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Technological Approaches To Streamline Vaccination Schedules, Progressing Towards Single-Dose Vaccines
Technological Approaches To Streamline Vaccination Schedules, Progressing Towards Single-Dose Vaccines
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Vaccines represent the most successful medical intervention in history, with billions of lives saved. Although multiple doses of the
same vaccine are typically required to reach an adequate level of protection, it would be advantageous to develop vaccines that
induce protective immunity with fewer doses, ideally just one. Single-dose vaccines would be ideal to maximize vaccination
coverage, help stakeholders to greatly reduce the costs associated with vaccination, and improve patient convenience. Here we
describe past attempts to develop potent single dose vaccines and explore the reasons they failed. Then, we review key
immunological mechanisms of the vaccine-specific immune responses, and how innovative technologies and approaches are
guiding the preclinical and clinical development of potent single-dose vaccines. By modulating the spatio-temporal delivery of the
vaccine components, by providing the appropriate stimuli to the innate immunity, and by designing better antigens, the new
technologies and approaches leverage our current knowledge of the immune system and may synergize to enable the rational
design of next-generation vaccination strategies. This review provides a rational perspective on the possible development of future
single-dose vaccines.
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GSK, Slaoui Center for Vaccines Research, Rockville, MD 20850, USA. ✉email: [email protected]
1
modulate those mechanisms are reviewed. into technological innovation for a rational design of potent
single-dose vaccines. This approach so far has shown to be
problematic, mostly because of the technical challenges that
VALUE OF SINGLE-DOSE VACCINES vaccinologists face when trying to elicit strong immune responses
In general, the design of single-administration vaccines aims to with few vaccine doses, all of which is discussed in the next
achieve three major objectives: (1) to improve the likelihood of sections.
worldwide coverage of vaccination, (2) to decrease the costs
associated with multi-dose regimens, and (3) improve patient
convenience. Incomplete immunization coverage is a major cause CLINICAL EVIDENCE FOR OPTIMIZED VACCINATION
of preventable illness and death in both high-income and low- SCHEDULES
income countries (3,6). A study on vaccination coverage among Vaccination schedules are typically based on series of 3–4 doses
US children under 2 years old has reported that only 30% receive within 4–6 months, with some vaccines requiring a booster dose
all the six recommended vaccinations on time, indicating that too through lifetime. Multi-dose series are used for at least two
many children do not receive vaccines at age-appropriate times or reasons: (1) to drive desired immune response in an individual to
never complete the recommended vaccine schedules3,4. Several an adequate level of protection and (2) equally if not more
socioeconomic factors have been associated with low country important from a public health perspective, to increase the
vaccination coverage including government health spending, percentage of the vaccinated population that are no longer
difficulty to deliver the vaccines and educational variables4,7,8, susceptible and achieve an “herd effect”. When trying to optimize
indicating that additional country-specific policy, educational, and the vaccination schedules, it is often difficult to determine
clinical interventions—not only technological innovation—are whether fewer doses of the same vaccine would still confer
required to facilitate vaccine uptake. The complexities in achieving protection, because for most vaccines the immune correlates are
global vaccination coverage and immunization equity are many not well defined, making it difficult to predict the level of
and have been discussed elsewhere9–11. Clearly, reducing the protection that would be reached with a vaccine schedule that is
number of vaccine injections may maximize vaccination coverage different from the one assessed during the clinical trials. None-
and improve vaccine effectiveness as ease of vaccine uptake may theless, post-marketing epidemiology studies, meta-analysis and
sometimes help to compensate for vaccines with less-than-ideal ad-hoc clinical trials comparing different schedules side-by-side,
efficacy12,13, especially for those pathogens for which a highly have provided important evidence that certain vaccines can be
effective vaccine is difficult to design such as the influenza protective after fewer doses and some regulatory authorities have
virus14,15. Single-administration vaccines might also help to updated their recommended vaccine schedules accordingly. Here
decrease the costs associated with multi-dose regimens and are a few examples. The schedule of the AS04-adjuvanted HPV
improve patient convenience, as fewer injections and healthcare vaccine (Cervarix from GSK), initially approved with a three-dose
visits will be needed to provide immunity, especially in certain schedule, was updated with a two-dose schedule following several
target populations that require quick and protective immune post-marketing studies in 9–25-year-old women in which the level
responses, including travelers, patients expecting imminent of safety and immunogenicity was observed to be similar between
surgery, and during outbreaks. the two vaccine regimens23–26. The adjuvant AS04, which has a
Historically, a great approach to simplify immunization sche- composition of aluminum hydroxide and 3-O-desacyl4′-monopho-
dules and improve vaccine coverage has relied on the adoption of sphoryl lipid A (MPL-A), is believed to be responsible for the high
combination vaccines. Two of the most used combination level of vaccine-induced antibody titers, a surrogate of protection
vaccines are the Diphtheriae, Tetanus and acellular Pertussis for HPV, and persist for several years in vaccinated women, even
(DTaP), a subunit vaccine, and the Measles, Mumps and Rubella after a single vaccine dose24–27; Cervarix is currently recom-
(MMR) a live attenuated vaccine. Although combination vaccines mended with a two-dose schedule as well as another HPV vaccine,
have helped a lot in improving vaccine coverage and extending Gardasil (Merck), which contains aluminum hydroxy phosphate
efficacy against the pathogens included in the vaccines16, not all sulfate as an adjuvant. Another example of an updated
npj Vaccines (2020) 88 Published in partnership with the Sealy Center for Vaccine Development
G. Lofano et al.
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vaccination schedule is Prevnar 13 (Pfizer), an aluminum- demonstrating that controlled antigen release can provide a
adjuvanted pneumococcal vaccine, for which it has been shown successful approach in small animal models.
that for 9 of the 13 serotypes included in the vaccine, the post- Although preclinical testing provided promising results for the
booster responses after a single priming are equivalent or superior use of PLG nanoparticles, technical limitations in the manufactur-
to those seen following the standard 2 priming + 1 boost ability of such vaccines arose. Unfortunately, the process to
schedule, indicating that a schedule with fewer doses still confers prepare PLG particles typically damaged the stability of the
protection28. Another recent example is represented by Heplisav- antigen and impaired its immunogenicity37,38. In most groups, PLG
B, a CpG ODN-adjuvanted vaccine against Hepatitis B virus, which microparticles were prepared through a process of antigen
is administered with a two-dose schedule and shows similar safety encapsulation that involved the use of organic solvents, agitation,
and immunogenicity profile to three doses of other HBV and the creation of interfaces. Moreover, upon degradation
vaccines29. A key commonality among all those vaccines is that in vivo, the polymer created a low pH environment, all of which
they use well-optimized adjuvanted formulation, all of which led to protein unfolding and aggregation, with subsequent loss of
highlights the importance of using vaccine adjuvants not only to functional epitopes and antigenicity39–42. Furthermore, clinical
modulate the vaccine-specific immune responses, but also to manufacturing of vaccines requires the product to be either
reduce the number of doses required to reach protection with produced under aseptic conditions or terminally sterilized, all of
subunit-based vaccines. Interestingly, Cervarix, Gardasil, and which may endanger antigen stability in PLG nanoparticles.
Heplisav-B also present a nanoparticle-based antigen structure, Several research groups in the past decades have tried to address
suggesting that a combination of multicopy antigen display and these manufacturability issues and have made many attempts to
immune adjuvant formulations may provide key strategies for the stabilize entrapped antigens, with limited success. As an
rational design of streamlined vaccination schedules. alternative approach, Singh et al. successfully developed a way
Several decades ago, when vaccinologists began to dissect the to obviate the need for encapsulation, by preparing the PLG
mechanism of action of the first successful vaccines, it became particles separately and then adsorbing the antigen on the
clear that the development of new technologies and approaches particles; while the surface adsorption of antigen does not allow
would have been essential to design vaccination strategies with for controlled release of antigen, such an approach does take
fewer injections. Since then, extensive research has been advantage of the adjuvant properties of particulate antigen
generated, new technologies and approaches have been delivery43–45. In another study, Malyala et al. developed a two-
explored, but no potent single-dose vaccine has yet reached the stage process in which PLG microparticles were first sterilized by
clinic. In the next sections we discuss major historical accomplish- γ-irradiation, avoiding the need for aseptic manufacturing, and
ments and challenges for vaccinologists, and we summarize the then incubated with reconstituted, sterile antigens to allow
new technologies that are enabling the preclinical development of surface adsorption; the adsorbed PLG vaccines induced potent
single-dose vaccines. immune responses46. Most recently, Tzeng et al. showed a process
for PLG encapsulation of an inactivated polio vaccine that
involved the use of excipients to stabilize the formalin-fixed
HISTORY OF ATTEMPTS TO DEVELOP SINGLE-DOSE VACCINES antigens and helped to preserve its stability47. Nevertheless,
The concept of single-dose vaccines dates back nearly 40 years although this is an important advance which could contribute to
and received most attention with the WHO Special Program for the eventual eradication of polio, it should be appreciated that the
Vaccine Development initiated in the 1980s30. The WHO initiative complex formulation challenges required to stabilize a multi-
focused on the development of single-dose vaccines for the low- component combination vaccine in PLG microparticles still
income countries and a key strategy involved the use of controlled represent a potentially insurmountable problem. However, biode-
release technologies for a slow release of vaccine antigens, with gradable PLG-based vaccines remain attractive and if the
the scope of mimicking multi-dose regimes or natural infections. technology can be combined with other new approaches for
The primary target to employ the new release technologies was the development of potent and efficient single-dose vaccines,
neonatal tetanus, at that time a major threat for public health promise remains.
because of the hurdles to reach full compliance with the multi-
dose vaccine schedule required to confer protection30,31. The
WHO initiative led to the evaluation of biodegradable polymer- BETTER ANTIGEN DESIGN
based microparticles for the delivery of antigens including tetanus The design of optimal vaccination strategies begins with the
toxoid (TT), diphtheria toxoid, hepatitis B antigen and HIV selection of the right antigen. In the past, vaccine antigens were
envelope glycoprotein gp12031. typically selected through an empirical approach in which the
In the late 1970s Langer et al. first showed that a single pathogen (or a component of it) was isolated, inactivated and
vaccination with a polymer-based vaccine formulation could injected to induce protective immunity; many vaccines in use
induce long-lasting antibody titers in animal models32,33. The today have been developed with this empirical approach. In the
polymer was bio-erodible and allowed for slow and sustained last two decades a new and rational approach, named reverse
antigen release, whilst also providing an adjuvant effect. Subse- vaccinology48, has enabled the development of new vaccines for
quently, extensive research has focused on the development of which an empirical approach had not been successful, including a
single-dose vaccines, mostly using the established biodegradable licensed vaccine against serogroup B meningococcus and many
polymer, poly(lactide-co-glycolide) (PLG) nanoparticle technolo- others are in clinical development48,49. By combining the most
gies for controlled antigen delivery. O’Hagan et al. showed long- advanced technologies in genome sequencing, proteomics and
term vaccine-specific antibody responses in mice following bioinformatics, the reverse vaccinology approach has provided a
subcutaneous immunization with ovalbumin entrapped in PLG- framework to select the right vaccine antigens starting from the
based biodegradable nanoparticles34. In another study Men et al.35 pathogen’s genome. Reverse vaccinology has pioneered new
prepared nanoparticles with entrapped TT that induced high TT- ways of thinking about vaccine development, including a new
specific antibody and T cell responses in mice following a single structural vaccinology approach, and represents a milestone in the
immunization, with TT-specific IgG titers similar to those observed history of vaccinology50,51.
after three injections of TT adsorbed to alum35. Many other groups Structure-based antigen design, also known as structural
reported similar findings with antigens, such as diphtheria toxoid, vaccinology, is today a key driving force in vaccine innova-
hepatitis B antigen, and HIV envelope glycoprotein gp120 tion48,49,52. It relies on bioinformatics and computational tools to
(reviewed by Siddhartha, J. et al. and O’Hagan, D.T. et al.33,36) design optimized protein antigens for an efficient display of
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G. Lofano et al.
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protective epitopes to B cells. The structural vaccinology approach vaccines; other adjuvants, including MF59, AS03, and QS-21,
has been used in at least three new vaccine design strategies. One activate tissue inflammation pathways with mechanisms that
strategy aims to stabilize proteins or polypeptides in a protective involve DAMPs71,72. We refer to other reviews for a comprehensive
antibody-inducing conformation, typically a conformation that is description of the mechanisms of action of adjuvants, but we
not very stable by itself, but it can be stabilized by modifying the would like to highlight that a key advantage offered by vaccine
sequence of the protein; this approach has led to the develop- adjuvants is that they can be used not only to increase the
ment of an RSV candidate vaccine and is helping in the quantity, but also to modulate the quality of the vaccine-specific
development of the first universal influenza vaccine and HIV immune response. For example, it is well known that the oil-in-
vaccine53–55. A second approach aims to merge multiple variants water emulsions MF59 and AS03 may help to expand the antigen-
of the same protective epitope into a single chimeric protein or specific antibody repertoire, whereas, for example, AS01 adjuvant
polypeptide chain; such approach has been successful to provide may help to stimulate both humoral and cell-mediated immu-
vaccine candidates including Factor H binding protein (FHbp) nity73–78. Although there are limited data on the immune
from serogroup B meningococcus and the backbone protein of responses measured after just a single dose of adjuvanted
pilus from serogroup B streptococcus52,56. A third approach aims vaccines, it was shown that a single dose of an MF59-
to design self-assembling proteins (nanoparticles or virus-like adjuvanted H5N1 vaccine was enough to induce a 3-fold increase
particles) that display multiple copies of protective epitopes, in the frequency of virus-specific total CD4+ T cells which
which is a very efficient way to induce potent B cell responses57–59. accurately predicted the rise of neutralizing antibodies against
Nanoparticles have been successfully used to develop HBV and pandemic influenza, supporting the use of MF59 adjuvant in
HPV vaccines and can be used as scaffolds to design highly single-dose pre-pandemic influenza vaccines79. Adjuvants help to
immunogenic multivariant antigens. For example, in the works modulate the vaccine-specific immune response toward a desired
from Yassine et al. and Kanekiyo et al., ferritin, a self-assembling immunological phenotype or a correlate of protection, and
protein nanoparticle with robust thermal and chemical stabilities, represent an outstanding tool for the designing of more potent
has been used as a scaffold to present a multivalent array of single-dose vaccines73,80,81.
influenza virus hemagglutinin (HA) with its native trimeric Viral vector-based vaccines mimic viral infections, display a
conformation intact; this structure-based design strategy has led constellation of immune evasion mechanisms, and activate PRRs
to the development of a potent and broad-coverage influenza that ultimately lead to potent immune activation82–98. As an
vaccine that is now being tested in a phase I clinical trial60–63. In example, most adenoviruses enter cells upon binding to the
another study from Marcandalli et al. a single-immunization with a coxsackievirus and adenovirus receptor (CAR)99, and the viral
nanoparticle-based RSV vaccine outperformed the non- double-stranded DNA is sensed by TLRs in the endosome and by
nanoparticle formulation in inducing T follicular helper (Tfh) cells NOD-like receptors in the cytoplasm leading to a pro-
and germinal center (GC) B cells64, highlighting the value of inflammatory cytokine response50,52. However, in some cases
adopting a strategy of multi-copy antigen display in the designing innate immunity is less important than antigen persistence in
of next-generation vaccines. More recently, a study by Tokatlian driving potent vector-induced immunity, as shown in the work of
et al. has shown that nanoparticles induce potent GC responses in Quinn et al. in which adenoviral vaccine potency was independent
an immunogen glycan-dependent manner, suggesting that heavy of IFN and STING signaling100. Not only viral vectors, but also DNA
glycosylated nanoparticles may have an advantage over the less and RNA vaccines can be sensed by PRRs on innate immune cells
glycosylated ones in promoting strong B cells responses65. Overall, and bring an intrinsic adjuvant effect that contributes to the
a wide body of evidence indicates that nanoparticle-based potent immune responses observed with these vaccines101–103.
antigens are more immunogenic than monovalent recombinant
antigens. Nonetheless, it is unlikely that a single dose of a
nanoparticle antigen would be adequate to induce robust and MODULATING THE RELEASE KINETICS OF THE VACCINE
long-lasting immune responses in a clinical setting, especially in COMPONENTS
naïve subjects; however, a combination of all the strategies Two observations led scientists to speculate that antigen
presented below may succeed66–68. persistence after vaccination is an important factor for potent
vaccine-specific immune responses. One observation came from
studies on viral infections or live attenuated vaccines in which viral
STIMULATING THE INNATE IMMUNITY replication makes the antigen available for extended time and this
Potent and long-lasting vaccine-specific adaptive immune is typically associated with strong antibody responses104–108. A
responses occur only after adequate stimulation of the innate second observation came from studies on the mechanisms of
immunity. In the past century, during the early studies on the action of alum, the most widely deployed vaccine adjuvant, which
mechanisms of action of live attenuated vaccines, it became clear was initially thought to deliver a slow release of the antigen, a
that a key aspect of the robust immunogenicity of these vaccines mechanism called “depot” effect71,109–111. Although more recent
was their ability to stimulate innate immune cells through pattern studies have challenged this paradigm for alum mechanism of
recognition receptors (PRRs). PRRs are a series of germline- action, today it is widely accepted that prolonged antigen
encoded host proteins, mostly expressed by innate immune cells, exposure leads to strong vaccine-specific immune
that may recognize pathogen-associated molecular patterns responses82,112–117. Many recent works have provided further
(PAMPs), which are molecules associated with microbial patho- evidence: antigen exposure in draining lymph nodes over periods
gens, or damage-associated molecular patterns (DAMPs), which of at least several days induces an optimal cytokine profile118,
are molecules associated with components of host’s cells that are enhances the differentiation of Tfh cells119–121, stimulates potent
released during cell damage or death. In the past 30 years, there germinal center responses and improves the quality of the
has been a revolution in our understanding of the cells, receptors, antibodies114,120,122–124. For example, a recent study from Cirelli
and molecules that contribute to innate immunity and in the ways et al., in which an HIV antigen was delivered with osmotic pumps
that the innate response directs the subsequent adaptive immune over up to 4 weeks under the skin of non-human primates, shows
responses69,70, all of which has led to design immunological that persistent antigen availability induces improved HIV-specific
adjuvants to include in subunit vaccines and induce potent GC responses and antibody quality, probably with a mechanism
antigen-specific immune responses. Most adjuvants directly target that involves enhanced immune complex deposition on follicular
PRRs, including TLR-agonists such as monophosphoryl lipid A dendritic cells114,124. All of this provides a better understanding of
(MPL-A), a component of AS04-adjuvanted and AS01-adjuvanted the immunological mechanisms after vaccination and raises key
npj Vaccines (2020) 88 Published in partnership with the Sealy Center for Vaccine Development
G. Lofano et al.
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questions on how to translate preclinical concepts into clinical array of solid pyramidal or cylindrical projections of few microns in
applications. size and are designed to mechanically perforate the stratum
Multiple materials and technologies, each coming with specific corneum to enter the epidermis and/or upper dermis upon
biophysical properties and antigen release kinetics, are currently application to the skin. Microneedles can be coated with dried
being explored in preclinical settings to enable a programmed vaccine formulations, or the microneedles themselves can
antigen release after a single vaccination. How long antigen comprise dissolving polymers that release the vaccine with
persistence should be, and if there is an optimal release kinetic, programmable kinetics. Vaccine administration via microneedles
are still unclear points. Evidence suggests that antigen persistence provides several key advantages over traditional intramuscular
should be maintained for more than 2–3 weeks in order to injections by allowing for minimal pain and discomfort, by
appreciate an improved immune response when compared to a targeting the myriad of innate immune cells in the skin141, and
single bolus injection: there seems to be a “lag” phase in which by providing a way to encapsulate the bioactive molecules in a
the presence of extra antigen does not make the immune stable and lyophilized state prior to use142. Several types of
responses stronger; this phase seems to last for around 14 days microneedle patches that can tailor vaccine kinetics have been
after the first antigen exposure, until the peak of the GC designed and tested in mice, NHPs, and early clinical trials,
reactions69,112,114. Instead, 2–3 weeks of continuous antigen including microneedles composed of polymers that swell or
exposure not only is associated with stronger immune responses dissolve at controlled rates when applied to the skin, releasing
but also may help to preserve subdominant protective epitopes in encapsulated vaccines136,143–145. For example, a type of dissolving
the appropriate conformations124. The kinetic of antigen persis- microneedle patch made of carboxylmethylcellulose and trehalose
tence also plays an important role: “exponential increase” outper- was used to deliver cell-culture-derived influenza vaccine146;
forms a “nearly constant” release kinetic112. Although it is still another type of patch used silk protein-based microneedles to
difficult to design a vaccine that may allow for an exponentially release vaccines with extended kinetics in the skin, promoting T
increased delivery of the antigen in vivo, some materials, such as cell and humoral responses following vaccination136.
PLGA microparticles, can be programmed with a “pulsatile” release
kinetic, in which the antigen is released in multiple sequential
waves after a single vaccination, somehow mimicking a multi- ALTERNATIVE APPROACHES TO PROMOTE PERSISTING
bolus schedule. Alternatively, most of the technologies enable a ANTIGEN AVAILABILITY
“zero-order kinetic” in which a nearly constant release of the Other than biodegradable materials, at least three more
antigen persists for days or weeks. The most relevant materials approaches can be used to induce a programmable delivery of
and technologies that are being evaluated for the development of the antigen after vaccination; those approaches are based on the
single dose vaccines are described below. use of nucleic acids, viral vectors, or adjuvants. Although each of
Although a variety of materials have been approved in humans them has complex mechanisms of action, here we focus on
for controlled release of small drugs and hormones125,126, none reviewing their properties as antigen delivery systems.
has been approved for controlled release of vaccines. Major For many years, DNA and RNA have been used in vaccination
obstacles that have limited the adoption of selected materials for strategies, indeed nucleic acids can be efficiently engineered with
vaccine development include: (a) difficulties in maintaining the sequence of an antigen of interest and used to induce long-
stability of the antigen (on-shelf and in vivo after injection); (b) term in vivo expression of the antigen101,102,147. Mode of action
challenges in controlling desired release kinetics; and (c) studies of DNA-based and RNA-based vaccines have been
manufacturing constraints36,82,127. Nonetheless, a variety of extensively reviewed102,103,148 and are outside the scope of this
materials and devices have been tested in preclinical studies review, however it is very important to note that both DNA-based
and seem promising for clinical applications. PLG was the first and RNA-based vaccines potentially induce long-term antigen
biopolymer to be tested in the attempts to develop single-dose expression in vivo with kinetics that can resemble a zero-order
vaccines. As discussed above, although several technical limita- antigen release, and may generate strong immune responses after
tions so far have prevented widespread clinical application of PLG- a single dose; for example studies with luciferase-encoding DNA
based vaccines, PLG microspheres have been widely published have shown that the luciferase can be detected for at least
and clinically validated for the controlled release of proteins, 3–4 weeks after a single vaccine injection149–155, suggesting that a
peptides, and small molecules36,82,126,128, with the potential of co- DNA vaccine would be suitable to replace at least the second dose
delivering antigens and adjuvants129–131. A single-dose vaccine of in a two-dose, 1-month apart vaccination schedule. Nevertheless,
stabilized PLG microparticles may allow for a pulsatile release of after its initial emergence in the 1990s, the level of interest in DNA
the antigen and the timing of the “bursts” can be potentially as a vaccine modality gradually decreased as it became clear that
modulated by changing the biochemical and biophysical proper- the technology lacked potency when used in human subjects.
ties of the PLG microparticles36,132; however, a continuous release RNA vaccines are a more recent and more potent tool to induce
kinetic is also possible133–135. Other relevant materials that offer an persistent antigen expression and several works have been
alternative to the use of the PLG polymer are silica, dextran, published in the recent years showing that one immunization of
collagen, chitosan, hydrogels, and fibroin from silk82,136–139; these RNA vaccines may be sufficient to induce strong and protective
materials are biodegradable, appear safe, can encapsulate a immunity. For example, using a nucleoside-modified RNA plat-
protein antigen and typically release it with zero-order release form, Pardi et al. have shown that a single dose RNA vaccine can
kinetics that can last for weeks after a single dose injection82. As protect from Zika virus infection156; in another work, by using a
examples, collagen minipellets can induce continuous antigen different platform with lipid-nanoparticle RNA vaccine, Bahl et al.
release for up to 14 days140, chitosan up to 35 days134. Although have shown that an influenza vaccine was very immunogenic after
very few preclinical immunogenicity studies have been reported just a single dose157,158; furthermore, recent clinical data have
so far with these materials, they are in the toolkit for the shown that a single vaccination with an mRNA-based vaccine
development of future vaccine formulations for controlled antigen boosted pre-existing serum neutralization titers against hMPV and
release. PIV3, two significant causes of severe respiratory diseases for
Although the polymers described above are mostly used in the infants and children. Another very promising RNA vaccine
form of microparticles for intramuscular antigen delivery, they can platform is based on self-amplifying mRNA (SAM) vaccines which
also be engineered to design devices that deliver the antigens employ a modified version of the alphavirus genome and encode
through the skin. The best example for this approach is for the RNA replication machinery together with the gene of the
represented by microneedle skin patches which encompass an antigen of interest103. SAM vaccines have several advantages over
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single-copy RNA vaccines, indeed SAM vaccines not only enable a system. Immune cell exhaustion defines a state in which immune
large amount of antigen production from an extremely small dose cells, typically T cells, following continuous and prolonged
of vaccine, but also allow for persistent antigen expression that exposure to the antigen, become less responsive and
seems to last for many weeks after a single vaccination159,160. SAM exhausted175,176. T cell exhaustion usually manifests with several
vaccines have been shown to provide strong B and T cell characteristic features, such as progressive and hierarchical loss of
immunity against a variety of pathogens including influenza, HIV, effector functions, sustained upregulation and co-expression of
RSV, Ebola, CMV, and Dengue161. Although RNA vaccines degrade multiple inhibitory receptors, altered expression and use of key
faster than DNA vaccines, several modifications on the RNA transcription factors, metabolic derangements, and homeostatic
structure and optimized delivery systems, including cationic hypo-responsiveness177. Although it is not clear how long the
nanoemulsions and lipid nanoparticles, can be used to improve exposure needs to occur before triggering an unfavorable
RNA stability, in vivo sustained protein translation and the exhausted immune phenotype, it is important that any vaccine
antigen-specific immune responses102. strategy designed to induce persistent antigen exposure carefully
Viral vectors represent the closest model to live attenuated evaluate and limit the occurrence of this phenomenon.
vaccines, a benchmark for vaccine potency. Viral vectors enable Vaccine adjuvants mediate immunostimulatory signals to the
potent stimulation of the innate immune system and persistent body’s immune system and promote potent vaccine-specific B
antigenic stimulation, all of which ultimately leads to potent and T cell responses. Although those signals are key for adjuvant
humoral and cell-mediated immunity82–90,100,162,163. Factors that mechanisms of action, we have evidence that certain adjuvants
have limited the wide-spread use of viral vectors for clinical may form an antigen “depot” at the site of injection hereby
applications include: (a) the presence of pre-existing immunity promoting antigen slow release after vaccination. Initially a “depot
against the viral vector which may limit its potency or give rise to effect” was hypothesized to explain the mechanism of action of
unwanted side effects164; (b) evidence suggests that years of alum-adjuvanted vaccines71,178; instead new findings of the past
antigen stimulation may lead to exhaustion of the immune decades have suggested that an antigen depot might be far less
responses165,166, (c) replication-competent vectors may lead to important than alum-induced inflammasome activation in the
chronic infections and pathogenesis167. In order to circumvent modulation of alum adjuvanticity71,109–111. Importantly, a recent
these potential limitations, several vectors have been developed paper from Moyer et al. has shown that if the absorption of the
for safe and effective use in humans including vectors based on antigen on alum is very strong, this may form antigen
replication-defective adenoviruses82–90,168, single-cycle adeno- nanoparticles that slowly traffic to the lymph nodes—peaking
viruses91–93, vesiculoviruses169, herpes viruses94, or adeno- up to 8 days after injection—and prolongs antigen bioavailability
associated viruses (AAV)95,170. The most used class of viral vectors in vivo; this mechanism seems to be very important in shaping
is represented by the replication-defective adenoviruses, small both the quantity and the quality of the antigen-specific immune
double-strand species-specific DNA viruses of human or non- responses of alum-adjuvanted vaccines179. A “depot effect” was
human origin that are unable to establish chronic infections and also hypothesized to be important for the mechanisms of action
may provide antigen persistence for months, depending on the of the oil-in-water emulsion MF59 adjuvant, but by using an
animal model or viral strain considered82–87,171. For example, in a immunization model with a labeled antigen, it was shown that the
mouse model of immunization with a viral vector encoding for the presence of MF59 did not significantly modify the distribution of
SIV-Gag antigen, the number of antigen transcripts has been the antigen132. Nonetheless, MF59 has been shown to induce
detected for at least 15 days after vaccination and most likely antigen-specific GC B cells for at least 4 months after last
persist for longer100. In another study from Tatsis et al. using the immunization180, suggesting that antigen is retained in its native
AdC68 vector, antigen sequences were detected at the site of form for long-term GC activation all of which might contribute to
injection for at least 1 year after administration83. In both these the strong and broad B cell responses typically seen with MF59-
studies a single immunization was sufficient to induce potent B aduvanted vaccines71. Adjuvants that may form an antigen depot
and T cell responses, with a mechanism that seems independent at the site of injection also include the cationic adjuvant
of the vector-induced activation of innate immunity100. All of this formulations (CAF platform) and the IC31 adjuvant. The principal
evidence strongly supports the use of replication-defective component of the CAF adjuvant platform is the surfactant
adenoviruses as one of the best tools to induce strong immune dioctadecylammonium (DDA) formulated into liposomes or
responses with a single immunization. The replication-competent emulsions, which modulate antigen biodistribution at the injec-
adeno-associated viruses (AAV) represent another class of tion site181; Schmidt et al. have shown that intramuscular injection
extremely attractive vaccine carriers as they are highly versatile, of a CAF-adjuvanted vaccine activates classical migratory dendritic
they can readily be produced to high titers, they only encode the cells that slowly transport the antigen to the draining lymph
transgene product and are appear tolerated, even if given at very nodes and elicit strong T cell responses182,183. IC31 is another
high doses94,95. AAVs have also been engineered as a gene adjuvant that combines the immunostimulatory effects of an
therapy tool to deliver antibodies or other functional proteins, antibacterial peptide with the biochemical properties of a
providing remarkable results in term of long-term gene expres- synthetic oligodeoxynucleotide (ODN1a, an agonist of the Toll-
sion. For example, AAVs have been shown to induce durable like receptor (TLR)-9) in order to induce potent antigen-specific
transgene expression for at least 12 months in mice170–172 and immune responses184; Schellack et al. reported that an OVA
4 months in rhesus macaques173. The only viral vector-based peptide co-administered with IC31 adjuvant formed a depot at the
vaccines that has reached approval for clinical use so far is the injection site, which was still detectable 58 days after injection and
rVSV-ZEBOV Ebola vaccine, which is based on the replication- was potentially responsible for the strong immune responses
competent vesicular stomatitis virus (VSV), a virus in the family typically observed with IC31-adjuvanted vaccines185. CAF and IC31
Rhabdoviridae; the rVSV-ZEBOV vaccine has been recently used adjuvants are currently in clinical trials for the development of a
during the Ebola outbreak in the Democratic Republic of Congo in tuberculosis vaccine.
2018, showing 97.5% vaccine effectiveness. We refer to a
reference from Lauer et al. for a more detailed discussion on viral
vectors used for clinical applications174. TARGETING SITE-SPECIFIC DELIVERY OF THE VACCINE
A major potential weakness in using viral vectors as delivery COMPONENTS
systems is linked to the possibility of inducing immune cell Although having a programmable system of antigen delivery is
exhaustion, a phenomenon sometime observed with chronic viral helpful, it might not be enough to efficiently stimulate all the
infections and in theory possible with any antigen persistence players of the vaccine-specific immune responses. Indeed,
npj Vaccines (2020) 88 Published in partnership with the Sealy Center for Vaccine Development
G. Lofano et al.
7
Hours Days
Lymph node
Neutrophil
Adjuvanted vaccine
HEV
Monocyte
Afferent lymphacs
B cell
follicle
10-100 nm
vaccine SCS macrophage Angen-specific B cell
components
Fig. 1 The journey of the vaccine components after injection. Intramuscular or subcutaneous injection triggers the recruitment of immune
cells to the site of injection, where they activate, capture the antigen and migrate to the draining lymph node. Antigens, adjuvants or other
components smaller than 10–100 nm may also diffuse in the lymphatic systems and reach the lymph node through the afferent lymphatics.
While smaller molecules (<70 kDa) may diffuse through fenestrae of the subcapsular sinus, the largest molecules are transferred to the B cells
with the help of subcapsular sinus macrophages. B cells and resident dendritic cells may also sense molecules in the conduits and transfer
them to the B cell area to initiate the germinal center responses.
appropriate stimuli—including the antigen—need to be displayed and exhibit distinct time frames of lymph node homing192. Many
not only at the right time, but also in the right place. In general, materials for targeting skin dendritic cells have been explored,
there are two compartments that are particularly important in the including hydrogels143,193 and large particulates194, with the
context of vaccination: the site of injection, typically the muscle, common aim of localizing the materials to the site of administra-
and its draining lymph node (Fig. 1). A potent single-dose vaccine tion to increase the likelihood of APC uptake and migration to the
is required to induce the appropriate stimuli in each of those two draining lymph node. Overall, the site of injection—skin or muscle
sites. —is a dynamic environment, where single-dose technologies may
To begin with, the skeletal muscle is typically where the vaccine play an important role in modulating the “when” and the “how”
first enters in the body: a resting muscle usually contains few antigens and adjuvants are released in order to trigger an efficient
immune cells, but vaccine administration triggers the recruitment transport of the antigen from the injection site to the draining
of tissue resident and infiltrating immune cells, including lymph node, where the adaptive response occurs.
professional antigen-presenting cells (APCs), which activate after Antigens and adjuvants may reach the draining lymph node in
vaccine administration186. With regard to vaccines platforms that two different ways: through diffusion in the lymphatic vessels, or
are based on DNA or RNA delivery systems, they employ the through cell-mediated transport. The lymphatic capillaries possess
expression machinery of the muscle cells in order to express the a fenestrated endothelium that allow for the passage of molecules
antigen at the injection site, which promotes the recruitment of 10–100 nm in diameter, including antigens and immune com-
immune cells102. With regard to subunit vaccines, antigens are plexes, that flow through the lymphatic systems until they reach
typically delivered with vaccine adjuvants, that help to induce a the subcapsular sinus (SCS) area of the lymph nodes195,196. Only
transient inflammation in the muscle and further promote molecules that are smaller than 70 kDa will be able to pass
immune cell recruitment and activation187. The persistence and through the SCS and directly access to the B cell follicles, while
the quality of the adjuvant-induced immune stimuli, including bigger molecules will require an active transport from specialized
cytokines and chemokines, represents a fingerprint of each cells, such as the poorly degradative SCS macrophages which
adjuvant and provide qualitatively unique immune responses at capture immune complexes and other big molecules to mediate
the injection site. For example mouse studies have shown that their transport to the follicles197–200. The different hydrodynamic
frequencies of neutrophils and monocytes peak, respectively, at properties of the lymphatic systems have been used in the past to
16 and 48 h in MF59-injected muscles, while the AS01 adjuvant design delivery systems that release molecules in specific sites of
induces faster kinetics as both neutrophil and monocyte numbers interest in the lymph node. For example, in a study by Lutz et al.
peak already at 6 h188–191; adjuvant mechanisms of action are a adjuvant-loaded particles were encapsulated in pH-degradable
complex and broad topic, and it will be briefly discussed below. hydrogel: the authors show that the hydrogel degraded over time
Another important injection site is the skin; indeed, some vaccines and slowly released 50 nm nanoparticles that diffused away from
are administered subcutaneously where they encounter a myriad the injection site and targeted dendritic cells in the draining
of skin-resident APCs. For example, skin-resident dendritic cells lymph node hence inducing stronger immune responses with a
and MHCII + Langerhans cells reside in different skin tissue layers mechanism that did not require cell-mediated transport201.
Published in partnership with the Sealy Center for Vaccine Development npj Vaccines (2020) 88
G. Lofano et al.
8
Site-specific delivery of the vaccine components
mAbs
Engaging innate
Multivalent immunity
antigens
Adjuvants
Structure-based design
DNA & RNA
Nanoparticles and VLPs
Viral vectors
Viral vectors
Adjuvants
Fig. 2 Approaches for the rational design of potent single-dose vaccines. A rational design of potent single-dose vaccines can be achieved
by modulating the spatial and temporal deliveries of the vaccine components, by designing multivalent antigens and by efficiently engaging
the innate immunity. The muscle and the draining lymph node are the two most important sites for targeted delivery of antigen and immune
stimuli, which can be achieved by modulating the biophysical and biochemical properties of the vaccine components. Antigen persistence in
germinal center regions of lymph nodes is essential to promote robust immune responses and can be modulated with controlled-release
materials, DNA, RNA, viral vectors, or adjuvants. Antigens can be designed as virus-like particles (VLPs) to present a multivalent conformation
and promote efficient B cell receptor engagement on the surface of B cells. Furthermore, activation of innate immune cells is essential to
induce strong antigen-specific immune responses and can be achieved by engaging pattern recognition receptors with adjuvants, nucleic
acids, and viral vectors vaccines.
In other studies, it has been shown that follicular B cells have also monoclonal antibody-coated microparticles can be engineered to
direct access to small soluble antigens202, even when those deliver a cargo directly to the high endothelial venules of lymph
antigens are initially administered in the form of large nanopar- nodes and modulate the immune responses211.
ticles203, suggesting that lymph proteases may release small Overall those studies highlight the importance of targeted
antigens from large carriers, resulting in a free diffusion of the in vivo delivery of the vaccine components, which display unique
small molecules and direct access to B cells in the lymph nodes. hydrodynamic and immunological properties. In designing next
For larger molecules, the story is different as they require active generation vaccines, we need to take into account the many
transport by immune cells. For example, immune complexes— possible journeys of the vaccine components in vivo, indeed an
vaccine antigens trapped by serum antigen-specific antibodies— optimal vaccine formulation may be specifically designed to target
are too big to get free access to the B cell follicles, instead they are different cell types, in different places and even at different times,
transported to the lymph node by migratory immune cells, or in order to induce potent immune responses with a single
diffuse in the subcapsular sinus (SCS) area until they are captured vaccination.
by SCS macrophages that transfer them to the B cells area204;
typically SCS macrophages also capture heavy glycosylated
antigens205, nanoparticles206, and liposomes. Importantly, lipo- SINGLE-DOSE VACCINES FOR THE CORONAVIRUS PANDEMIC
somes are commonly used carriers owing to their amphipathic The rapid expansion of the COVID-19 pandemic has made the
composition, which promotes internalization by endocytosis development of a SARS-CoV-2 vaccine a global health and
rather than scavenging by phagocytosis207. Once internalized, economic priority. Given the rapid evolution of the pandemic—
liposomes are processed by phospholipases, which disrupt their hence for the need to quickly make available millions of doses of a
structure, causing the intracellular release of encapsulated cargo, vaccine to as many as possible and to rapidly generate protective
including adjuvants and other small molecules208, and can be immunity—a single-dose vaccination strategy is considered a
used for targeted delivery to SCS macrophages. Regarding the hallmark of the target vaccine profile. At the time we write many
largest molecules, their transport from the injection site to the vaccines are being evaluated in preclinical and clinical settings,
draining lymph node is modulated by the active transport of some of which have already been shown to be safe and induce
migratory immune cells. For example, dendritic cells and MHCII + functional antibody titers after just a single vaccination. Those
Langerhans cells capture the antigen, migrate and localize in vaccines are designed to elicit protective immune responses
discrete draining lymph node locations192 to exert specific against the S protein of SARS-CoV-2 and use RNA or viral vectors
immunomodulatory functions209. Monocytes, neutrophils, myeloid as antigen delivery systems. At least one RNA vaccine has reached
dendritic cells, or plasmacytoid dendritic cells can also be phase III clinical testing, and encouraging data from a phase I trial
selectively recruited by different vaccine adjuvants and can have been published212. Although a two-doses schedule seems to
transport the antigen to the draining lymph node210. Finally, be required to reach the desirable functional antibody titers, just
npj Vaccines (2020) 88 Published in partnership with the Sealy Center for Vaccine Development
G. Lofano et al.
9
one dose of the RNA vaccine was safe and enough to induce 3. Kurosky, S. K., Davis, K. L. & Krishnarajah, G. Completion and compliance of
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