentley-Lukschal A,
Stremnitzer C, et al. Proof of concept study with an HER-2 mimotope
anticancer vaccinededuced from a novel AAV-mimotope library platform.
Oncoimmunology (2016) 5(7):e1171446. doi: 10.1080/2162402X.
242. Shukla S,Myers JT,Woods SE, Gong X, Czapar AE, Commandeur U, et al. Plant
viral nanoparticles-based HER2 vaccine: Immune response influenced by
differential transport, localization and cellular interactions of particulate
carriers. Biomaterials (2017) 121:15–27. doi: 10.1016/j.biomaterials.2016.12.030
243. Arab A, Nicastro J, Slavcev R, Razazan A, Barati N, Nikpoor AR, et al.
Lambda phage nanoparticles displaying HER2-derived E75 peptide induce
effective E75-CD8+ T response. Immunol Res (2018) 66:200–6. doi: 10.1007/
244. Palladini A, Thrane S, Janitzek CM, Pihl J, Clemmensen SB, de Jongh WA,
et al. Virus-like particle display of HER2 induces potent anticancerresponses.
Oncoimmunology (2018) 7(3):e1408749. doi: 10.1080/
245. Wiedermann U, Wiltschke C, Jasinska J, Kundi M, Zurbriggen R, Garner-
Spitzer E, et al. A virosomal formulated Her-2/neu multi-peptide vaccine
induces Her-2/neu-specific immune responses in patients with metastatic
breast cancer: A phase i study. Breast Cancer Res Treat (2010) 119:673–83.
doi: 10.1007/s10549-009-0666-9
246. Mason NJ, Gnanandarajah JS, Engiles JB, Gray F, Laughlin D, Gaurnier-
Hausser A, et al. Immunotherapy with a HER2-Targeting listeria induces
HER2-Specific immunity and demonstrates potential therapeutic effects in a
phase I trial in canine osteosarcoma. Clin Cancer Res (2016) 22:4380–90.
doi: 10.1158/1078-0432.CCR-16-0088
247. Lizotte PH, Wen AM, Sheen MR, Fields J, Rojanasopondist P, Steinmetz NF,
et al. In situ vaccination with cowpea mosaic virus nanoparticles suppresses
metastatic cancer. Nat Nanotechnol (2016) 11:295–303. doi: 10.1038/
248. Neek M, Il KT, Wang SW. Protein-based nanoparticles in cancer vaccine
development. Nanomed Nanotechnol Biol Med (2019) 15:164–74.
doi: 10.1016/j.nano.2018.09.004
249. World Health Organization. List of Blueprint priority diseases. Available at:
priority-diseases.html. (2018).
250. Isanaka S, Guindo O, Langendorf C, Matar Seck A, Plikaytis BD, Sayinzoga-
Makombe N, et al. Efficacy of a Low-Cost, Heat-Stable Oral Rotavirus
Vaccine in Niger. N Engl J Med (2017) 376:1121–30. doi: 10.1056/
251. Fda, Cber. . Available at:
(Accessed September 1, 2020).
252. Kis Z, Shattock R, Shah N, Kontoravdi C. Emerging Technologies for Low-
Cost, Rapid VaccineManufacture. Biotechnol J (2019) 14:1–14. doi: 10.1002/
253. Berlanda Scorza F, Tsvetnitsky V, Donnelly JJ. Universal influenza vaccines:
Shifting to better vaccines. Vaccine (2016) 34:2926–33. doi: 10.1016/
254. Gilbert SC, Warimwe GM. Rapid development of vaccines against emerging
pathogens: The replication-deficient simian adenovirus platform technology.
Vaccine (2017) 35:4461–4. doi: 10.1016/J.VACCINE.2017.04.085
255. Huang Y, Diazgranados C, Janes H, Huang Y, DeCamp AC, Metch B, et al.
Selection of HIV vaccine candidates for concurrent testing in an efficacy trial.
Curr Opin Virol (2016) 17:57–65. doi: 10.1016/j.coviro.2016.01.007
256. Warimwe GM, Gesharisha J, Carr BV, Otieno S, Otingah K, Wright D, et al.
Chimpanzee adenovirus vaccine provides multispecies protectionagainst rift
valley fever. Sci Rep (2016) 6:20617. doi: 10.1038/srep20617
257. Plotkin SA. Vaccines for epidemic infections and the role of CEPI. Hum Vaccin
Immunother (2017) 13:2755–62. doi: 10.1080/21645515.2017.1306615
258. Seib K, Pollard AJ, de Wals P, Andrews RM, Zhou F, Hatchett RJ, et al. Policy
making for vaccine use as a driver of vaccine innovation and development in
the developed world. Vaccine (2017) 35:1380–9. doi: 10.1016/
259. 172 countries & multiple candidate vaccines engaged in COVID-19 Vaccine
Global Access Facility – CEPI . Available at:
Brisse et al. Non-Viral Vaccine Technologies
Frontiers in Immunology | 20 September 2020 | Volume 11 | Article 583077
facility/ (Accessed September 1, 2020).
260. Wu NC, Lv H, Thompson AJ, Paulson JC, Mok CKP, Wilson IA. Preventing
an Antigenically Disruptive Mutation in Egg-Based H3N2 Seasonal
Influenza Vaccines by Mutational Incompatibility. Cell Host Microbe
(2019) 25:836–44. doi: 10.1016/j.chom.2019.04.013
261. Graham BS, Mascola JR, Fauci AS. Novel vaccine technologies: Essential
Components of an AdequateResponse to Emerging Viral Diseases. JAMA
(2018) 319:1431. doi: 10.1001/jama.2018.0345
262. Lurie N, Saville M, Hatchett R, Halton J. Developing Covid-19 Vaccines at
Pandemic Speed. N Engl J Med (2020) 382:1969–73. doi: 10.1056/
263. Chen WH, Strych U, Hotez PJ, Bottazzi ME. The SARS-CoV-2 Vaccine
Pipeline: an Overview. Curr Trop Med Rep (2020) 7:61–4. doi: 10.1007/
264. Amanat F, Krammer F. SARS-CoV-2 Vaccines: Status Report. Immunity
(2020) 52:583–9. doi: 10.1016/j.immuni.2020.03.007
265. Jiang S. Don’t rush to deploy COVID-19 vaccines and drugs without
sufficient safety guarantees. Nature (2020) 579:321. doi: 10.1038/d41586-
266. Lv H, Wu NC, Mok CKP. COVID-19 vaccines: knowing the unknown. EurJ
Immunol (2020) 50:939–43. doi: 10.1002/eji.202048663
267. Iwasaki A, Yang Y. The potential danger of suboptimal antibody responses in
COVID-19. Nat Rev Immunol (2020) 20:339–41. doi: 10.1038/s41577-020-
268. Usher AD. COVID-19 vaccines for all? Lancet (2020) 395:1822–3.
doi: 10.1016/s0140-6736(20)31354-4
269. Yamey G, SchäferhoffM, Hatchett R, Pate M, Zhao F, McDade KK. Ensuring
global access to COVID-19 vaccines. Lancet (2020) 395:1405–6. doi: 10.1016/
270. Survival of the Most: Why Multiple Vaccines May Be the Best Way to Stop the
COVID-19 Pandemic . Available at:
pandemic (Accessed August 30, 2020).
271. Multiple vaccines could be needed to fully stop COVID-19 pandemic, UW
vaccinologist says – GeekWire . Available at:
(Accessed August 30, 2020).
272. Safety and Immunogenicity Study of 2019-nCoV Vaccine (mRNA-1273) for
Prophylaxis of SARS-CoV-2 Infection (COVID-19) – Full Text View – Available at:
NCT04283461 (Accessed July 4, 2020).
273. A Study to Evaluate the Safety, Reactogenicity and Immunogenicity of Vaccine CVnCoV
in Healthy Adults – Full Text View – . Available at: https://www. (Accessed July 4, 2020).
274. A Trial Investigating the Safety and Effects of Four BNT162 Vaccines Against
COVID-2019 in Healthy Adults – Full Text View – .
Available at: (Accessed
July 4, 2020).
275. Mulligan MJ, Lyke KE, Kitchin N, Absalon J, Gurtman A, Lockhart SP, et al.
Phase 1/2 Study to Describe the Safety and Immunogenicity of aCOVID-19
RNA Vaccine Candidate (BNT162b1) in Adults 18 to 55 Years of Age:
Interim Report. medRxiv (2020) . doi: 10.1101/2020.06.30.20142570
276. Walsh EE, Frenck R, Falsey AR, Kitchin N, Absalon J, Gurtman A, et al. RNABased
COVID-19 Vaccine BNT162b2 Selected for a Pivotal Efficacy Study.
medRxiv Prepr Serv Heal Sci (2020). doi: 10.1101/2020.08.17.20176651.
277. Mulligan MJ, Lyke KE, Kitchin N, Absalon J, Gurtman A, Lockhart S, et al.
Phase 1/2 study of COVID-19 RNA vaccine BNT162b1 in adults. Nature
(2020), 1–8. doi: 10.1038/s41586-020-2639-4
278. ISRCTN. ISRCTN17072692: Clinical trial to assess the safety of a coronavirus
vaccine in healthy men and women . Available at:
ISRCTN17072692 (Accessed July 4, 2020).
279. Ascending Dose Study of Investigational SARS-CoV-2 Vaccine ARCT-021 in Healthy
Adult Subjects – Full Text View – . Available at: https:// (Accessed September 6, 2020).
280. A Phase I clinical trial to evaluate the safety, tolerance and preliminary
immunogenicity of different doses of a SARS-CoV-2 mRNA vaccine in population
aged 18-59 years and 60 years and above . Available at:
showprojen.aspx?proj=55524 (Accessed September 6, 2020).
281. Safety, Tolerability and Immunogenicity of INO-4800 for COVID-19 in
Healthy Volunteers – Full Text View – . Available at: (Accessed July 4, 2020).
282. INOVIO. Reports Positive Interim Phase I Data for COVID-19 DNA Vaccine,
Joins “Warp Speed” Primate Study . Available at: https://www.genengnews.
GEN+Daily+News+Highlights_20200701 (Accessed July 4, 2020).
283. Patel A, Walters J, Reuschel EL, Schultheis K, Parzych E, Gary EN, et al.
Intradermal-delivered DNA vaccine provides anamnestic protection in a
rhesus macaque SARS-CoV-2 challenge model. bioRxiv (2020). doi: 10.1101/
2020.07.28.225649. 2020.07.28.225649.
284. Safety and Immunogenicity Study of GX-19, a COVID-19 Preventive DNA
Vaccine in Healthy Adults – Full Text View – . Available at: (Accessed July 4, 2020).
285. Study of COVID-19 DNA Vaccine (AG0301-COVID19) – Full Text View – Available at:
NCT04463472 (Accessed September 6, 2020).
286. Study of COVID-19 DNA Vaccine (AG0302-COVID19) – Full Text View – Available at:
NCT04527081 (Accessed September 6, 2020).
287. A prospective, randomized, adaptive, phase I/II clinical study to evaluate the
safety andimmunogenicity of Novel Corona Virus -2019-nCov vaccine
candidate of M/s Cadila Healthcare Limitedby intradermal route in healthy
subjects. Available at:
288. Evaluating the Safety, Tolerability and Immunogenicity of bacTRL-Spike
Vaccine for Prevention of COVID-19 – Full Text View –
Available at: (Accessed
July 4, 2020).
289. Immunity and Safety of Covid-19 Synthetic Minigene Vaccine – Full Text
View – Available at:
NCT04276896 (Accessed July 4, 2020).
290. Safety and Immunity of Covid-19 aAPC Vaccine – Full Text View – Available at:
NCT04299724 (Accessed July 4, 2020).
291. Phase Ib-II Trial of Dendritic Cell Vaccine to Prevent COVID-19 in Adults –
Full Text View – . Available at:
show/NCT04386252 (Accessed July 4, 2020).
292. Evaluation of the Safety and Immunogenicity of a SARS-CoV-2 rS (COVID-
19) Nanoparticle Vaccine With/Without Matrix-M Adjuvant – Full Text View
– Available at:
NCT04368988?term=novavax&recrs=ab&draw=2&rank=1 (Accessed July
4, 2020).
293. SCB-2019 as COVID-19 Vaccine – Full Text View – .
Available at: (Accessed
July 4, 2020).
294. Therapeutic Vaccine Trial of COVID-19 for Severe Acute Respiratory
Syndrome Coronavirus 2 (SARS-CoV-2) Infection – Full Text View – Available at:
NCT04428073 (Accessed July 4, 2020).
295. A Phase I, Prospective, Open-Labeled Study to Evaluate the Safety and
Immunogenicity of MVC-COV1901 – Full Text View – .
Available at: (Accessed
September 6, 2020).
296. A Study to Evaluate the Safety and Immunogenicity of COVID-19 (AdimrSC-
2f) Vaccine – Full Text View – Available at: https:// (Accessed September 6, 2020).
297. An interventional study to evaluate the safety and immune response of a
vaccine against Severe Acute Respiratory Syndrome coronavirus 2 (SARSCoV-
2, the virus that causes COVID-19 infection) when given to healthy adult
participants . Available at:
trial/ACTRN12620000674932 (Accessed September 6, 2020).
298. A Study on the Safety, Tolerability and Immune Response of SARS-CoV-2
Sclamp (COVID-19) Vaccine in Healthy Adults – Full Text View –
Brisse et al. Non-Viral Vaccine Technologies
Frontiers in Immunology | 21 September 2020 | Volume 11 | Article 583077 Available at:
NCT04495933 (Accessed September 6, 2020).
299. Safety, Tolerability and Immunogenicinity of a Coronavirus-Like Particle
COVID-19 Vaccine in Adults Aged 18-55 Years. – Full Text View – Available at:
NCT04450004 (Accessed September 6, 2020).
300. Folegatti PM, Ewer KJ, Aley PK, Angus B, Becker S, Belij-Rammerstorfer S,
et al. Safety and immunogenicity of the ChAdOx1 nCoV-19 vaccine against
SARS-CoV-2: a preliminary report of a phase 1/2, single-blind, randomised
controlled trial. Lancet (2020) 396:467–78. doi: 10.1016/S0140-6736(20)
301. van Doremalen N, Lambe T, Spencer A, Belij-Rammerstorfer S,
Purushotham JN, Port JR, et al. ChAdOx1 nCoV-19 vaccine prevents
SARS-CoV-2 pneumonia in rhesus macaques. Nature (2020) 1–8.
doi: 10.1038/s41586-020-2608-y
302. Zhu FC, Li YH, Guan XH, Hou LH, Wang WJ, Li JX, et al. Safety, tolerability,
and immunogenicity of a recombinant adenovirustype-5 vectored COVID-
19 vaccine: a dose-escalation, open-label, non-randomised, first-inhumantrial.
Lancet (2020) 395:1845–54. doi: 10.1016/S0140-6736(20)
303. Zhu FC, Guan XH, Li YH, Huang JY, Jiang T, Hou LH, et al.
Immunogenicity and safety of a recombinant adenovirus type-5-vectored
COVID-19 vaccine in healthy adults aged 18 years or older: a randomised,
double-blind, placebo-controlled, phase 2 trial. Lancet (2020) 396:479–88.
doi: 10.1016/S0140-6736(20)31605-6
304. Mahase E. Covid-19: Where are we on immunity and vaccines? BMJ (2020)
370:m3096. doi: 10.1136/bmj.m3096
305. Mercado NB, Zahn R, Wegmann F, Loos C, Chandrashekar A, Yu J, et al.
Single-shot Ad26 vaccine protects against SARS-CoV-2 in rhesus macaques.
Nature (2020), 1–11. doi: 10.1038/s41586-020-2607-z
306. Mahase E. Covid-19: Russia approves vaccine without large scale testing or
published results. BMJ (2020) 370:m3205. doi: 10.1136/bmj.m3205
307. Clinical Trial to Evaluate the Safety and Immunogenicitiy of the COVID-19
Vaccine – Full Text View – Available at: https:// (Accessed September 6, 2020).
308. A Study to Assess Safety, Tolerability, and Immunogenicity of V591 (COVID-
19 Vaccine) in Healthy Participants (V591-001) – Full Text View – Available at:
NCT04498247 (Accessed [Accessed September 6, 2020]).
Conflict of Interest: The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a
potential conflict of interest.
Copyright © 2020 Brisse, Vrba, Kirk, Liang and Ly. This is an open-access article
distributed under the terms of the Creative Commons Attribution License (CC BY).
The use, distribution or reproduction in other forums is permitted, provided the
original author(s) and the copyright owner(s) are credited and that the original
publication in this journal is cited, in accordance with accepted academic practice. No
use, distribution or reproduction is permitted which does not comply with these terms.
Brisse et al. Non-Viral Vaccine Technologies
Frontiers in Immunology | 22 September 2020 | Volume 11 | Article 583077

Get Professional Assignment Help Cheaply

Buy Custom Essay

Are you busy and do not have time to handle your assignment? Are you scared that your paper will not make the grade? Do you have responsibilities that may hinder you from turning in your assignment on time? Are you tired and can barely handle your assignment? Are your grades inconsistent?

Whichever your reason is, it is valid! You can get professional academic help from our service at affordable rates. We have a team of professional academic writers who can handle all your assignments.

Why Choose Our Academic Writing Service?

  • Plagiarism free papers
  • Timely delivery
  • Any deadline
  • Skilled, Experienced Native English Writers
  • Subject-relevant academic writer
  • Adherence to paper instructions
  • Ability to tackle bulk assignments
  • Reasonable prices
  • 24/7 Customer Support
  • Get superb grades consistently

Online Academic Help With Different Subjects


Students barely have time to read. We got you! Have your literature essay or book review written without having the hassle of reading the book. You can get your literature paper custom-written for you by our literature specialists.


Do you struggle with finance? No need to torture yourself if finance is not your cup of tea. You can order your finance paper from our academic writing service and get 100% original work from competent finance experts.

Computer science

Computer science is a tough subject. Fortunately, our computer science experts are up to the match. No need to stress and have sleepless nights. Our academic writers will tackle all your computer science assignments and deliver them on time. Let us handle all your python, java, ruby, JavaScript, php , C+ assignments!


While psychology may be an interesting subject, you may lack sufficient time to handle your assignments. Don’t despair; by using our academic writing service, you can be assured of perfect grades. Moreover, your grades will be consistent.


Engineering is quite a demanding subject. Students face a lot of pressure and barely have enough time to do what they love to do. Our academic writing service got you covered! Our engineering specialists follow the paper instructions and ensure timely delivery of the paper.


In the nursing course, you may have difficulties with literature reviews, annotated bibliographies, critical essays, and other assignments. Our nursing assignment writers will offer you professional nursing paper help at low prices.


Truth be told, sociology papers can be quite exhausting. Our academic writing service relieves you of fatigue, pressure, and stress. You can relax and have peace of mind as our academic writers handle your sociology assignment.


We take pride in having some of the best business writers in the industry. Our business writers have a lot of experience in the field. They are reliable, and you can be assured of a high-grade paper. They are able to handle business papers of any subject, length, deadline, and difficulty!


We boast of having some of the most experienced statistics experts in the industry. Our statistics experts have diverse skills, expertise, and knowledge to handle any kind of assignment. They have access to all kinds of software to get your assignment done.


Writing a law essay may prove to be an insurmountable obstacle, especially when you need to know the peculiarities of the legislative framework. Take advantage of our top-notch law specialists and get superb grades and 100% satisfaction.

What discipline/subjects do you deal in?

We have highlighted some of the most popular subjects we handle above. Those are just a tip of the iceberg. We deal in all academic disciplines since our writers are as diverse. They have been drawn from across all disciplines, and orders are assigned to those writers believed to be the best in the field. In a nutshell, there is no task we cannot handle; all you need to do is place your order with us. As long as your instructions are clear, just trust we shall deliver irrespective of the discipline.

Are your writers competent enough to handle my paper?

Our essay writers are graduates with bachelor's, masters, Ph.D., and doctorate degrees in various subjects. The minimum requirement to be an essay writer with our essay writing service is to have a college degree. All our academic writers have a minimum of two years of academic writing. We have a stringent recruitment process to ensure that we get only the most competent essay writers in the industry. We also ensure that the writers are handsomely compensated for their value. The majority of our writers are native English speakers. As such, the fluency of language and grammar is impeccable.

What if I don’t like the paper?

There is a very low likelihood that you won’t like the paper.

Reasons being:

  • When assigning your order, we match the paper’s discipline with the writer’s field/specialization. Since all our writers are graduates, we match the paper’s subject with the field the writer studied. For instance, if it’s a nursing paper, only a nursing graduate and writer will handle it. Furthermore, all our writers have academic writing experience and top-notch research skills.
  • We have a quality assurance that reviews the paper before it gets to you. As such, we ensure that you get a paper that meets the required standard and will most definitely make the grade.

In the event that you don’t like your paper:

  • The writer will revise the paper up to your pleasing. You have unlimited revisions. You simply need to highlight what specifically you don’t like about the paper, and the writer will make the amendments. The paper will be revised until you are satisfied. Revisions are free of charge
  • We will have a different writer write the paper from scratch.
  • Last resort, if the above does not work, we will refund your money.

Will the professor find out I didn’t write the paper myself?

Not at all. All papers are written from scratch. There is no way your tutor or instructor will realize that you did not write the paper yourself. In fact, we recommend using our assignment help services for consistent results.

What if the paper is plagiarized?

We check all papers for plagiarism before we submit them. We use powerful plagiarism checking software such as SafeAssign, LopesWrite, and Turnitin. We also upload the plagiarism report so that you can review it. We understand that plagiarism is academic suicide. We would not take the risk of submitting plagiarized work and jeopardize your academic journey. Furthermore, we do not sell or use prewritten papers, and each paper is written from scratch.

When will I get my paper?

You determine when you get the paper by setting the deadline when placing the order. All papers are delivered within the deadline. We are well aware that we operate in a time-sensitive industry. As such, we have laid out strategies to ensure that the client receives the paper on time and they never miss the deadline. We understand that papers that are submitted late have some points deducted. We do not want you to miss any points due to late submission. We work on beating deadlines by huge margins in order to ensure that you have ample time to review the paper before you submit it.

Will anyone find out that I used your services?

We have a privacy and confidentiality policy that guides our work. We NEVER share any customer information with third parties. Noone will ever know that you used our assignment help services. It’s only between you and us. We are bound by our policies to protect the customer’s identity and information. All your information, such as your names, phone number, email, order information, and so on, are protected. We have robust security systems that ensure that your data is protected. Hacking our systems is close to impossible, and it has never happened.

How our Assignment Help Service Works

1. Place an order

You fill all the paper instructions in the order form. Make sure you include all the helpful materials so that our academic writers can deliver the perfect paper. It will also help to eliminate unnecessary revisions.

2. Pay for the order

Proceed to pay for the paper so that it can be assigned to one of our expert academic writers. The paper subject is matched with the writer’s area of specialization.

3. Track the progress

You communicate with the writer and know about the progress of the paper. The client can ask the writer for drafts of the paper. The client can upload extra material and include additional instructions from the lecturer. Receive a paper.

4. Download the paper

The paper is sent to your email and uploaded to your personal account. You also get a plagiarism report attached to your paper.

smile and order essay GET A PERFECT SCORE!!! smile and order essay Buy Custom Essay

Place your order
(550 words)

Approximate price: $22

Calculate the price of your order

550 words
We'll send you the first draft for approval by September 11, 2018 at 10:52 AM
Total price:
The price is based on these factors:
Academic level
Number of pages
Basic features
  • Free title page and bibliography
  • Unlimited revisions
  • Plagiarism-free guarantee
  • Money-back guarantee
  • 24/7 support
On-demand options
  • Writer’s samples
  • Part-by-part delivery
  • Overnight delivery
  • Copies of used sources
  • Expert Proofreading
Paper format
  • 275 words per page
  • 12 pt Arial/Times New Roman
  • Double line spacing
  • Any citation style (APA, MLA, Chicago/Turabian, Harvard)

Our guarantees

Delivering a high-quality product at a reasonable price is not enough anymore.
That’s why we have developed 5 beneficial guarantees that will make your experience with our service enjoyable, easy, and safe.

Money-back guarantee

You have to be 100% sure of the quality of your product to give a money-back guarantee. This describes us perfectly. Make sure that this guarantee is totally transparent.

Read more

Zero-plagiarism guarantee

Each paper is composed from scratch, according to your instructions. It is then checked by our plagiarism-detection software. There is no gap where plagiarism could squeeze in.

Read more

Free-revision policy

Thanks to our free revisions, there is no way for you to be unsatisfied. We will work on your paper until you are completely happy with the result.

Read more

Privacy policy

Your email is safe, as we store it according to international data protection rules. Your bank details are secure, as we use only reliable payment systems.

Read more

Fair-cooperation guarantee

By sending us your money, you buy the service we provide. Check out our terms and conditions if you prefer business talks to be laid out in official language.

Read more
error: Content is protected !!
Open chat
Need assignment help? You can contact our live agent via WhatsApp using +1 718 717 2861

Feel free to ask questions, clarifications, or discounts available when placing an order.
  +1 718 717 2861           + 44 161 818 7126           [email protected]
  +1 718 717 2861         [email protected]