Mario Freedom's Space: Engineering Antiviral Vaccines

La terra è un bel posto peccato ci siano gli umani...

The earth is a beautiful place, too bad there are humans...

Source:

https://pubs.acs.org/doi/10.1021/acsnano.0c06109

Engineering Antiviral Vaccines

Xingwu Zhou,● Xing Jiang,● Moyuan Qu,● George E. Aninwene, II, Vadim Jucaud, James J. Moon,

Zhen Gu, Wujin Sun,* and Ali Khademhosseini*

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ABSTRACT: Despite the vital role of vaccines in ﬁghting viral pathogens, eﬀective

vaccines are still unavailable for many infectious diseases. The importance of vaccines

cannot be overstated during the outbreak of a pandemic, such as the coronavirus disease

2019 (COVID-19) pandemic. The understanding of genomics, structural biology, and

innate/adaptive immunity have expanded the toolkits available for current vaccine

development. However, sudden outbreaks and the requirement of population-level

immunization still pose great challenges in today’s vaccine designs. Well-established

vaccine development protocols from previous experiences are in place to guide the

pipelines of vaccine development for emerging viral diseases. Nevertheless, vaccine

development may follow diﬀerent paradigms during a pandemic. For example, multiple

vaccine candidates must be pushed into clinical trials simultaneously, and manufacturing

capability must be scaled up in early stages. Factors from essential features of safety,

eﬃcacy, manufacturing, and distributions to administration approaches are taken into

consideration based on advances in materials science and engineering technologies. In

this review, we present recent advances in vaccine development by focusing on vaccine discovery, formulation, and delivery

devices enabled by alternative administration approaches. We hope to shed light on developing better solutions for faster and

better vaccine development strategies through the use of biomaterials, biomolecular engineering, nanotechnology, and

microfabrication techniques.

KEYWORDS: COVID-19, pandemics, infectious disease, vaccine, immunotherapy, drug discovery, drug delivery, biomedical devices

he appearance of severe acute respiratory syndrome-

coronavirus 2 (SARS-CoV-2) has caused unprece-

dented global disruption to health, economy, and

social stability.1 SARS-CoV-2 causes coronavirus disease 2019

(COVID-19) in which individuals can suﬀer from mild to

severe symptoms once infected.2 Global scientiﬁc communities

are working together to ﬁght the crisis caused by this

pandemic. The initial eﬀorts to combat this pandemic included

identifying infected patients and ramping up clinical trials for

repurposing existing drugs.3 However, the development of

eﬀective vaccines to prevent SARS-CoV-2 infection is believed

by many to be the most eﬀective long-term solution.4

The pathway from identifying a virus to having a vaccine is

lengthy and expensive. After billions of dollars spent and

multiple years of trials, researchers generally face high rates of

failure in the traditional vaccine development paradigms

(Figure 1a).4 Despite rapid responses from the scientiﬁc

community and the pharmaceutical industry, vaccines have

historically not been ready even after an epidemic becomes

manageable, such as severe acute respiratory syndrome

(SARS), Zika, and Ebola.5 Considering economic factors,

funding for vaccine development may be reallocated after a

substantial drop in infection cases is observed, as in the 2015−

2016 Zika and SARS epidemics. The 2014−2016 Ebola

outbreak in Africa resulted in over 28,000 cases and 11,000

deaths.6 After continuous research funding support, in

December 2019, the FDA granted the ﬁrst approval to Ervebo

(a single-dose, live attenuated virus vaccine from Merck) for

Ebola virus prevention.7 Beneﬁting from egg- and cell-based

platforms, vaccine approval for the most recent 2009 H1N1

pandemic was relatively fast following the peak of the outbreak,

and the vaccine was ultimately incorporated in the seasonal

inﬂuenza vaccine.8 Since the initiation and ending of epidemics

are highly unpredictable, the rapid deployment of vaccine

development may still be too slow for a sudden outbreak.4,5

To tackle a pandemic, a diﬀerent vaccine development

process is needed to rapidly produce safe and eﬀective vaccines

against novel viruses. This process is characterized by

overlapping phases, multiple potential vaccine candidates,

Received: July 21, 2020

Accepted: September 18, 2020

Published: October 1, 2020

Review

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These permissions are granted for the duration of the World Health Organization (WHO)

declaration of COVID-19 as a global pandemic.

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and early ramp-up of manufacturing capacities (Figure 1a).4

After the initial release of the genetic sequence of SARS-CoV-

2, researchers worldwide started to develop vaccines for the

prevention of COVID-19. The ﬁrst dose was administered into

humans by Moderna Inc. on March 16, 2020.9 This method

utilized lipid nanoparticles (LNPs) with a formulated mRNA

vaccine.9 This work was fast-tracked at an unprecedented pace,

even though mRNA-based vaccines have never been licensed

before.9 A snapshot of the landscape of COVID-19 vaccines

(Figure 1b) shows that around 31 vaccines have entered

clinical trials as of August 25, 2020.10 A high diversity of

vaccine candidates is seen in these forerunners, which includes

six mRNA-based, ﬁve inactivated virus-based, ﬁve nonreplicat-

ing viral vector-based, one replicating viral vector-based, four

DNA-based, and one cell culture-based vaccines as well as nine

protein-based vaccines (Figure 1b,c). The incomplete under-

standing of the SARS-CoV-2 interactions with the human

immune system became one of the major obstacles in vaccine

development. A recent report on a second COVID-19

infection in the same patient further raises the question of

protective immunity and immune memory. For instance, it is

still unknown how durable the immune memory response

induced by SARS-CoV-2 could be or what is the threshold of

antibodies titers that can protect the patients against

reinfection.11,12 In addition, protective immunity against

SARS-CoV-2 should also be comprehensively studied, where

the balance of cellular and humoral immunity, the ratio of

eﬀector to memory T cells, the maintenance of memory B

cells, and functional features of activated T cells should be

characterized.11,12 These aspects could ultimately facilitate

vaccine designs and evaluations.

In this review, we aim to provide engineering insights on

vaccine development by covering vaccine discovery, vaccine

formulations in terms of diﬀerent materials (lipid, polymer, and

inorganic particles), and vaccine delivery devices. We will

present how some of the existing challenges could potentially

be addressed by nano/micro/macroscale engineering ap-

proaches. We will also discuss some emerging technologies

that may contribute to future vaccine development pipelines.

VACCINE DISCOVERY

After a virus is identiﬁed, diﬀerent categories of vaccines can be

developed, which includes virus-, viral vector-, nucleic acid-,

protein/peptide-, and/or cell-based vaccines (Figure 1c).13

Virus-based vaccines commonly require patient-derived

viruses, while other types can utilize the viral genomic

sequences to accelerate vaccine development. Previously

approved vaccines mostly fall into the virus-based and the

recombinant protein-based categories, but not into the nucleic

acid-based one (Figure 2a).14 The discovery process of each

vaccine type is distinct with corresponding pros and cons

(Figure 2b−d).

Virus-Based Vaccines. Virus-based vaccines, including the

weakened and inactivated types, are still the most widely used

to date. The eﬀective prevention of the polio epidemic by

vaccination in the 20th century represented a landmark success

in medical history.18 This vaccination regimen included the use

of the inactivated polio vaccine (IPV) and oral polio vaccine

Figure 1. Summary of vaccine development paradigms and major types of vaccines. (a) Paradigms of vaccine development in a traditional

condition vs during a pandemic.5 (b) Snapshot of the vaccine landscape for COVID-19 with the diﬀerent types of vaccine candidates in

various clinical stages as of August 25, 2020.10 (c) Schematic of major types of vaccines, including virus-based (weakened and inactivated),

viral vector-based (replicating and nonreplicating), nucleic acid-based (DNA and mRNA), protein and peptide-based, and cell-based.13

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(OPV).18 The IPV was made by inactivating lab-cultured

poliovirus by formalin. The OPV was weakened live polio-

virus.18 Weakened viruses are highly immunogenic and are

commonly developed by the deletion of viral genes. Never-

theless, mutations could cause virulence reversion as observed

for vaccine-derived polioviruses that have been responsible for

recent polio outbreaks.19 This represents an obstacle for polio

eradication and, more importantly, a concern for the weakened

virus-based vaccines in general. Further genetic manipulation

strategies are therefore required to prevent this issue. For

instance, modifying the 5′ untranslated region (UTR), 2C

coding region, and 3D polymerase region resulted in a

genetically stabilized OPV strain that was less likely to regain

virulence and exhibited limited viral adaptability.20

On the contrary, inactivated virus vaccines generally do not

have virulence reversion issues. They also do not rely on

extensive genetic manipulations, but the inactivation process

may cause the loss of antigenicity of viral immunogens. This

vaccine type has been quickly adapted to cope with COVID-

19, where patient-derived viruses were inactivated with β-

propiolactone, followed with puriﬁcation by chromatography,

and formulated with Al(OH)3 as the adjuvant.15 The vaccine

protected nonhuman primates from SARS-CoV-2 challenges

with no notable side eﬀects.15 Advantages of these virus-based

methods include well-developed methodologies, well-estab-

lished regulation policies, and existing manufacturing facilities

from previous practices. Nevertheless, the requirement of virus

seeding and culture along with unknown safety issues

Figure 2. Vaccine discovery processes for diﬀerent types of vaccines. (a) Timeline of previously approved antiviral vaccines.14 (b) Virus-

based vaccines require the seeding and culture of speciﬁc viruses derived from patients. By manipulating the genetic sequences of the virus

or inactivating the virus directly with chemicals, weakened and inactivated virus vaccines can be produced.15 (c) Viral vector-based and

nucleic acid-based vaccines are independent of virus culture and rely on the genetic sequence of the virus and/or the selection of

immunogenic sequences of the virus. By selecting commonly developed vectors, virus-speciﬁc sequences can be inserted.16 Nucleic acid

vaccines require further formulations for optimal eﬃcacy. (d) Structure-based understanding of native viral proteins can validate the

expressed recombinant proteins and predict the peptide sequences desirable as vaccines. Protein/peptide vaccines require further

formulations for optimal eﬃcacy.17

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necessitates alternative practices for the development of other

vaccines.

Viral Vector-Based Vaccines. Viral vector-based vaccines

work by inserting viral antigen-encoding DNA sequences into

host cells, which leads to the expression of viral antigens by the

host cells.16 The rationale is based on the capability of viruses

to infect cells eﬃciently through viral genome integration

mechanisms.16 This method could also be problematic since

the genome of the host could be altered and cause other

diseases. The selection of viruses is therefore crucial in

balancing the eﬃcacy (eﬃcient infectivity) and safety (limited

pathogenicity). Another common obstacle is the host’s pre-

existing immunity to certain vectors that would mitigate the

eﬃcacy of a vaccine. The selected viral vectors could be further

genetically modiﬁed to be nonreplicable by disrupting genes

responsible for replication. Adenoviruses (Ad) are the most

extensively used vectors for vaccines because of several

advantages: infectious to various cell types, eﬃcient transgene

expression, high in vitro growth, lack of genome integration,

and genetic stability.21 For instance, Johnson & Johnson (J &

J) deployed the Ad26 vector to deliver gene encoding surface

proteins of the SARS-CoV-2, and CanSino Biological used the

Ad5 vector.22 The spike (S) glycoprotein of SARS-CoV-2 was

cloned with the tissue plasminogen activator signal peptide

gene in the early region 1 (E1) and early region 3 (E3) deleted

Ad5 vector. The E1 and E3 deletions render the Ad5 vector

nonreplicating. Even though rapid humoral and T cell

responses were induced by the vaccine, pre-existing anti-Ad5

immunity partially diminished the immune responses among

healthy adults.22 To address the pre-existing immunity to Ad,

chimpanzee Ad (ChAdOX1) represents an alternative solution

that provided broad protective immunity against the Middle

East Respiratory Syndrome Coronavirus (MERS-CoV).23 An

investigational vaccine for SARS-CoV-2 from the University of

Oxford is based on this vector.23 Their preclinical study

showed the prevention of SARS-CoV-2 pneumonia in rhesus

macaques with no evidence of subsequent immune-enhanced

diseases.23 However, viral loads were more signiﬁcantly

reduced in the lower respiratory tract but not in the nasal

swabs. In general, because of previous vaccine programs, the

manufacturing capacity and protocols for this method are

already well-established; therefore, less eﬀort may be needed to

shift toward the large-scale production of vector-based

vaccines. However, both the pre-existing immunity toward

certain vectors and previously demonstrated safety concerns

over the increased risk of human immunodeﬁcient virus type 1

(HIV-1) acquisition should be carefully evaluated.22

Nucleic Acid-Based Vaccines. DNA- and mRNA-based

vaccines have the greatest potential for rapid development

because of their synthetic nature that circumvents the need for

cell culture or fermentation of viruses. The synthetic nature of

this method also results in a high safety proﬁle and rapid

manufacturing ability.24 However, this method generally

requires further delivery formulations or better delivery devices

to boost vaccine eﬃcacy.24 In contrast to RNA, the increased

stability of DNA decreases the need for frozen storage and low-

temperature transportation.24 One of the forerunners among

COVID-19 vaccines is DNA-based (Inovio).10 Similar DNA-

based vaccines have had promising results for the prevention of

MERS.25 In the discovery phase of DNA-based vaccines,

strategies such as codon/RNA optimization and the addition

of highly eﬃcient immunoglobulin leader sequences are

needed to enhance the magnitude and breadth of the immune

response.26 For instance, Muthumani et al. designed a

consensus DNA sequence for the S protein by analyzing S

protein sequences, where sequences from clades A and B can

be both involved.26 To enhance the in vivo expression, the

immunoglobulin E (IgE) leader sequence was added to the

immunogen sequence. The insert was further incorporated into

the pVax1 vector, which allowed a high-level transient

expression of proteins of interest in mammalian cells.26 The

strategies of adding the IgE leader sequence and the use of the

pVax1 vector could be quickly adapted to the diﬀerent virus

sequences as well.

Due to their high potency, rapid development, and low cost

for manufacturing, mRNA vaccines have emerged as a

promising alternative vaccination development strategy for

various infectious diseases and cancers.27 No mRNA-based

vaccine is currently FDA-approved. Several strategies have

been proposed to solve the common drawbacks of mRNA-

based vaccines, which include mRNA instability and high

innate immunogenicity.28 The 5′ and 3′ UTR of mRNA has

signiﬁcant regulatory inﬂuences on both stability and trans-

latability.28 Half-life and expression can be enhanced when

these sequences come from viral genes: A 5′ cap is essential for

eﬃcient protein expression, and a poly(A) tail aﬀects mRNA

translation and stability.29 Furthermore, the replacement of

rare codons with frequently used synonymous codons also

enhances protein expression.30 Additional optimization strat-

egies include increasing G:C content and introducing modiﬁed

nucleosides.31,32 In spite of the innate immunostimulatory

eﬀect of exogenous or unpuriﬁed mRNA, further investigations

are required to assess the advantages of their use for vaccines

speciﬁcally.33 Innate immune sensing mechanisms of mRNA

have resulted in the inhibition of antigen expression and a

decreased immune response.34 Therefore, further formulations

for vaccine delivery could potentially reduce innate mRNA-

associated immunogenicity and favor immune activation.

Subunit Protein/Peptide Vaccines. Unlike nucleic acid-

based vaccines, which produce viral protein after admin-

istration, the direct application of viral protein antigens may be

a more straightforward method to trigger immunity. This

approach currently represents the largest category of COVID-

19 vaccines under preclinical investigation.4,10 Full-length

proteins are advantageous because they can induce the

development of antibodies against multiple epitopes. Impor-

tantly, there is a higher probability that the developed

antibodies could bind to the native conformation of the viral

protein. However, this also increases the chance of inducing

nonspeciﬁc cross-reactive antibodies.35 In addition to the high

cost of recombinant protein technologies, the recombinant

protein products may not fully match all the molecular features

of the corresponding viral proteins, which may lead to

ineﬀective induction of broad neutralizing antibodies

(bNAb).35 Optimizing peptide sequences in the discovery

phase can improve the stabilization of epitopes associated with

bNAb while reducing the antigenicity of non-NAb epitopes.17

Given that only speciﬁc amino acid sequences of the full-

length protein antigens are responsible for eﬀective immune

responses, minimally immunogenic peptides, mimicking B and

T cell epitopes, have been proposed for vaccines.17 Early

approaches used short peptide sequences (8−10 amino acids)

that could potentially be presented by major histocompatibility

complex (MHC) I molecules. Importantly, the memory

immune response of cytotoxic T cells could not be eﬃciently

induced with such T cell epitope-mimicking peptides.36 One

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proposed rationale is that in vivo direct loading of short

peptides occurs for all cells expressing MHC molecules,

including T and B cells. Unlike DCs, they are incapable of

generating an eﬀective immune response.37 These early

ﬁndings beneﬁtted the design of synthetic long peptide

(SLP) vaccines where both helper T cells (TH) peptides and

toll-like receptor (TLR) ligand or peptides could be hybridized

to enhance the immune response. For example, Jackson et al.

demonstrated one single SLP vaccine that can target TLR 2

can serve as self-adjuvant and contain both MHC I- and II-

speciﬁc peptides (T cell epitopes).38 SLPs cannot directly bind

with MHC I and have to be internalized by DCs for further

presentation. Furthermore, in silico computer-based ap-

proaches can be used to beneﬁt the discovery of peptide

vaccines by enabling quicker screening and more accurate

predictions of peptide sequences with high immunogenicity

and binding aﬃnities to MHC I and II molecules.39 These

approaches have been used to accelerate the development

pipeline of vaccines, and particularly for emerging viral diseases

such as COVID-19. These computational approaches can

predict the binding aﬃnity of speciﬁc peptides sequences to

either MHC I and II molecules or B cell receptors/antibodies.

In this regard, theoretical B and T cell epitopes speciﬁc to

SARS-CoV-2 were synthesized to generate a multiepitope

protein.39 The antigenicity, allergenicity, physiochemical

parameter, and secondary and tertiary structures of these

multiepitope proteins were determined in silico. However, the

processing in the endosomal/lysosomal compartments of the

Figure 3. Action mechanisms of vaccine formulations. (a) Vaccines generate humoral and cellular immunity within lymph nodes (LNs): I.

DCs can process the antigens and present the peptide fragments via both MHC class I and class II molecules. II. B cells can directly

recognize the antigens via BCRs and present the antigenic peptide fragments by MHC class II to helper T cells (CD4+). Stimulated B cells

can subsequently initiate a humoral immune response. III. Cytotoxic T cells (CD8+) can recognize the antigenic peptide fragments presented

by MHC class I through TCRs and trigger the cellular immune response.12,45 (b) Intracellular response of DCs to antigen presentation for

diﬀerent types of vaccines through PRRs. I. Vaccine formulations can be eﬀectively internalized by cells, followed by II. Endosomal release.

Before III. MHC loading of antigen peptide, peptide vaccine undergoes enzymatic processing, DNA vaccine undergoes transcription and

translation, and mRNA vaccine undergoes translation.24,27,37

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designed multiepitope SARS-CoV-2 antigens remains to be

addressed.

Cell-Based Vaccines. DCs are the most potent antigen-

presenting cells (APCs) at the interface of innate and adaptive

immunity to sense the “danger signals”, process the antigens,

and present the nonself-antigens.40 Unlike other types of

vaccines, DC vaccines are manipulated ex vivo to be antigen-

speciﬁc, and thus they are ready to initiate immune responses

after being reinfused in vivo. The DC vaccine is quality

controllable and natively able to traﬃc eﬃciently in vivo.

Diﬀerent methodologies have been developed to produce

modiﬁed DC vaccines ex vivo, including loading DCs with

immunogenic peptides or proteins (subunit DCs), viral

transduction to express immunogenic peptides,41 or using

mRNA-based DCs.42 Diﬀerent strategies may produce DC

vaccines with diﬀerent potency, for instance, genetically

modiﬁed DCs may be more eﬃcacious in immune activation

than subunit DC vaccines.41 In addition, supplementary

immunomodulatory genes could be incorporated to enhance

the potency. However, the process of producing cell-based

vaccines is labor-intensive and expensive.43 Thus, this method

could be the most challenging to be scaled up as a universal

vaccine for a global pandemic.43

VACCINE FORMULATIONS

Action Mechanisms. Vaccines are commonly adminis-

tered intramuscularly, making it less eﬃcient in interacting

with the abundant immune cells present within the skin.

Alternative strategies, including oral or intranasal deliveries, are

also widely studied because of their ease of use.44 The

eﬀectiveness of alternative strategies is based on mucosal

immunity.44 Nonetheless, diﬀerent strategies rely on the

activation of the adaptive immune system, which involves the

interactions between T cells and APCs (DCs and B cells).12 As

shown in Figure 3a, APCs can internalize exogenous antigens

and process them into antigenic peptide fragments. The

peptide fragments can subsequently be loaded onto the MHC

II molecules and displayed on the surface of the APCs.12,45

CD4+ helper T cells could recognize peptides presented by

MHC class II through T cell receptors (TCRs), which could

induce the production of cytokines, such as IL-2, that are

essential to the normal function of immune cells.12,45 B cells

can directly capture antigens through B-cell receptors (BCRs).

The antigens can be internalized and subsequently processed

into antigenic peptides for presentation by MHC class II to

CD4+ helper T cells. This process is required for the clonal

expansion and diﬀerentiation of B cells into IgG antibody-

secreting plasma cells and memory B cells, which is classiﬁed as

humoral immunity.12,45 Generally, only endogenous antigens

could be degraded into peptides and loaded onto MHC class I

molecules for presentation to CD8+ T cells.12 Cross-

presentation is the ability of certain APCs to uptake, process,

and present extracellular antigens in an MHC class I-

dependent manner to CD8+ T cells. Cross-presentation can

be achieved by several cell types, including DCs (the primary

cell type), neutrophils, macrophages, and endothelial cells.12

The MHC class I presented peptide fragments can activate

CD8+ cytotoxic T cells through TCR recognition. Antigen-

speciﬁc CD8+ cytotoxic T cells can then recognize these

peptide fragments presented by target cells and kill them,

which refers to cellular immunity.12,45 Lastly, DCs are also

capable of detecting “danger signals” via an array of receptors

called pattern recognition receptors (PRRs). PRRs are capable

of recognizing pathogenic molecules as part of the innate

immunity for a response to virus infection by identifying

pathogen-associated molecular patterns (PAMPs) from viruses

or microbes. The understanding of innate immunity could

beneﬁt the development of adjuvants to enhance the immune

response further. Together, the understanding of the action

mechanisms facilitates the design of vaccine formulations to

boost their eﬃcacy.46

Requirement of Formulations. Based on the current

understanding of eliciting optimal immune responses, materi-

Figure 4. Materials-based vaccine formulations for important immunological functions. (a) Vaccine formulations that protect the active

“cargo” and enhance cellular uptake may be based on lipids, polymeric materials, and/or inorganic particles. (b) Vaccine formulations that

allow LN targeting by forming extra small-sized albumin-based vaccine complexes, tuning the net charge to be negative and incorporating

targeting ligands. (c) Vaccine formulations that promote immunostimulatory eﬀects by using microbial wall-derived polysaccharides as

nanocarriers, co-delivering adjuvants with peptide antigens, and exploring innate immunostimulatory lipids.

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als-based nanoengineering approaches could improve the

eﬃcacy of vaccines. In this regard, diﬀerent methodologies

could be applied based on diﬀerent types of vaccines, with

either distinct or shared requirements. Virus and viral vector-

based vaccines generally do not require further formulations,

and their eﬃcacy and safety proﬁles are largely dependent on

the puriﬁcation and inactivation strategies, the optimization of

culturing of viral particles, and the choice of vectors.47 They

have eﬃcient innate advantages in delivery due to their small

sizes, and they are easily recognized by the immune system.

Although inactivated or weakened virus-based vaccines still

make up most of the eﬀective vaccines today, they are not

eﬀective for all pathogens.47 Similarly, the potency of DC

vaccines is mostly dependent on how DCs are manipulated ex

vivo. Strategies in further potentiating cell-based immuno-

therapies, such as the decoration with nanoparticles (NPs),48

are mainly developed for cancer immunotherapies, but they

may be less suitable for large-scale manufacturing that is

needed for the production of antiviral vaccines.

In contrast, synthetic vaccines (nucleic acid-based and

protein/peptide-based ones) generally require additional

formulation designs for optimal eﬃcacy.49 These synthetic

molecules all have essential secondary or higher-order

structures that could be disrupted in physiological con-

ditions.12 Various endogenous enzymes (such as nuclease or

protease) can digest these synthetic molecules before reaching

immunocompetent cells, such as APCs, or immunocompetent

sites, such as secondary lymphoid organs.12 Furthermore,

physiochemical features of these synthetic molecules may pose

challenges for cells to eﬃciently uptake and process them. For

instance, DNA and mRNA are too large to directly diﬀuse

across the cellular membrane and their dense negative charge

repels them from cell membranes, which further prevents

eﬃcient cellular uptake.27 Even after penetrating the

membrane, the vaccine formulations need to reach the

endosomal compartments either to be processed into peptides

(protein/peptide-based vaccines), released for further tran-

scription (DNA-based vaccines), or translation (mRNA-based

vaccines). The resulting peptide fragments can be further

loaded onto MHC molecules and exported to the membrane

surface for antigen presentation to T cells (Figure 3b).50

We refer vaccine formulations to engineering approaches

that could facilitate the in vivo delivery of vaccines and enhance

immune responses.51 We categorize the vaccine formulations

in terms of commonly used materials: lipids, polymeric

materials, and inorganic particles (Figure 4). Commonly

pursued feature goals include protection of cargo, improving

loading capacity, and enhancing cellular uptake (Figure 4a).52

In addition, the ideal microenvironment for initiating and

amplifying immune responses is secondary lymphoid organs

Table 1. List of the Representative Formulations Used for Vaccines

materials formulations cargos (vaccine types) functions refs

lipids

ionizable lipid mRNA (glycoprotein 100 (gp100) and tyrosinase-

related protein 2 (TRP 2)) and adjuvants

lipopolysaccharide (LPS)

stabilization, enhanced cellular uptake 54

covalently cross-linked lipid

bilayers

protein/adjuvants (Monophosphoryl lipid A

(MPLA))

stabilization, sustained delivery, co-delivery 55

net negative charged formulations

by tuning cationic lipids and

mRNA ratio

mRNA (inﬂuenza virus hemagglutinin (HA) and

OVA)

LN targeting, stabilization 56

nanodiscs via sHDL peptide neoantigens/adjuvants (CpG); dual

adjuvants (CpG and MPLA)

co-delivery, enhanced uptake 57, 58

lipids with cyclic amino headgroup mRNA (OVA and E7) immunostimulatory function 59

polymers

modiﬁed dendrimer mRNA (H1N1 inﬂuenza, Ebola viruses, and

Toxoplasma gondii parasite)

enhanced loading, cellular uptake, and

endosomal release

stearic acid conjugated low MW

PEI

peptide (TRP 2) and mRNA (HIV-1) lowered charge density, higher

biocompatibility

61, 62

cyclodextrin conjugated PEI mRNA (HIV) lowered charge density, higher

biocompatibility

PLGA NPs protein (OVA) controlled antigen release 64

PEI/chitosan/poly lysine coated

PLGA/PLA

protein (HBV surface antigen) enhanced loading and cellular uptake,

intrinsic immunostimulatory function

65−67

PBAE and PLGA plasmid DNA (Luciferase) enhanced loading and lower cytotoxicity 68

ionizable amino polyester mRNA (luciferase) spleen targeting 69

oligopeptide-modiﬁed PBAE mRNA (enhanced green ﬂuorescent protein) APC targeting 70

albumin and albumin-binding

lipids

peptide (OVA) /adjuvant (CpG) LN targeting 71

polysaccharide from microbial cell

wall and PEI

mRNA (OVA) immunostimulatory, enhanced loading and

endosomal release

inorganic

particles

aluminum hydroxide phosphoserine conjugated protein antigen (HIV

trimer)

immunostimulatory, sustained release 73

chemically functionalized Au NPs

with hydrophobicity

− intrinsic immunostimulatory function 74

aspect ratio optimized and surface

modiﬁed Au NRs

plasmid DNA (HIV-1) enhanced cellular uptake, intrinsic

immunostimulatory function

75, 76

sized optimized Au NPs peptide (OVA) and adjuvant (CpG) enhanced DC activation 77

mesoporous Si NPs adjuvant; protein (E2 of diarrhea virus) intrinsic immunostimulatory function 78, 79

antigen density tunable/Size

controllable QDs

peptide (self-antigen from multiple sclerosis) LN homing and higher density of antigens

leads to more eﬀective tolerance, intrinsic

antiviral eﬀects

80, 81

oxidized multiwalled CNTs protein (NY-ESO-1) and adjuvant (CpG) co-delivery, enhanced uptake 82

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where APCs, T cells, and B cells are in close proximity for cell-

to-cell interactions. Therefore, targeting DC or LN can be one

type of approach to pursue (Figure 4b).53 To further boost

immune responses, immunostimulatory components or

adjuvants are usually required, and coformulation approaches

have been widely explored for developing immunostimulatory

vaccine formulations (Figure 4c). Relevant studies have been

summarized in Table 1.

Lipids. Lipid molecules are suitable for vaccine formula-

tions because of their high safety proﬁle and tunable

physiochemical features.55,59 These advantages also make

them the leading platform in developing vaccines quickly.

Currently, there are two mRNA vaccine forerunners in

development for COVID-19 that are based on lipid

formulations (Moderna and BioNTech).10 Lipids are a class

of amphiphiles that contain hydrophilic head groups and

hydrophobic tails. Interactions between those molecules can

drive the formation of spherical vesicles to encapsulate vaccine

formulations either with one (unilamellar) or more (multi-

lamellar) lipids bilayers.55 Lipid molecules have also been

explored to achieve distinct biological functions.59 For

instance, cationic lipids are commonly used for eﬃcient

cellular uptake and increase the loading of negatively charged

mRNA and DNA.56 DOTMA, DOTAP, and DOPE are

common examples.56 However, cationic lipids are commonly

associated with high toxicity and neutralization by anionic

serum proteins, which reduce the delivery eﬃcacy.83 There-

fore, ionizable lipids that can change their charge state at

diﬀerent pH conditions can be utilized to maintain eﬃcacy, to

reduce toxicity, and to facilitate the endosomal release of

cargos.84 Some common strategies to improve the eﬃcacy of

LNPs include enhancing cellular uptake and promoting

endosomal escape by incorporating cholesterol, polyethylene

glycol (PEG) lipids, or helper lipids DOPE.56 Hydrophobic

cholesterol can ﬁll the gaps between lipid tails to stabilize the

vesicle. PEG lipids can shield the vesicles and protect them

from clearance when systemically delivered.56 Helper lipids

have unsaturated bonds to form an unstable hexagonal lamellar

phase that enhances the endosomal escape.85 In addition, good

biocompatibility of lipids makes it possible for expedited FDA

approvals.86 Therefore, LNPs can be optimized in vaccine

formulations to stabilize and enhance uptake of cargo, to

improve immunostimulatory functions for antigens, and for

targeted delivery. Oberli et al. utilized an ionizable lipid, a

phospholipid, cholesterol, and a PEG anchored lipid as LNP

formulations for mRNA vaccine delivery, which resulted in

stable nanoformulations, eﬃcient antigen presentation, and

elicitation of a strong T cell response.54 However, the in vivo

potency of LNP-based vaccines could be limited by their

suboptimal physiological stability, which can cause the fast

release of antigens before initiating any immune response.

Moon et al. developed a hyperstabilized lipid-based vaccine

formulation by covalently cross-linking two lipid molecule

layers between their head groups using a mild chemical

condition that is compatible with loaded antigens.55 The

resulting vesicles are stabilized multilamellar structures that

could trap high levels of protein antigens. Combined with

PEGylation as a well-established stabilization strategy, the

hyperstabilized NPs can sustain the release of antigens up to 30

days in the presence of serum, which signiﬁcantly enhanced the

humoral immune response.87 By adding the lipid-like mono-

phosphoryl lipid A (MPLA) as adjuvants, the vaccine

formulation can further enhance T cell-mediated humoral

responses.

In addition, LNPs formulations can be optimized for LN

targeting, thereby enhancing the immune response in situ. One

notable study by Kranz et al. demonstrated that a designed

mRNA-lipoplexes (RNA-LPX) could target DCs in vivo simply

by using lipid molecules.56 They established a library of RNA

lipoplexes by tuning the lipid to RNA ratios. Cationic lipids

DOTMA or DOTAP and the helper lipid DOPE or cholesterol

were used. The charge ratio, size, ζ potential, colloidal

properties, and RNA stability also varied with the shifting of

lipid to RNA ratios. Interestingly and unexpectedly, all

negatively charged particles displayed selective targeting to

the spleen. The negatively charged formulation (1.3:2

lipid:RNA) displayed eﬀective targeting of DCs and formed

monodisperse NPs (∼300 nm) that protected encapsulated

mRNA. The authors demonstrated the in vivo eﬃcacy using

RNA-LPX encoding for the inﬂuenza virus hemagglutinin

(HA), where DCs, NK, B, and T cells were activated by a

single intravenous injection of HA-LPX. They also observed an

enhanced IFN-α production by vaccination, which is a typical

hallmark of APCs sensing for antiviral environment develop-

ment induced by RNA virus infections through TLR3 and

TLR7.88

To enhance immunostimulatory eﬀects, a common strategy

is to co-deliver adjuvants to stimulate immune responses. Kuai

et al. designed an antigen and adjuvant co-delivery nanodisc

platform based on synthetic high-density lipoprotein

(sHDL).89 One of the striking features of the nanodisc is its

extra-small size: ∼10 nm in diameter, which contributed to the

enhanced traﬃcking to LNs.57 Exploiting the endogenous role

of HDL as a cholesterol carrier, adjuvant CpG was modiﬁed

with cholesterol to eﬃciently insert into preformed nanodiscs.

This strategy also allowed the co-loading of more than one

adjuvant, which promoted a synergistic immune activation.58

Together, these features contributed to the sustained antigen

presentation by DCs. Recently, there is a growing interest in

exploring innate immunostimulatory functions of lipid

molecules. To expand the diversity of lipid molecules, Miao

et al. developed a library of lipids through a one-step three-

component reaction that included amines, isocyanides, and

alkyl ketones.59 This strategy signiﬁcantly increased the

diversity of synthesized lipids compared to traditional two-

component reactions.59 They showed that the immunostimu-

latory eﬀect of lipids was dependent on the head groups. The

identiﬁed lipids with cyclic amino head groups displayed

immune cell activation eﬃcacy through the intracellular

stimulator of interferon genes (STING) pathway. The lipids

could be further formulated with mRNA to form LNPs as

mRNA vaccine formulations to eﬃciently deliver the

oligonucleotide products. Another advantage is that the

formulation could activate the mRNA-independent intra-

cellular STING pathway rather than TLRs, which could

reduce systemic toxicity. In addition, inhibition of the antigen

expression related to the TLRs binding by exogenous mRNA

was prevented to favor an optimal immune response.27

Polymers. Aside from the lipids system, polymers have

been widely explored for the delivery of diﬀerent vaccine

candidates. Speciﬁcally, cationic and ionizable polymers are

promising in nucleic acid-based vaccine formulations.

Positively charged polymers can condense nucleic acids as

nanocomplexes for eﬃcient cellular uptake and endosomal

release. In addition, early studies showed that cationic

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materials tend to induce an inﬂammatory response.90 One

commonly applied polymer category is polyamines, such as

polyethylene imine (PEI) and its derivatives.91 Chahal et al.

utilized modiﬁed dendrimer for antigen-encoding mRNA

replicon complexation to form monodisperse NPs.60 This

delivery system, with self-amplifying mRNA, enabled immuni-

zation in mice, which protected them from various virus

challenges, including H1N1 inﬂuenza, Ebola virus, and the

Toxoplasma gondii parasite.60 However, their high molecular

weight (MW) and branched polymer structure (needed to

allow a massive loading of vaccine therapeutics) also lead to

severe cytotoxicity, which limits wider applications.92 Zhao et

al. developed a low MW PEI with modiﬁcations (stearic acid

and 2 kDa PEI conjugates) to form self-assembled micelles for

delivering mRNA encoding HIV-1 group-speciﬁc antigen

(gag).62 Li et al. used 2 kDa PEI conjugated with cyclodextrin

for intranasal delivery of HIV antigen-encoding mRNA that

was able to stimulate HIV-speciﬁc immune response.63

Cyclodextrin modiﬁcation provided mucosal permeation and

mitigated cytotoxicity by lowering the charge density. Other

commonly used polymers include polyester-based, such as

poly(lactic-co-glycolic acid) (PLGA) and poly(lactic acid)

(PLA), because of their biocompatibility and biodegradabil-

ity.93 Several studies have demonstrated the delivery of protein

antigens in PLGA NPs with enhanced immune responses as

compared to soluble formulations.94,95 In addition, such solid

polymer particles can control the kinetics of antigens released

from the polymer core based on diﬀerent biodegradation

proﬁles.64,96 However, nonmodiﬁed PLGA is generally

negatively charged, and as a result, it has a limited cellular

uptake eﬃciency and loading of anionic antigens.66 Therefore,

various coating or modiﬁcation strategies (such as PEI,65,97

chitosan,66 and poly-L-lysine (PLL) coatings) have been

demonstrated to enable the delivery of vaccines and promote

interactions with negatively charged cellular membranes.67

Notably, the cationic polymeric delivery carrier itself could

induce T helper cell responses through TLR-4 dependent IL-

12 secretion.67,98 Poly(β-amino esters) (PBAE) is also

extensively used in delivering plasmids DNA or mRNA

vaccines because of its nontoxic and biodegradable nature.99

Fields et al. developed NPs formulations for plasmid DNA

delivery by combining PLGA and PBAE to reconcile the

drawbacks from each component: the low DNA loading

eﬃciency of PLGA and the cytotoxicity of PBAE.68 Other

polymeric materials have also been explored, such as

polysaccharides100 and block copolymers.101

In terms of targeted delivery to immune cells, versatility in

polymer chemistry shows great potential in developing tissue-

selective formulations. For instance, a large library of PBAE-

based polymers could be easily synthesized and screened for

desirable target polymers.69 Kowalski et al. designed an

ionizable amino polyester (APE) for mRNA delivery through

the ring opening of lactones with amino alcohols.69 Tissue-

selective delivery was achieved by diﬀerent APE candidates,

including APCs within the spleen.69 Oligopeptides have been

used to modify PBAE to allow the tuning of the charge status

of NPs.102 Initially, this was done to optimize cargo loading

and mitigate cytotoxicity. Later, Fornaguera et al. demon-

strated that speciﬁc oligopeptide modiﬁcations lead to APCs

targeting within the spleen and exhibited an eﬃcient

transfection of mRNA.70 Inspirations also come from using

native LN traﬃcking molecules. Clinically, visualization of

sentinel LNs is achieved by injecting dyes that bind to albumin

because of the LN traﬃcking capability of albumin particles.103

Inspired by the endogenous role of albumin, Liu et al. designed

a vaccine by conjugating the adjuvants or antigens to the

albumin-binding lipid tail.71 The vaccines displayed a notable

increased accumulation in the LNs, which decreased the

systemic distribution of the vaccines and prolonged the vaccine

bioavailability. These features limited the systemic toxicity

while increasing the potency of vaccination for OVA antigen,

HIV antigen, and human papillomavirus (HPV) antigen.71

Some polymeric delivery carriers have been demonstrated to

have immunostimulatory eﬀects, such as polymers with

cationic charge and hydrophobic domains.104,105 In addition,

inspired by microbes’ immunostimulatory eﬀect, Son et al.

designed sugar capsules that were derived from microbial cell

walls for vaccine NPs coating.72 The coating process was

achieved by using silicate NPs (Si NPs) as a template, followed

by its removal after the mRNA was loaded and the sugar

capsules were entirely coated. Notably, the removal of Si NPs

renders relatively ﬂexible nanostructures that are beneﬁcial for

lymphatic drainage and accumulation.72 The mRNA cargos

can be eﬃciently loaded by PEI coating due to electrostatic

interactions. The PEI coating was also expected to facilitate the

endosomal release for mRNA translation and antigen

presentation. The sugar capsule itself functioned as a strong

DC activator by eliciting the production of a variety of pro-

inﬂammatory factors, including IL-6, TNF-α, and IL-12 p40,

without the use of exogenous adjuvants.72

Inorganic Particles. Inorganic materials have been used in

vaccine formulations for a long time. Back in 1926, an

aluminum compound (alum) was discovered to be able to

precipitate the toxoid to enhance the immunogenicity.106 Alum

is still the most widely used adjuvant today. In spite of over 80

years of development, the underlying mechanisms for alum as

an adjuvant are still controversial.107 Popular explanations

include the “depot theory” for extended antigen release106 and

enhanced uptake by APCs through antigen absorption on

alum.108 Various immunity signaling and activation pathways

have also been identiﬁed to contribute to the function of the

adjuvants.109 Recently, Moyer et al. utilized the interaction

between alum and phosphoserine (pSer) to engineer a short

peptide-immunogen conjugate to signiﬁcantly prolong the

immunogen release from pSer-immunogen:alum complexes.73

The complexes could form NPs for eﬃcient traﬃcking to LNs

and initiated antigen processing and presentation by APCs.73

Site-speciﬁc pSer modiﬁcation on the base of the HIV trimer

could enable the immunogen (HIV trimer) to be displayed

with a speciﬁc orientation, which could prevent the induction

of undesired NAbs.73 Apart from alum, other inorganic

materials have been proposed that may stimulate vaccine-

speciﬁc immune responses, such as silica crystal110 and calcium

phosphate.109

In addition to the traditional use as immunostimulatory

adjuvants, a wide range of biocompatible inorganic particles

have also been developed as vaccine formulations,111 including

quantum dots (QDs),80 gold nanoparticles (Au NPs),77

carbon nanotubes (CNTs),82 and Si NPs.79 The physical

properties of formulations, such as surface charge, hydro-

phobicity, and aspect ratios, are closely associated with the

eﬃcacy of vaccines.112 Inorganic materials have a high degree

of tunability to allow them to fulﬁll the requirements of vaccine

formulations. Easy preparation and good storage stability are

additional advantages of inorganic particles.113 For example,

Au NPs have been chemically functionalized to investigate the

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relationship between surface hydrophobicity and the associated

immune activity.74 It is also expected that other functional

groups may bring a pathway-dependent immune activation

that is speciﬁcally desirable for antiviral defense. Xu et al.

described surface-engineered Au nanorods (NRs) for plasmid

DNA vaccine delivery.75 With certain surface modiﬁcations

and aspect ratios, cellular toxicity of Au NRs can be lowered,

internalization can be enhanced, and intrinsic adjuvant features

can be utilized.76 In vivo vaccination with HIV-1 envelope

glycoprotein (Env) plasmids demonstrated enhanced cellular

and humoral immunity.75 Zhou et al. synthesized a series of Au

NPs (15 to 80 nm) for co-delivery of antigens (OVA peptides)

and adjuvants (CpG) by surface conjugation.77 Particles of 60

and 80 nm outperformed other sizes (15, 30, and 40 nm) in

activating DCs; this was most likely due to a higher payload of

antigens. Notably, they promoted DC homing to the LNs and

induced potent cellular immune response.77 High tunability is

also achievable in other organic particles, such as adjuvant-

based mesoporous Si NPs,78 mesoporous Si NP-based

antigen/adjuvant co-delivery systems,79 antigen density tuna-

ble QDs,80 size-controlled antiviral QDs,81 and surface

oxidized antigen/adjuvant-based multiwall CNTs.82

VACCINE DELIVERY DEVICES

If the vaccine formulations are considered as utilizing

nanoscale engineering, then vaccine delivery devices utilize

micro/macroscale engineering to improve vaccine eﬃciency

and eﬃcacy. Beyond the optimization of the vaccine itself and

designing appropriate delivery carriers, the eﬃcacy of a vaccine

also depends on pharmacokinetics (PK) of the antigens after

administration.114 When the body is exposed to an exogenous

antigen, both humoral and cellular immune responses are

initiated to neutralize it, which also leads to immunological

memory.12 During a virus infection, the replication process

typically takes one to several weeks to sustainably stimulate the

immune system with a continuous supplement of antigens.115

In contrast, typical vaccines show a rapid clearance and can

only be detected in LNs within days.114 Although still poorly

Figure 5. Summary and characteristics of various vaccine delivery devices. (a) Drug delivery devices developed for vaccine administration: I.

Traditional vaccine-containing solutions for bolus injection. II. Minimally invasive microneedle devices (including fast-dissolving, sustained-

released, and liquid-eluting hollow microneedles). III. Injectable drug delivery devices (such as microparticles, solid scaﬀolds, and hydrogel-

based). IV. Other devices for enhancing vaccine eﬃcacy (such as electroporation and iontophoresis). (b) Schematic for representative PKs

of vaccine and administration features for diﬀerent vaccine delivery devices: I. Traditional bolus injection is invasive and may require

multiple doses. II. Fast dissolving or liquid eluting MNs can be self-administrable and minimally invasive but still require multiple doses.125

III. Sustained released MNs can be both minimally invasive, with only one dose necessary,122 while scaﬀold and gel vaccines could be

injectable to avoid implantation and also only require one dose. IV. Pulse-released microparticles can mimic a multiple-dose regimen with

only one injection.127,128 (c) Schematic of representative mechanisms for vaccine delivery devices. I. Scaﬀold vaccines: The scaﬀold could

sustainably release recruiting factors to home DCs, and encapsulated antigens could further be processed by DCs in situ.129 II. Gel vaccines:

Hydrogel microenvironment can be engineered to support encapsulated cell vaccines (such as DC vaccines) by incorporating cell adhesive

peptides and supplementary factors.136

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understood, the PK of a vaccine, which refers to the dynamics

of the antigens presence during immunization, has a profound

impact on immune response.87 One study indicated that

increasing the dosage of vaccines over 2 weeks at both priming

and boost stages induced a durable immune response and

increased antibody production compared to traditional bolus

immunization.87 Also, computational models suggested that

antigens can be better captured in germinal centers (GCs) if

antigen availability is extended.116 In addition to the

advantages of extended antigen availability, traditional

vaccination strategies often fail to yield the desired optimized

immune responses in a single dose. Furthermore, vaccine

regimes are often designed to be multiple shots, thus achieving

an even longer immune protection by rechallenging the

immune system with the same antigens in secondary

“boosting” shots administered after the primary shots.117

Therefore, vaccine delivery devices have been widely

investigated, to develop single-dose vaccines, minimal

invasively administered vaccines, and self-administrable

vaccines (Figure 5a,b). However, its potential can only be

realized if various design principles are met (Table 2).

Microneedles Vaccines. Transdermal microneedles

(MNs) patches have been proposed as a promising drug

delivery device for immunotherapies144−147 based on the

minimally invasive nature of MNs and the highly active

immune environment of the skin. Additionally, since MNs

patch-based vaccines can be easily administered without the

need for signiﬁcant medical training, these vaccines can be

easily deployed both in areas with limited medical resources,

such as developing countries and during times of signiﬁcant

medical crisis, as seen during an outbreak or pandemic. Early

studies include the use of vaccine-coated metal MNs118 and

fast-dissolving polymer-based MNs.119 Recently, the dissolving

MNs (carboxymethyl cellulose-based) were used for adjuvants

and Ad5-based vaccine co-delivery in an attempt to address

COVID-19.147 A 1 month storage of MNs vaccine at 4 °C

maintained the immunogenicity of Ad, where no signiﬁcant

loss in antibody responses was shown in mice studies.147

Similar dissolving MNs are also being investigated to deliver

the SARS-CoV-2 subunit spike protein (S1).120 Potent virus-

speciﬁc antibody responses were elicited in mice as early as 2

weeks after the immunization with MNs. The dissolvable MNs

for inﬂuenza vaccine have been previously evaluated in a phase

I clinical trial compared to the traditional intramuscular

hypodermic injection of a single-dose vaccine.121 The MN

patch was well-tolerated and immunogenic after a single-dose

vaccination. In addition, self-administration exhibited eﬃcacies

comparable to the administration by medical workers. The

dissolving feature leaves no sharp wastes, and self-admin-

istration could signiﬁcantly relieve the pressure on medical

resources, which could be especially critical in coping with a

pandemic. Beyond these milestones, advances in micro-

fabrication, materials engineering, and vaccine formulation

optimization have provided additional opportunities to

enhance vaccine eﬃcacies.148

Recent advances in microfabrication, such as 3D printing

and stereolithography, can allow for more sophisticated MNs

designs.125 Liquid-based vaccine formulations are still the most

widely used.125 However, liquid-based vaccine formulations are

incompatible with most solid-based MNs forms. Inspired by

the capability of snake-fangs to eﬃciently inject venom, Bae et

al. used an exposure lithography-based strategy to produce

MNs patches with snake-fang architectures for eﬃcient liquid

Table 2. Summary of Typical Designs, Advantages, and Limitations of Diﬀerent Vaccine Delivery Devices

delivery devices designs advantages limitations refs

MNs vaccine (minimally invasive; minimal require-

ment on medical professionals and medical

equipment)

fast-dissolving

MNs

fast-dissolving polymers as MNs matrix fast and enhanced transdermal delivery; could be cold-chain free multiple administrations; vaccine for-

mulations may get denatured from

MNs fabrication

118−121

sustained-re-

lease MNs

controlled biodegradable materials as

MNs matrix

potential to be single shot; protect the vaccine formulations in the

physiological environment; could be cold-chain free

vaccine formulations may get dena-

tured from MNs fabrication

122−124

liquid-eluting

MNs

hollow MNs with diﬀerent architectures compatible with liquid vaccine formulation; fast and enhanced

transdermal delivery

multiple administrations; requirement

of cold-chain transport

125

injectable materials assisted vaccine (potential for

single-shot vaccines to reduce the requirement of

medical resources)

pulse-released

microparticle

materials with diﬀerent biodegradation

proﬁles

optimal release for a potent immune responses; release kinetic can

be optimized by materials engineering

vaccine formulations may get dena-

tured during the fabrication

126−128

scaﬀold vac-

cine

solid scaﬀolds that could tune the release

of diﬀerent immunomodulatory factors

continuously modulate the immune response in situ injectability need to be achieved to

avoid surgical implantation

129−134

hydrogel vac-

cine

biomimicking gellable materials with

tunable biophysical properties and high

cytocompatibility

compatible with various types of vaccines, including cell-based

vaccines; simple fabrication process and the water environment

help protect the cargos

in situ gelation may be weak to achieve

sustained release

135−143

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formulations delivery.125 The design was compatible with most

existing vaccine formulations, and the patch can be applied

with the pressure of the thumb and achieve an eﬃcient

transdermal delivery.125 Nevertheless, solid-based vaccines,

including MNs-based ones, also attracted signiﬁcant attention

because of the decreased cost of vaccine manufacturing,

transport, and storage when compared to the liquid-based

formulations.149 One of the key challenges faced by these MNs

patches is the loss of vaccine activity during the drying or

heating process of MNs patch fabrication,150 where molecular

structures of vaccines may be disrupted.151 Diﬀerent strategies

can be adopted, such as the use of lyoprotective polymers as

MNs matrices, which include silk ﬁbroin,122 gelatin, dextran,

and PVP,152 or adding sugar molecules as stabilizing excipients,

including trehalose and sucrose.147,152 In the vaccine discovery

phase, the introduction of certain mutations can improve the

thermal stability of vaccines as well,148 which can be further

applied to speciﬁc MNs formulations. Additionally, a hyper-

stabilized vaccine formulation, such as the interbilayer-cross-

linked multilamellar vesicles, helped protect vaccines during

the drying process of MNs fabrication and have outperformed

other traditional delivery strategies in inducing a potent

antigen-speciﬁc immune response.123

MNs also provide advantages in tuning optimal release

proﬁles of vaccines. As mentioned earlier, the sustained

availability of vaccines is a desirable feature for immunization.

Vaccines targeting diﬀerent infectious diseases may vary in

terms of optimal release proﬁles based on the unique features

of each virus. Initially, MNs for vaccination were mainly an

upgrade to traditional bolus delivery by using a fast-dissolving

MNs matrix,119 which requires multiple doses for suﬃcient

immunity. With the exploration of the desired PK of vaccines,

MNs can play a much more important role. For example, a silk

matrix with crystallized structures could be used as an MNs

matrix to sustainably release loaded vaccines over 2 weeks and

to elicit potent immune responses with one single admin-

istration.122 In addition, biodegradable polymers with known

degradation kinetics can be used to encapsulate the vaccines

and tune antigen release proﬁles. These polymers include

biodegradable cationic PBAE123 and PLGA.124

Injectable Material-Assisted Vaccines. Macroscale

biomaterials strategies have recently boosted the ﬁeld of

immunotherapy.153 There are many advantages in controlled

release and encapsulation of various cargos, ranging from small

molecules to proteins and cells.153 These varied cargo elements

can help to meet the need for diverse types of vaccine

candidates. In terms of controlling PKs of vaccines, both

sustained and pulsatile releases could be possibly achieved.153

In addition, materials engineering can enable devices that do

not require surgically invasive administrations.153 Together,

they could minimize the requirement of medical resources

without mitigating the vaccine eﬃcacy.

PLGA has also been developed for a single-shot vaccine

based on the tunability of its biodegradation proﬁle.126 In the

case of polio vaccines, two to three administrations over several

months are required. Tzeng et al. demonstrated that IPV could

be encapsulated within PLGA microspheres stably and released

through two bursts that were one-month apart.126 They

examined the eﬀect of adding cationic excipients (Eudragit E,

PLL, and branched PEI) to stabilize IPV antigens, protecting

them from denaturation by acidic byproducts, and modulate

degradation proﬁles of PLGA microspheres.126 Notably, a

single shot of PLGA microsphere-based vaccines could elicit

the production of NAb as potent as two bolus injections.126

Combined with the advances in microfabrication and additive

manufacturing, McHugh et al. fabricated microscale (∼400

μm) hollow PLGA-based microparticles that were inject-

able.127 The microparticles can be ﬁlled with vaccine

formulations, and the degradation rate of the microparticle

shell (a lactic/glycolic copolymer) can be tuned to achieve

controlled release spanning from a few days to a few

months.127 Such a design can accomplish the goals of general

transdermal vaccine injection and achieve temporally con-

trolled pulsed delivery of antigens in a single injection.127

Recently, a similar PLGA-based microparticles platform was

applied to release a STING agonist (cyclic guanosine

monophosphate-adenosine monophosphate (cGAMP)) in a

pulsatile manner over a long period (∼16 days).128 One single

injection of the pulsatile drug-releasing microparticles induced

potent immunity as eﬀective as three separate injections. These

engineered platforms are versatile in terms of encapsulating

diﬀerent cargos required by vaccine formulations and tuning

release kinetics correspondingly. However, even though the

use of FDA-approved PLGA could facilitate the clinical

translation to a certain degree, several design limitations

need to be overcome for translating PLGA-based drug delivery

devices into clinical applications. For instance, the pulsatile

release was achieved with hollow microparticles that have thick

PLGA shells to encapsulate the drugs in the hollow core for

controlled release. This design signiﬁcantly sacriﬁced their

loading eﬃciency due to the limited volume of the core.

Clinically, patients need a high drug dosage, and this

formulation needs further optimization to meet the dosage

requirement. In addition, PLGA can create an acidic

microenvironment during biodegradation, damaging the

encapsulated drugs.126 Furthermore, the encapsulated vaccines

need to survive the fabrication process; brief heating requires

thermally stable vaccines.127,128 All these aspects could

compromise their applications for a broader collection of

drugs or vaccines.

Traditionally, ex vivo modiﬁcation of antigen-speciﬁc DC

vaccines is a multistep process associated with high costs and

high levels of complexity.43 It was proposed that in situ DC

recruitment, activation, and further traﬃcking to LNs would be

more desirable compared to the ex vivo modiﬁcation strategy

(Figure 5c).129 The early study by Ali et al. demonstrated the

use of macroporous scaﬀold vaccines based on PLGA, which

presented cytokines (GM-CSF), danger signals (CpG), and

antigens in a spatiotemporally deﬁned manner. GM-CSF can

be sustainably released from the scaﬀolds over 20 days for

eﬀective DC recruitment.129 By co-loading the CpG through

PEI-mediated electrostatic interactions, CpG was immobilized

on the scaﬀolds for local internalization by the recruited DCs.

This process mimicked an infection and was able to

continuously recruit and activate DCs.129 This speciﬁc scaﬀold

vaccine was surgically implanted; nevertheless, it encouraged

further development of injectable vaccines that work similarly.

Kim et al. demonstrated the use of mesoporous silica rods

(MSRs) with a high-aspect-ratio that could be injected with a

syringe needle.130 They could self-assemble in vivo as

macroporous scaﬀolds to initiate potent immune responses.130

The DCs could be recruited into the scaﬀolds where the

inﬂammatory signals (GM-CSF), adjuvants (CpG), and

antigens (OVA) were sustainably released from the scaﬀolds

for in situ DC activation and maturation. It was also

demonstrated that locally injected scaﬀold vaccines based on

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MSRs could elicit strong systemic immune responses with the

increase in levels of antigen-speciﬁc B cells, helper T cells, and

cytotoxic T cells.130 A PEI coating was later added to the

design to enable antigen loading through adsorption for

enhanced immunogenicity.131 This speciﬁc system was

recently utilized for synthetic peptide vaccines and small

antigens.132 This strategy was able to bypass multiple

immunization requirements but sustained a more robust

antibody response, compared to traditional bolus or alum

vaccine formulations.132

Scaﬀold-based vaccines have displayed eﬃcacy in inducing

systemic immune response through in situ immune modu-

lations. In vivo self-assembled scaﬀolds provide a good

alternative to bypass surgically implantation; however, the

self-assembling process is commonly undeﬁned in terms of

how long it could take for self-assembling.133 Materials

engineering facilitates the fabrication of scaﬀolds maintaining

injectability. Bencherif et al. described a strategy for fabricating

injectable scaﬀolds with shape memory based on cryotropic

gelation.134 The cryogels were based on alginate methacrylate,

and they were capable of being reversibly deformed, over 90%

strain, after being chemically cross-linked in a frozen condition

(−20 °C).134 The ice crystals hindered the cross-linking in

frozen spots and therefore formed the macroporous pores that

not only enabled the shape memory for the gel but also

allowed the DCs inﬁltration.134

Hydrogels have been extensively explored in the ﬁeld of

immunotherapy.153 They have advantages in terms of control-

lable delivery of multiple cargos, in situ immunomodulation,

and mild fabrication process for various vaccine formulations

(Figure 5c). Early pioneering work includes the loading of ex

vivo antigen-primed DC vaccines within Ca2+ cross-linked

alginate gel by Hori et al.135 Activated DCs have a short

lifespan, and continuous immune responses may be suppressed

if no suﬃcient host DCs and T cells could be recruited and

primed in vivo.135 Therefore, cell-based vaccines delivered by

gels are expected to improve cell survival and prolong the

presence of DCs.136 Both host DCs and cytotoxic T cells can

be recruited to the injection site by cytokines and chemokines

secreted from the injected DCs. In addition, the enhanced

eﬃcacy of transplanted cells can be enhanced by supplement-

ing the gels with other immunomodulatory molecules, such as

cytokines (IL-2) or adjuvants (CpG).137 The increased

persistence of transplanted cells could be achieved by

designing gels mimicking ECM by the incorporation of cell-

adhesive peptides, such as RGD.154

Apart from cell-based vaccines, other types of vaccines could

also be incorporated into gel systems, where the release proﬁles

can be tuned. Roy et al. utilized the in situ cross-linking

mechanism of tetrafunctional poly(ethylene oxide) amine and

poly(ethylene oxide) succinimidyl glutarate to encapsulate

plasmid DNA.138 The gelation did not interfere with the

supercoiled structure of plasmid DNA, and the gelling ability

can be tuned by adjusting the concentrations of these two

components. Notably, gene expression levels were comparable

to bare DNA, but the duration of expression was signiﬁcantly

longer. Other in situ gel systems explored for vaccination

include temperature-responsive gels139 and Michael-addition-

type hydrogels.140 Singh et al. engineered dextran and PEG-

based hydrogels that could be cross-linked in situ for antigen/

adjuvant delivery.141,142 Umeki et al. designed an immunosti-

mulatory CpG DNA-based hydrogel for antigen delivery.143

To achieve a sustained release of antigens, they proposed to

cationize the antigens by ethylenediamine conjugation where

antigens could form complexes with a negatively charged DNA

hydrogel matrix. The innate immunostimulatory function of

gel and prolonged antigen presence leads to an enhanced

antigen-speciﬁc immune response with less toxicity.143 In situ

gelling strategies or physically cross-linked gels from these

studies generally take minutes to hours, which renders limited

control on cargos release and material properties during the

gelling period.143 Therefore, the development of gel systems

that are both injectable and display deﬁned cargo release/

biodegradation proﬁles could be desirable for vaccine

administration.

Other Delivery Devices. Many more investigations are

currently ongoing to engineer alternative drug delivery devices.

For instance, to promote the uptake of DNA vaccine by cells,

electroporation is one of the traditional and eﬀective strategies

to enhance the uptake of plasmids.155 Diﬀerent parameters,

such as electrical features (voltage, current, and frequency) and

length of the electrode (intradermal or intramuscular targets),

need to be optimized to ensure eﬃcacy and safety and to

minimize pain. This process has recently demonstrated

promising clinical phase I results for MERS.25 The same

electroporation device (CELLECTRA) is also being inves-

tigated for a DNA vaccine of SARS-CoV-2 from Inovio.156 The

S protein of SARS-CoV-2 encoding DNA vaccine demon-

strated potent antigen-speciﬁc T cell responses and the

production of neutralization antibodies in preclinical mice

and guinea pigs studies.156 In order to incorporate the

advantages of MNs, electroactive MNs devices have also

been developed for the electroporation of DNA vaccine in a

minimally invasive manner.157 Similar devices using iontopho-

resis can also enhance the transdermal delivery of vaccines.158

Recently, Tadros et al. designed millimeter-scale star-shaped

particles, termed STAR particles, based on aluminum oxide for

topical vaccine delivery,159 and the minimally invasive STAR

particles elicited an immune response comparable to intra-

muscular injection.159 Even though STAR particles are simple

to manufacture and well-tolerated by the immune system, a

much higher dosage was administered compared to intra-

muscular injection. In addition, potential human-to-human

variability in administration and drug leftovers on the skin may

result in diﬀerential eﬃcacy and undesirable toxicity.

In terms of reducing the demands for medical resources,

vaccines that could be intranasally or orally administered are

desired if their eﬃcacy is comparable to transdermal delivery.44

The main strategy for intranasal or oral vaccine development is

currently limited to weakened viral vaccines mimicking natural

infections. Nonliving vaccines are safer but less immunogenic

and thus may beneﬁt greatly from drug delivery formulations

and devices.160 A variety of drug delivery devices have been

proposed for oral vaccination delivery. For instance, the

micromotor-driven microparticles for oral delivery of anti-

gens.161,162 The micromotor vaccine can eﬃciently enter the

intestine to enhance the antigen uptake. Aran et al. designed a

needle-free oral microjet where the high-pressure liquid jet of

the vaccine was produced by a self-contained gas-producing

reaction.163 It was shown that a buccal immunity response was

enhanced, and this method was able to elicit a potent humoral

immune response both locally and systemically.163 Abramson

et al. engineered a self-orienting system for oral delivery that

could attach to the gastric wall by autonomously positioning to

maximize the delivery eﬃciency.164 Innovations in drug

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delivery devices will signiﬁcantly transform traditional

vaccination regimes.

SUMMARY AND FUTURE DIRECTIONS

The devastating disruptions caused by the sudden outbreak of

COVID-19 has urged vaccine development at an unprece-

dented speed.4 Within 70 days of the ﬁrst release of the genetic

information on SARS-CoV-2, the ﬁrst dose of a vaccine was

administered to human participants.9 As of August 25, 2020,

31 vaccine candidates are in various stages of clinical trials, and

around 142 candidates are in preclinical trials.10 Apart from the

high diversity of the vaccine candidates, many candidates are

based on diﬀerent technological platforms, such as nano-

technology delivered mRNA vaccine, electroporation device-

enabled DNA vaccine, and MN-delivered vaccine. The

COVID-19 pandemic has accelerated the deployment of

diﬀerent vaccine formulations and delivery devices. It is

reasonable to expect that more eﬀorts will be dedicated to

further optimize the vaccine designs to help ﬁght future

outbreaks.

Here, we reviewed recent research eﬀorts in the ﬁeld of

vaccine discovery, vaccine formulation development, and

vaccine delivery device designs. Because of the deployment

of various engineering technologies, it is expected that future

vaccines will not only need to demonstrate safety and eﬃcacy

but also to be relatively convenient and simple to use for better

coping with population-level vaccinations or sudden outbreaks

worldwide. Nanoengineered formulations could enhance the

immune response by delivering the required components

together and maximizing their uptake by immunocompetent

cells. To expedite the future preparedness of vaccines for the

occurrence of a pandemic, synthetic vaccines have demon-

strated great potential for rapid clinical applications because

they are molecularly deﬁned and independent of the time-

consuming culture of viruses, which can accelerate the initial

vaccine development. However, synthetic vaccines are also the

ones that need to be formulated the most, which constitutes

the major part of their time costs. To accelerate the

development of synthetic vaccines, the nanoengineered

formulations need to be simple and robust to manufacture in

terms of chemistry or the selection of raw materials.165,166 In

terms of vaccine delivery devices, self-administrable vaccines,

single-shot vaccines, and solid MN-based vaccines could

further enhance the availability of vaccines to a broader

population. For instance, self-administrable vaccines can be

mailed to patients at home without the need of medical

experts.165 Single-shot vaccines can be much more convenient

compared to the traditional immunization regimens that

require multiple shots. Furthermore, solid MN-based vaccines

that are cold chain free could simplify the vaccine trans-

portation and distribution process. All of these aspects could

be enabled by engineering approaches to accelerate the

development of vaccines at a pandemic speed.165,166 Although

combining the advantages of vaccine formulations and vaccine

delivery devices is promising, micro/macro-engineering still

needs to develop solutions that do not require “harsh”

conditions to be compatible with nanoengineered formula-

tions.167 Expectedly, platform technologies that can be quickly

adapted to address emerging viral diseases could speed up the

pace of vaccine development.

However, several challenges remain before these goals can

be fully achieved. First, a diﬀerently identiﬁed virus is unknown

in terms of its interactions with our immune system and which

type of immune responses are most desirable to trigger

prolonged immunity. An improved understanding of our

immune system and its interactions with viruses are the basis

for designing an eﬀective vaccine, which also guides the steps

for designing corresponding vaccine administration regimens.

Second, innovative formulations or delivery devices for

vaccines are mostly in the preclinical stage, which have not

been approved by the FDA yet. For example, MN-based

vaccines showed encouraging results from a phase I trial of

dissolvable MN-based inﬂuenza vaccine and indicated a

promise of using it as an alternative strategy for vaccination.121

However, patient compliance with this approach and its

eﬃcacy needs to be tested in a larger population, considering

only 100 human subjects are currently involved. Also, the

eﬃcacy of vaccines by self-administration should be further

validated in comparison to those performed by medical

experts. In addition, the proposed formulations and delivery

devices should be easy to manufacture with minimal batch-to-

batch variability to simplify the quality control process. The

raw materials used could prioritize the FDA-approved ones to

simplify the regulatory process, and the required chemistry is

preferred to be simple and robust. Even though advanced

manufacturing technologies excel in reducing batch-to-batch

variability, a high cost may be associated. The improved

eﬃcacy of a vaccine from these formulations and delivery

devices should not be complicated by the fabrication process

so that it can facilitate the clinical translation and regulatory

approvals.165

Additional challenges for vaccine development also exist

during a pandemic. Multiple vaccine candidates ﬂush into

diﬀerent stages of clinical trials that could burden the

regulatory process by the FDA for selecting promising

candidates and deciding ﬁnal ones.4 Furthermore, human

trials are starting even before the most suitable animal models

for safety and eﬃcacy are deﬁned.168 Based on previous

successful preclinical and early stage clinical data for a similar

platform, animal studies are even skipped directly for human

administrations.10 Other engineering approaches such as

organs-on-a-chip169−171 or screening using organoids172 may

serve as a promising tool in future clinical trials for eﬃcacy and

toxicity determination. Ethically controversial human challenge

trials by deliberately infecting humans with a virus have been

proposed and pursued to accelerate the process of vaccine

development.173 Therefore, potentially we should have vaccine

clinical trials speciﬁcally designed for diﬀerent scenarios by

incorporating engineering advances and regulatory guidelines

readily outlined for unusual trials. In conclusion, human health

and societal stability are constantly challenged by sudden virus

outbreaks, and the rapid development of eﬀective vaccines is

believed to be an ultimate solution. We expect to observe faster

and more eﬃcient vaccine development paradigms in the

future by combining the advances in diﬀerent engineering

ﬁelds.

AUTHOR INFORMATION

Corresponding Authors

Ali Khademhosseini −Department of Bioengineering, Center for

Minimally Invasive Therapeutics, Department of Chemical and

Biomolecular Engineering, California NanoSystems Institute,

Jonsson Comprehensive Cancer Center, and Department of

Radiological Sciences, University of California, Los Angeles, Los

Angeles, California 90095, United States; Terasaki Institute for

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ACS Nano 2020, 14, 12370−12389

12383

Biomedical Innovation, Los Angeles, California 90064, United

States; Email: khademh@terasaki.org

Wujin Sun −Department of Bioengineering, Center for

Minimally Invasive Therapeutics, and California NanoSystems

Institute, University of California, Los Angeles, Los Angeles,

California 90095, United States; Terasaki Institute for

Biomedical Innovation, Los Angeles, California 90064, United

States; orcid.org/0000-0002-3167-111X; Email: wsun@

terasaki.org

Authors

Xingwu Zhou −Department of Bioengineering and Center for

Minimally Invasive Therapeutics, University of California, Los

Angeles, Los Angeles, California 90095, United States;

Department of Pharmaceutical Sciences, University of Michigan,

Ann Arbor, Michigan 48109, United States

Xing Jiang −School of Nursing, Nanjing University of Chinese

Medicine, Nanjing 210023, China

Moyuan Qu −The Aﬃliated Stomatology Hospital, Zhejiang

University School of Medicine, Key Laboratory of Oral

Biomedical Research of Zhejiang Province, Zhejiang University

School of Stomatology, Hangzhou 310006, China

George E. Aninwene, II −Department of Bioengineering and

Center for Minimally Invasive Therapeutics, University of

California, Los Angeles, Los Angeles, California 90095, United

States

Vadim Jucaud −Terasaki Institute for Biomedical Innovation,

Los Angeles, California 90064, United States

James J. Moon −Department of Pharmaceutical Sciences,

University of Michigan, Ann Arbor, Michigan 48109, United

States

Zhen Gu −Department of Bioengineering, Center for Minimally

Invasive Therapeutics, California NanoSystems Institute, and

Jonsson Comprehensive Cancer Center, University of California,

Los Angeles, Los Angeles, California 90095, United States

Complete contact information is available at:

https://pubs.acs.org/10.1021/acsnano.0c06109

Author Contributions

●These authors contributed equally to this work.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

This work was supported by National Institute of Health

(CA214411, GM126831, and AR069564).

VOCABULARY

Coronavirus disease 2019 (COVID-19), a disease caused by

severe acute respiratory syndrome coronavirus 2 (SARS-CoV-

2) and has been declared as a pandemic by WHO since March

11, 2020; antiviral vaccine, a biological preventive preparation

strategy to train the immune systems to ﬁght against viral

pathogens; vaccine formulations, biomaterial engineering

strategies aiming to boost the eﬃcacy or reduce side eﬀects

of vaccines through co-delivering adjuvants, targeting immune

cells, or protecting vaccines activities; vaccine delivery

devices, tools for administering the vaccine formulations, and

they can be used by medical workers or the patients

themselves; pattern recognition receptors (PRRs), proteins

that can recognize pathogenic molecules as part of innate

immunity in response to virus infection, including identifying

pathogen-associated molecular patterns from a virus; adju-

vants, supplementary agents that could be co-delivered with

vaccines to boost the immune response, minimize the dosage

of antigens, and maintain longer immunity; lymph nodes,

round or bean-shaped clusters of cells containing abundant

immune cells that can initiate the robust immune response

toward viral infections

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