Microneedle Array Delivered Recombinant Coronavirus Vaccines Immunogenicity and Rapid Translational Development Eun Kim Download 2024 Full Chapter

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JID: EBIOM
ARTICLE IN PRESS [m5G;April 1, 2020;16:51]
EBioMedicine 000 (2020) 102743
Contents lists available at ScienceDirect
EBioMedicine
journal homepage: www.elsevier.com/locate/ebiom
Research paper
Microneedle array delivered recombinant coronavirus vaccines:

Immunogenicity and rapid translational development
Eun Kima, Geza Erdosb, Shaohua Huanga, Thomas W. Kennistona, Stephen C. Balmertb,
Cara Donahue Careyb, V. Stalin Raje,1, Michael W. Epperlyc, William B. Klimstrad,
Bart L. Haagmanse, Emrullah Korkmazb,f, Louis D. Falo Jr.b,f,g,h,*, Andrea Gambottoa,**
a
Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, W1148 Biomedical Science Tower, 200 Lothrop St., Pennsylvania, PA 15213, USA
b
Department of Dermatology, University of Pittsburgh School of Medicine, W1150 Biomedical Science Tower, 200 Lothrop St., Pittsburgh, PA 15213, USA
c
Department of Radiation Oncology, University of Pittsburgh, Pittsburgh, PA 15213, USA
d
Center for Vaccine Research, Department of Immunology, University of Pittsburgh, Pittsburgh, PA 15213, USA
e
Department of Viroscience, Erasmus Medical Center Rotterdam, Rotterdam, the Netherlands
f
Department of Bioengineering, Swanson School of Engineering, University of Pittsburgh, Pittsburgh, PA 15231, USA
g
Clinical and Translational Science Institute, University of Pittsburgh, Pittsburgh, PA 15213, USA
h
The McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA 15219, USA
A R T I C L E I N F O A B S T R A C T
Article History: Background: Coronaviruses pose a serious threat to global health as evidenced by Severe Acute Respiratory Syn-
Received 16 March 2020 drome (SARS), Middle East Respiratory Syndrome (MERS), and COVID-19. SARS Coronavirus (SARS-CoV), MERS
Revised 18 March 2020 Coronavirus (MERS-CoV), and the novel coronavirus, previously dubbed 2019-nCoV, and now officially
Accepted 18 March 2020
named SARS-CoV-2, are the causative agents of the SARS, MERS, and COVID-19 disease outbreaks, respectively.
Available online xxx
Safe vaccines that rapidly induce potent and long-lasting virus-specific immune responses against these infectious
agents are urgently needed. The coronavirus spike (S) protein, a characteristic structural component of the viral
Keywords:
envelope, is considered a key target for vaccines for the prevention of coronavirus infection.
Subunit vaccines
Trimerization
Methods: We first generated codon optimized MERS-S1 subunit vaccines fused with a foldon trimerization
Microneedle array domain to mimic the native viral structure. In variant constructs, we engineered immune stimulants (RS09 or fla-
MERS-CoV S1 gellin, as TLR4 or TLR5 agonists, respectively) into this trimeric design. We comprehensively tested the pre-clini-
SARS-CoV-2 cal immunogenicity of MERS-CoV vaccines in mice when delivered subcutaneously by traditional needle
COVID-19 injection, or intracutaneously by dissolving microneedle arrays (MNAs) by evaluating virus specific IgG antibodies
in the serum of vaccinated mice by ELISA and using virus neutralization assays. Driven by the urgent need for
COVID-19 vaccines, we utilized this strategy to rapidly develop MNA SARS-CoV-2 subunit vaccines and tested
their pre-clinical immunogenicity in vivo by exploiting our substantial experience with MNA MERS-CoV vaccines.
Findings: Here we describe the development of MNA delivered MERS-CoV vaccines and their pre-clinical immuno-
genicity. Specifically, MNA delivered MERS-S1 subunit vaccines elicited strong and long-lasting antigen-specific
antibody responses. Building on our ongoing efforts to develop MERS-CoV vaccines, promising immunogenicity of
MNA-delivered MERS-CoV vaccines, and our experience with MNA fabrication and delivery, including clinical trials,
we rapidly designed and produced clinically-translatable MNA SARS-CoV-2 subunit vaccines within 4 weeks of the
identification of the SARS-CoV-2 S1 sequence. Most importantly, these MNA delivered SARS-CoV-2 S1 subunit vac-
cines elicited potent antigen-specific antibody responses that were evident beginning 2 weeks after immunization.
Interpretation: MNA delivery of coronaviruses-S1 subunit vaccines is a promising immunization strategy
against coronavirus infection. Progressive scientific and technological efforts enable quicker responses to
emerging pandemics. Our ongoing efforts to develop MNA-MERS-S1 subunit vaccines enabled us to rapidly
design and produce MNA SARS-CoV-2 subunit vaccines capable of inducing potent virus-specific antibody
responses. Collectively, our results support the clinical development of MNA delivered recombinant protein
subunit vaccines against SARS, MERS, COVID-19, and other emerging infectious diseases.
© 2020 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license.
(http://creativecommons.org/licenses/by/4.0/)
* Corresponding author at: Department of Dermatology, University of Pittsburgh School of Medicine, W1150 Biomedical Science Tower, 200 Lothrop St., Pittsburgh, PA 15213, USA.
** Corresponding author at: Department of Surgery, University of Pittsburgh, W1148 Biomedical Science Tower, 200 Lothrop St., Pittsburgh, PA 15213, USA.
E-mail addresses: lof2@pitt.edu (L.D. Falo), gambottoa@upmc.edu (A. Gambotto).
1
Present address: School of Biology, Indian Institute of Science Education and Research Thiruvananthapuram (IISER-TVM), Kerala, India
https://doi.org/10.1016/j.ebiom.2020.102743
2352-3964/© 2020 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license. (http://creativecommons.org/licenses/by/4.0/)
Please cite this article as: E. Kim et al., Microneedle array delivered recombinant coronavirus vaccines: Immunogenicity and rapid
translational development, EBioMedicine (2020), https://doi.org/10.1016/j.ebiom.2020.102743
JID: EBIOM
2 E. Kim et al. / EBioMedicine 00 (2020) 102743
HCoV was suspected, and on January 9, 2020, the Chinese Center for
Research in context
Diseases Control reported that a novel coronavirus was the causative
agent. Shortly thereafter the genomic sequences of several isolates
Evidence before this study
became publicly available. On February 10, 2020, the WHO named
Coronavirus is an emerging pathogen with exponentially the disease, caused by the new coronavirus, COVID-19, and the new
increasing significance due to the high case fatality rate, the virus was named severe acute respiratory syndrome coronavirus 2
large distribution of reservoir, and the lack of medical counter- (SARS-CoV-2). Genomic analyses indicated that SARS-CoV-2 shares
measures. The public health emergencies caused by Coronavi- genomic similarities with SARS-CoV within the receptor-binding
ruses, including SARS-CoV, MERS-CoV, and now SARS-CoV-2, motif that directly contacts the human receptor ACE2. This has
clearly demonstrate the urgency to evaluate candidate vaccines important implications for vaccine design and for predicting pan-
to combat these outbreaks. Continuous research efforts from demic potential. Human-to-human transmission of SARS-CoV-2 has
previous epidemics contribute to the efforts of investigators to now been established, and the situation with SARS-CoV-2 is evolving
quickly develop safe vaccines against these emerging infec- rapidly with the numbers of verified cases growing into the thou-
tions; however, the recent COVID-19 pandemic highlights an sands. Accordingly, The World Health Organization (WHO) has
important continuing need for the rapid design, production, declared COVID-19 a global pandemic.
testing, and clinical translation of candidate vaccines. Another recently emerged coronavirus, the Middle East Respiratory
Syndrome Coronavirus (MERS-CoV) was first isolated in 2012 in Saudi
Added value of this study Arabia [1]. As of January 31st, 2020 the World Health Organization
(WHO) has been notified of 2,513 laboratory-confirmed cases of infec-
These studies demonstrate the rapid development and immu-
tion with MERS-CoV that include 864 related deaths with a fatality
nogenicity of novel microneedle array (MNA) delivered recom-
rate of 34.4% [2]. Cases have arisen in 27 countries globally, including a
binant coronavirus (SARS-CoV-2) vaccines. MNA delivered
severe outbreak in the Republic of Korea in 2015 [3,4]. MERS-CoV is
MERS-S1 subunit vaccines induced potent and long-lasting
still endemic in the Middle East, most particularly in Saudi Arabia,
antigen antigen-specific immune responses. Notably, MNA
where more than 80% of cases have occurred. No efficacious drugs or
delivery of these vaccines generated significantly stronger
vaccines are currently approved for human use [2,5]. The WHO
immune responses than those administered by traditional sub-
included MERS-CoV on the list of priority blueprint diseases for which
cutaneous needle injection, indicating the improved immuno-
there is an urgent need for accelerated research and development of a
genicity by skin-targeted delivery. These efforts with MNA
safe and efficacious vaccine [5,6].
MERS-S1 subunit vaccines enabled the rapid design and pro-
For both these coronaviruses, the spike (S) protein is crucial for
duction of MNA SARS-CoV-2 vaccines, capable of eliciting
viral transmission and infection, and determines the tropism of the
potent virus-specific antibody responses that were evident as
virus and host cell entry. SARS-CoV-2 binds the ACE2 receptor as
early as 2 weeks after immunization. Rapid design and produc-
MERS-S binds to the cellular receptor dipeptidyl peptidase 4 (DPP4)
tion of MNA-embedded SARS-CoV-2-S1 subunit vaccines using
via the receptor-binding domain (RBD) in the N-terminal surface sub-
clinically applicable MNA fabrication methods supports the
unit (S1), and then employs its C-terminal transmembrane subunit
development of MNA delivered recombinant coronavirus vac-
(S2) to fuse with the host cell membrane. [7-11] Due to this vital
cines for clinical vaccine applications. Collectively, our studies
functional property and established antigenicity, the S protein is an
support the clinical development of MNA protein subunit vac-
important target for vaccine development. Our previous studies have
cines for COVID-19 and other emerging infectious diseases.
shown that adenoviral vaccine candidates expressing SARS-CoV-S1
and MERS-S1 subunits induced more efficacious antibody-mediated
Implications of the available evidence
neutralizing activity than full-length S1, suggesting the subunit
MNA delivery of coronaviruses-S1 subunit vaccines is a promising immunogen as an ideal vaccine candidate [12]. The native S proteins
immunization strategy against MERS-CoV and SARS-CoV-2 infec- exist as a trimeric form on the surface of the virus. However, when its
tion and can be adapted for other subunit vaccines against a broad ectodomain or S1 is expressed as a recombinant protein in eukaryotic
range of infectious diseases. The recent advances in recombinant systems, the protein exists predominantly in a monomeric form
DNA technology and vaccine delivery strategies enable quick [13,14]. Here, to make trimeric recombinant codon optimized subunit
design, production, and testing of vaccines against emerging proteins mimicking the native viral structures, we have fused the
infections. We plan to evaluate these SARS-CoV-2 vaccines for MERS-CoV-S1 and SARS-CoV-2-S1 segments to a 27-amino acid
other important predictors of vaccine efficacy in humans, includ- sequence called the foldon domain, which is a natural trimerization
ing the induction of neutralizing antibodies and for their ability to domain derived from the C terminus of bacteriophage T4 fibritin
prevent infection in animal challenge models, when these assays [15,16]. Inclusion of a foldon trimerization domain promotes a tri-
and preclinical models become available. Thus far, our studies meric structure and has been shown to increase the immunogenicity
suggest that it may now be possible to produce clinical grade vac- of several viral antigens including Influenza virus hemagglutinin,
cines against novel pathogens for human testing and subsequent HIV-1 glycoprotein, and SARS-CoV S1/S-ectodomain, supporting the
global distribution in time to significantly impact the spread of importance of mimicking native viral trimeric structures [14,17,18].
disease. Further, recent work has shown that a recombinant trimeric recep-
tor-binding domain (RBD) of MERS-CoV, made by fusing RBD with
foldon domain (RBD-Fd), elicits potent RBD-specific neutralizing anti-
bodies and protects hDPP4 transgenic mice from viral infection [19].
To potentially improve the immunogenicity of these vaccines, we
also constructed S1 subunits with integrated Toll-like receptor (TLR)
1. Introduction
agonist sequences, specifically RS09 and flagellin C. RS09 is a syn-
thetic form of an LPS mimic 7-mer peptide, which binds to TLR4 and
On December 31, 2019, Chinese authorities in Wuhan announced
induces nuclear localization of the transcription factor NF-kB, result-
a cluster of pneumonia cases of unknown etiology, most of which
ing in transcription of inflammatory cytokines and secretion of che-
included patients who reported exposure to Wuhan’s Huanan Sea-
mokines [20]. Flagellin is a highly conserved natural bacterial ligand
food wholesale market. Emergence of another pathogenic zoonotic
that binds to TLR5 to activate TLR5 expressing dendritic cells,
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E. Kim et al. / EBioMedicine 00 (2020) 102743 3
neutrophils, lung epithelial cells and pneumonocytes, augmenting (Lonza), at HindIII/NotI sites. For the construction of pmax/SARS-CoV-
immunogenicity in several vaccine models [21 23]. 2-S1fRS09, BamH I-6H-Not I of pmax/SARS-CoV-2-S1 was replaced
The skin is an ideal target for immunization. It contains a rich popu- with codon optimized BamH I-f-RS09(APPHALS)-Tp6H-Not I.
lation of antigen presenting and immune accessory cells capable of
inducing a proinflammatory microenvironment favoring the induction 2.2. Purification of recombinant proteins
of potent and durable adaptive immunity [24,25]. Of several emerging
skin-targeted drug delivery methods, dissolving MNAs have emerged as The recombinant proteins named rMERS-S1f, rMERS-S1fRS09, and
a minimally-invasive intracutaneous approach [26 28]. MNAs used rMERS-S1ffliC were expressed in Human Embryonic Kidney (HEK)
here are developed from mechanically strong water-soluble polymers 293 cells infected with Ad5.MERS-S1f, Ad5.MERS-S1fRS09, and Ad5.
to physically breach the outermost layer of skin (stratum corneum) and MERS-S1ffliC, respectively, and purified by His60 Ni Superflow Resin
then rapidly dissolve in the underlying viable epidermis and dermis to (Clontech) under native conditions from the supernatant as described
deliver cargos to skin microenvironments [27,29]. previously [40]. Briefly, the purified recombinant proteins were
Importantly, MNA delivery results in high concentrations of vaccine treated with AcTEV protease (Life Technology) followed by affinity
components in the local skin microenvironment, thereby contributing chromatography on a His60 Ni Superflow Resin to remove six-histi-
to a dose-sparing effect that could decrease the required vaccine doses dine tags and poly-histidine tagged protease from the cleavage reac-
for efficacious immunization and substantially reduce both cost and tion. The cleaved native recombinant proteins were collected from
toxicity [27,30-32]. Although the stability of each vaccine candidate in the flow-through fraction. The eluted solution was concentrated and
MNAs needs to be validated at different temperatures for varying stor- desalted with phosphate buffered saline (PBS) in an Amicon Ultra
age periods, the available literature indicates that MNA-embedded centrifugal filter devices (Millipore).
vaccines have the potential to remain stable for an extended period of For expression of recombinant proteins, rSARS-CoV-2-S1 and
time without expensive “cold chain” requirements [33,34]. Further, rSARS-CoV-2-S1fRS09, 293HEK cells were transfected by electropora-
previous animal and clinical studies suggest that MNAs could provide tion (Celetrix). Briefly, 293HEK cells were counted and suspended with
a safe and well-tolerated delivery platform for efficacious immuniza- plasmids in the electroporation buffer at 5 £ 107 cells, 200mg/ml. The
tion strategies [35 39]. mixture containing cells and plasmids was transferred into 1ml Cele-
Here, we demonstrate the rapid development of MNA SARS-CoV-2 trix electroporation tube with 25 mm distance between the electrodes,
S1 subunit vaccines and their promising immunogenicity in mice devel- and immediately subjected to electroporation under 930V and 30ms,
oped using our evolving experience with MERS-CoV vaccines and and then incubated for 72 hrs at 37°C in a humidified atmosphere
MNAs. First, we evaluate the immunogenicity of trimeric form of MERS- with 5% CO2. The recombinant proteins, rSARS-CoV-2-S1 and rSARS-
S1 subunit vaccines delivered subcutaneously by traditional needle CoV-2-S1fRS09, were purified by His60 Ni Superflow Resin (Clontech)
injection, or intracutaneously by MNAs. Driven by the promising immu- under native conditions from the supernatant as described previously
nogenicity of MNA-MERS-S1 vaccines and the urgent need to respond [40]. The concentrations of the purified recombinant proteins were
to the recent coronavirus pandemic (COVID-19), we rapidly (within determined by the Bradford assay using bovine serum albumin (BSA)
4 weeks of the identification of the SARS-CoV-2 S1 sequence) designed as a protein standard.
and produced MNA SARS-CoV-2 S1 vaccines and tested their immuno-
genicity in mice. We describe the rapid development of MNA embedded 2.3. SDS-PAGE and Western blot
SARS-CoV-2-S1 subunit vaccines using clinically-applicable MNA pro-
duction methods by relying on our experience with clinical trials utiliz- To evaluate the recombinant adenoviruses, A549 cells were trans-
ing MNA delivery (ClinicalTrials.gov Identifier: NCT02192021). Taken duced with ten multiplicity of infection (MOI) of Ad5.MERS-S1f, Ad5.
together, our results support the development of MNA delivered recom- MERS-S1fRS09, and Ad5.MERS-S1ffliC. At six hours after infection,
binant coronavirus vaccines for clinical applications. cells were washed three times with PBS, and added with serum-free
media, and incubated for 48 h. The supernatant of A549 infected with
2. Materials and methods adenoviruses were subjected to sodium dodecyl sulfate polyacryl-
amide gel electrophoresis (SDS-PAGE) and Western blot. Briefly, after
2.1. Construction of adenoviral vectors and plasmids the supernatants were boiled in Laemmli sample buffer containing
2% SDS, or native sample buffer, with or without beta-mercaptoetha-
The codon-optimized gene encoding MERS-S1 (amino acids 1 to nol (b-ME), the proteins were separated by Tris-Glycine SDS-PAGE
725 of full-length MERS S, according to the GeneBank JX869059) gels and then transferred to polyvinylidene fluoride (PVDF) mem-
lacking stop codon flanked with SalI & BamHI was amplified by PCR brane. After blocking for 1h at room temperature with 5% non-fat
from pAd/MERS-S1 12 and was used to replace the ZIKV-E in in pAd/ milk in PBST, anti-6xHis monoclonal antibody (1:50000) (Invitrogen)
ZIKV-Efl 40 at SalI & BamHI sites fused with BamH I-linked T4 fibritin were added and incubated overnight with at 4 °C as primary anti-
foldon trimerization domain (f), Tobacco Etch Virus Protease (Tp), body, and horseradish peroxidase (HRP)-conjugated goat anti-mouse
and six histidine tag (6H), generating plasmid pAd/MERS-S1f. For IgG (1:10000) (SantaCruz) was added and incubated at RT for 2 h as
the construction of pAd/MERS-S1fRS09 or pAd/MERS-S1ffliC, BamH secondary antibody. After washing three times with PBS, the signals
I-fTp6H-Not I of pAd/MERS-S1f was replaced with codon optimized were visualized using ECL Western blot substrate reagents and Amer-
BamH I-f-RS09(APPHALS)-Tp6H-Not I or BamH I-f-fliC (GenBank sham Hyperfilm (GE Healthcare).
ACY88831)-Tp6H-Not I. Subsequently, replication-defective human
adenovirus serotype 5, designated as Ad5.MERS-S1f, Ad5.MERS- 2.4. Fabrication of dissolvable microneedle arrays
S1fRS09, and Ad5.MERS-S1ffliC, were made by loxP homologous
recombination and purified and stored as described previously Dissolvable microneedle arrays (MNAs) incorporating the protein
[41 43]. MERS-S1f, MERS-S1fRS09, MERS-S1ffliC, SARS-CoV-2-S1, or SARS-
For the construction of pmax/SARS-CoV-2-S1, the codon-optimized CoV-2-S1fRS09) were fabricated from carboxymethyl cellulose (CMC,
gene encoding SARS-CoV-2-S1 amino acids 1 to 661 of full-length 90 kDa MW) at room temperature (22°C) using our previously
from BetaCoV/Wuhan/IPBCAMS-WH-05/2020 (GISAID accession id. described three-stage MNA manufacturing strategy [29,32]. Briefly,
EPI_ISL_403928) lacking stop codon flanked with HindIII-SalI & the production molds were prepared by casting Polydimethylsiloxane
BamHI-6H-NotI was synthesized (GenScript) and generated by sub- (PDMS, SYLGARDÒ 184) elastomer onto the micromachined MNA mas-
cloning the codon-optimized SARS-CoV-2-S1 gene into pmaxCloning ter molds that include 100 obelisk-shaped microneedles in a 10 £ 10
JID: EBIOM
array. The height, width, and apex angle of microneedles were chosen EDTA, for 2-3 min at 37°C, followed by trypsin inactivation by adding 5
to be 750 mm, 225 mm, and 30°, respectively. Next, CMC-based MNA- volumes of 10% FBS media, then washed with phosphate buffered
rMERS-S1f, MNA-rMERS-S1fRS09, MNA-rMERS-S1ffliC, MNA-rSARS- saline (PBS), and incubated with the mouse antiserum from each group
CoV-2-S1, or MNA-rSARS-CoV-2-S1fRS09 vaccines were produced followed by staining with a PE-conjugated anti-mouse secondary IgG
through a two-step spin-drying technique. First, 20 ml of purified pro- antibody (Jackson Immuno Research). Data acquisition and analysis
tein was dispensed onto each MNA production mold and centrifuged were performed using LSRII (BD) and FlowJo (Tree Star) software.
for 1 min at 2500 g to fill the microneedle cavities. Subsequently, the
excess solution was removed, and the residual protein solution was 2.7. ELISA
spin-dried into the tip of the microneedle cavities of the mold by cen-
trifugation at 2500 g for 30 min. The tip-loaded protein in the produc- Sera from the animals were collected every two weeks and tested
tion molds was overlayered with 75 mg of CMC hydrogel (20% w/w) to for MERS-S1 protein-specific IgG by conventional ELISA as previously
form the mechanically strong microneedles and the backing layer of described [12]. For long-lasting immunity, sera from the animals were
the MNAs. The molds loaded with the CMC-hydrogel were then centri- collected at week 23 and week 55 after primary inoculation and tested
fuged for 3 h at 2500 g to obtain final dissolvable MNA vaccines. The for MERS-S1 protein-specific IgG by ELISA. Briefly, 96-well plates were
theoretical loading of each MNA-rMERS-S1f, MNA-rMERS-S1fRS09, coated with the serum-free media from A549 cells infected with 10
MNA-rMERS-S1ffliC, SARS-CoV-2-S1, or SARS-CoV-2-S1fRS09 was 25 MOI of Ad5.MERS-S1 for 48 h overnight at 4°C in carbonating buffer
mg, 25 mg, 40 mg, 20 mg, or 20 mg proteins, respectively. The geomet- (pH 9.5) and then blocked with PBS containing 0.05% Tween 20 (PBS-
ric integrity of the fabricated MNAs was confirmed through optical T) and 2% bovine serum albumin (BSA) for 1 hr. Mouse sera were
stereomicroscopy imaging before the experiments. Importantly, this diluted 1:200 in PBS-T with 1% BSA and incubated for two hours. After
MNA design and fabrication strategy has also been used for MNAs uti- the plates were washed, anti-mouse IgG-horseradish peroxidase
lized in our ongoing Phase 1 clinical trials (ClinicalTrials.gov Identifier: (HRP) (1:2000, SantaCruz) were added to each well and incubated for
NCT02192021). In line with our efforts toward clinical production of one hour. The plates were washed three times and developed with
MNA SARS-CoV-2 subunit vaccines, we gamma irradiation (25 kGy) 3,3’5,5’-tetramethylbenzidine, and the reaction was stopped with 1M
sterilized a small group of these MNA SARS-CoV-2 subunit vaccines to H2SO4 and absorbance at 450 nm was determined using an ELISA
determine clinically relevant sterilizing conditions and any effect on reader (PerkinElmer 2030). For the binding of the recombinant pro-
immunogenicity. teins and mouse sera against Ad5.MERS-S1, the 96-well plates were
coated with 200 ng of the purified recombinant proteins per well over-
2.5. Animals and Immunization night at 4°C in carbonate coating buffer (pH 9.5), followed by addition
of diluted pre-immunized sera and sera from the mouse infected with
BABL/c mice (five animals per group) were inoculated subcutane- AdC5 or Ad5.MERS-S1 [12], and carried out sequentially based on a
ously with 25 mg of MERS-S1f only, 25 mg of MERS-S1f plus 20 mg of protocol similar to that described above. For ELISA of SARS-CoV-2-S1
MPLA (monophosphoryl lipid A; TLR-4 agonist), 25 mg of MERS- antibody, the 96-well plates were coated with 200 ng of the purified
S1fRS09, and 40 mg of MERS-S1ffliC or PBS as a negative control and rSARS-CoV-2-S1 per well overnight at 4°C in carbonate coating buffer
1011vp of Ad5.MERS-S1 as a positive control. It is noted that MPLA was (pH 9.5), followed by addition of 1:100 diluted mouse sera, and carried
added in a single control group at similar doses as used previously to out sequentially based on a protocol similar to that described above.
establish a positive control for the adjuvanted subunit vaccines [44].
The MNA rMERS-S1f, rMERS-S1fRS09, or rMERS-S1ffliC vaccines were 2.8. MERS-CoV neutralization assay
applied to the abdomen of anesthetized mice and removed after
10 min. Two weeks after the primary immunization, mice were We tested the MERS-CoV neutralization activity of sera derived
boosted intranasally (i.n.) or intracutaneously (MNAs) with the same from mice immunized with rMERS-S1f only, rMERS-S1fRS09, rMERS-
dose of the corresponding immunogens. S1ffliC, MERS-S1f with MPLA, MNA-MERS-S1f, MNA-MERS-S1fRS09,
For SARS-CoV-2 study, C57BL/6 female mice (five animals per MNA-MERS-S1ffliC. Ad5.MERS-S1, and PBS as previously described
group) were inoculated intracutaneously with MNAs loaded with of [12]. Mouse sera were obtained from the retro-orbital plexus every
SARS-CoV-2-S1 or SARS-CoV-2-S1fRS09 protein, or PBS as a negative two weeks for six weeks and tested for their ability to neutralize
control. At two weeks after the primary immunization, mice were sim- MERS-CoV (EMC isolate). Briefly, virus (200 PFU) was premixed 1:1
ilarly boosted with the same dose of the corresponding immunogens. with serial dilutions of sera from animal groups prior to inoculation
To preliminarily evaluate the effects of sterilizing gamma irradiation onto Vero cells, and viral infection was monitored by the occurrence
on MNA-SARS-CoV-2 vaccines, 2 mice per group were irradiated using of a cytopathic effect at 72 h post-infection. Virus neutralization titers
clinical sterilizing conditions. Mice were bled from the saphenous vein (VNTs) were determined as the highest serum dilutions that showed
at week 0, 1, 2, 3, and 4, and serum samples were evaluated for SARS- full protection against the cytopathic effect of MERS-CoV.
CoV-2-S1 antibody by enzyme-linked immunosorbent assay (ELISA).
Mice were maintained under specific pathogen-free conditions at 2.9. Statistical analysis
the University of Pittsburgh, and all experiments with mice were con-
ducted in accordance with animal use guidelines and protocols Statistical analyses were performed using GraphPad Prism (San
approved by the University of Pittsburgh’s Institutional Animal Care Diego, CA). Data were analyzed by one-way ANOVA, followed by Tukey's
and Use (IACUC) Committee. post-hoc testing. Differences were considered significant if p < 0.05.
2.6. FACS analysis 3. Results
Two weeks after the prime immunization, pooled sera were 3.1. Construction and characterization of recombinant proteins
obtained from all mice and screened for MERS-S-specific antibodies
using fluorescence-activated cell sorter (FACS) analysis of Human To construct the recombinant trimeric MERS-S1 proteins, codon-
Embryonic Kidney (HEK) 293 cells transfected with either pAd/MERS- optimized MERS-S1 was fused to the T4 fibritin foldon trimerization
S or pAd control using Lipofectamine 2000 (Invitrogen) as described domain. Two additional variants were engineered by integrating the
previously [12]. Briefly, after 36 h at 37°C, the transfected cells were TLR4 agonist peptide (RS09) or Salmonella typhimurium flagellin C
harvested, trypsinized by incubation with 0.25% Trypsin, 2.21mM (fliC) fragment at the C-terminal of the foldon. Moreover, all three
JID: EBIOM
antigens were designed with a 6-histidine tag and a Tobacco Etch PBS. Antibodies were not detected bound to cells transfected with
Virus (TEV) protease cleavage sequence at the carboxy terminus to pAd without the immunogen insert (data not shown). These data
allow for metal chelating affinity purification and to facilitate down- indicate that MERS-S1f protein with MPLA adjuvant and MNA deliv-
stream large-scale, purification compatible with clinical manufactur- ered rMERS-S1f, rMERS-S1fRS09, and rMERS-S1ffliC induced strong
ing (Fig. 1A). A shuttle vector (pAd/MERS-S1f) was generated and a antibody response to membrane-bound MERS-S two weeks after a
replication-defective adenovirus 5, designated as Ad5.MERS-S1f, was single immunization.
produced by loxP homologous recombination to use as a positive
control. To determine whether MERS-S1f, MERS-S1fRS09, and MERS- 3.3. Induction of humoral immune response to MERS-S1
S1ffliC would form trimeric structures, the serum-free supernatants
from A549 cells infected with each adenovirus were characterized by To investigate the immunogenicity of trimeric MERS-S1f proteins,
SDS-PAGE and Western blot analysis. Three recombinant proteins at two and four weeks after a boosting immunization, sera were
designed with a 6-histidine tag at the carboxy-terminal ends were obtained from all mice and screened for MERS-S1 specific antibodies
recognized by a monoclonal anti-6His antibody at the expected gly- by ELISA. As shown in Fig. 3A, following s.c. vaccination only Ad5.
cosylated monomer molecular weights of about 130 kDa, 135 kDa, MERS-S1 elicited a MERS-S1-specific IgG antibody response (p<0.05,
and 200 kDa under the denaturing reduced (with b-ME) conditions. one-way ANOVA, followed by Tukey's post-hoc testing) at week 2,
The higher molecular-weight bands (dimers or trimers) were evident while no antibody response was detectable in the mouse sera of
under the denatured non-reduced (without b-ME) condition, and rMERS-S1f, rMERS-S1fRS09, and rMERS-S1ffliC vaccinated mice when
were 2 or 3 times that of the reduced proteins (monomer), as compared with PBS-immunized mouse sera. However, 2 weeks after
expected (Fig. 1B). [45,46] After being boiled in the sample buffer, the boosting, antigen-specific IgG was detected in the sera of all s.c. immu-
proteins still migrated to the position of the trimer, indicating the nized mice, although the antibody titers were generally low. As an
remarkable stability of these proteins. These results demonstrate that exception to this, s.c. delivered MERS-S1fRS09 induced a relatively
recombinant MERS-S1 formed a trimeric conformation in the pres- strong MERS-S1-specific IgG antibody response (p<0.05, one-way
ence of the Fd trimerization motif. In addition, to delete the six-histi- ANOVA, followed by Tukey's post-hoc testing, at weeks 4 and 6).
dine tag, the purified recombinant proteins were treated with AcTEV Importantly, mice immunized with each of the vaccine variants by
protease (Life Technology) followed by affinity chromatography on a MNA delivery demonstrated significantly higher levels of antigen-spe-
nickel chelating resin to remove the six-histidine tags and poly-histi- cific IgG (p< 0.05, one-way ANOVA, followed by Tukey's post-hoc test-
dine tagged protease from the cleavage reaction. The cleaved native ing) as compared to mice immunized with PBS, and there was with no
recombinant proteins were collected from the flow-through fraction significant differences between the MNA vaccine variants (Fig. 3B).
and used as coating antigens followed by detection with mouse To further demonstrate the immunogenicity of MERS-CoV subunit
serum. Three purified recombinant proteins were detected using vaccines, mouse sera were also tested for their ability to neutralize
serum from mice immunized with Ad5.MERS-S1 (p<0.05, one-way MERS-CoV (EMC isolate). As shown in Fig. 3C, there were no detectable
ANOVA, followed by Tukey's post-hoc testing) prepared in our labo- MERS-CoV-neutralizing antibodies in the sera of mice immunized s.c.
ratory previously [12], while no specific antibody binding activity with MERS-S1f-, MERS-S1fRS09-, or MERS-S1ffliC at week 4. However,
was detected in the serum of pre-immunized naïve mice or from con- at week 6, sera of animals immunized with rMERS-S1fRS09, rMERS-
trol mice inoculated with AdC5 (Fig. 1C). ELISA data revealed that the S1ffliC, or rMERS-S1f + MPLA had significant and comparable levels of
three recombinant MERS-S1 proteins had strong reactivity with virus neutralizing activity, with geometric mean neutralizing titers of
MERS-S1-specific antibodies. 104, 50.8, and 168, respectively. These titers were 5.4, 2.6, and 8.8 fold
higher than that of sera from the mice immunized with MERS-S1f only
3.2. Detection of membrane-bound MERS-S-specific antibodies (VNT mean, 19.2). Most importantly, as shown in Fig. 3D, at week 6 all
groups of MNA immunized mice developed significant levels of neu-
We next determined whether these recombinant subunit vaccines tralizing antibodies (p< 0.05, one-way ANOVA, followed by Tukey's
could elicit antigen-specific immune responses in vivo. BALB/c mice post-hoc testing). Animals immunized with MNA-rMERS-S1fRS09,
were inoculated subcutaneously with 25 mg of MERS-S1f, 25 mg of MNA-rMERS-S1ffliC, and Ad5.MERS-S1 had geometric mean neutraliz-
MERS-S1fRS09, 40 mg of MERS-S1ffliC, or 25 mg of MERS-S1f plus 20 ing titers of 960, 832, and 3328, respectively. As expected, no neutral-
mg of the MPLA adjuvant to deliver the same molar ratio of immuno- izing activity was detected in the sera of mice immunized with PBS.
gen, or PBS as a negative control on day 0. The experimental schedule These results suggest that MNA delivery of these candidate subunit
is shown in Fig. 2A. Two weeks after immunization, we obtained sera vaccines generates strong antibody-mediated neutralizing activity that
and examined binding to membrane-bound MERS-S by measuring approaches that induced by live adenovector immunization and
the reactivity against 293 cells transfected with pAd/MERS-S or pAd exceeds that observed by s.c. delivery, even when a potent exogenous
using flow cytometry. As shown in Fig. 2B, in the presence of MPLA adjuvant is added to the s.c. vaccine. The promising immunogenicity
adjuvant, rMERS-S1f induced a strong membrane-bound MERS-S- of MNA MERS-CoV vaccines without chemical adjuvants makes them
specific IgG antibody response (p<0.05, one-way ANOVA, followed an ideal candidate for future clinical studies.
by Tukey's post-hoc testing), while no significant antibody response
was detectable in the mouse sera of rMERS-S1f, rMERS-S1fRS09, and 3.4. Longevity of the immune response in mice vaccinated with subunit
rMERS-S1ffliC, when compared with PBS-immunized mouse sera. On vaccines
the other hand, in the absence of the adjuvant sequence, MNA intra-
cutaneous delivery of MERS-S1f, MERS-S1fRS09, or MERS-S1ffliC, at To evaluate the persistence of MERS-S1-specific immunity, mouse
the same molar ratio, elicited potent IgG antibody responses (p<0.05, sera was collected at weeks 23 and 55 after immunization and evalu-
one-way ANOVA, followed by Tukey's post-hoc testing) that were ated for the presence of MERS-S1-specific IgG by ELISA. All animal
significantly stronger than those seen in s.c. immunized mice with groups immunized s.c. with recombinant subunit vaccine demon-
MPLA adjuvant (p<0.05, one-way ANOVA, followed by Tukey's post- strated the same or more levels of MERS-S1 IgG specific antibodies at
hoc testing), indicating the potential of MNAs as an effective recombi- the later time points as those observed at week 6. Surprisingly, mice
nant coronavirus vaccine delivery platform. (Fig. 2B). Mice immu- immunized s.c. with rMERS-S1fRS09 had significantly higher levels of
nized with Ad5.MERS-S1 as a positive control developed significantly IgG (p< 0.05, one-way ANOVA, followed by Tukey's post-hoc testing)
higher levels of IgG antibody against cell membrane-bound MERS-S at week 23 compared with those observed at week 6 (Fig. 4A), and
as compared to negligible IgG levels in control mice inoculated with this was sustained through 55 weeks. Importantly, MNA delivered
JID: EBIOM
Fig. 1. Construction of recombinant MERS-S1 Foldon subunit vaccines. (A) Schematic diagram of rMERS-S1s. The positions of the RBD (small dots) and transmembrane domain (stripes)
are indicated and S is divided into two subdomains, S1 and S2, at position 751. The vector was used to generate recombinant replication-deficient adenoviruses by homologous recom-
bination with the adenoviral genomic DNA. Arrows showed the cleavage site by TEV protease. Abbreviations are as follows: ITR, inverted terminal repeat; RBD, receptor binding
domain;F, T4 fibritin foldon trimerization domain; Tp, Tobacco Etch Virus (TEV) protease; fliC; Salmonella typhimurium flagellin C (B) Western blot of the supernatant of A549 cells
infected (10 MOI) with mock (lane 1 and 5), Ad5.MERS-S1f (lane 2 and 6), Ad5.MERS-S1fRS09 (lane 3 and 7), or Ad5.MERS-S1ffliC (lane 4 and 8), respectively, with anti-6His monoclonal
antibody. The supernatants were resolved on SDS-4~20% polyacrylamide gel after being boiled in 2% SDS sample buffer with or without b-ME. (C) Detection of purified rMERS-S1fs with
mouse sera against Ad5.MERS-S1. The recombinant MERS-S1f proteins were purified using His60 Ni Superflow Resin under native conditions. After cleavage of the fusion protein by
TEV protease, 6xHis tag and protease were removed from the cleavage reaction by affinity chromatography on a nickel chelating resin. Three rMERS-S1fs were coated in 96-well plate
with 200ng/well ELISA and detected with mouse sera against Ad5.MERS-S1. Significance was determined by one-way ANOVA followed by Tukey’s test. *p < 0.05.
JID: EBIOM
Fig. 2. MNA delivered subunit vaccines elicit antibodies recognizing cell surface bound MERS-S proteins. (A) The experimental schedule representing the timeline for the
immunizations. (B) Flow cytometry assay of 293 cells expressing MERS-S at the cell surface. HEK 293 cells were transfected with pAd/MERS-S (gray bar) or control pAd (white bar).
At 36 h post-transfection, binding to MERS-S at the cell surface was analyzed by incubation with mice sera obtained at week 2 after immunization followed by staining with PE-con-
jugated anti-mouse IgG. St Significance was determined by one-way ANOVA followed by Tukey’s test. *p < 0.05.
vaccines demonstrated generally increasing (statistically significant) specific antibodies by ELISA as previously described. As shown in
levels of MERS-S1-specific antibody through the 23- and 55-week Fig. 5C, significantly high (p< 0.05, one-way ANOVA, followed by
time points (Fig. 4B). No significant difference was observed in the Tukey's post-hoc testing) SARS-CoV-2 IgG responses were detected
mice immunized with PBS or Ad5.MERS-S1 at any time point evalu- as early as week 2 for all subunit vaccines as compared to control
ated. These results indicate that MNA-delivered subunit vaccines groups. The inclusion of the RS09 ligand in the trimer subunit
induce potent and long-lasting MERS-S1 specific antibody responses resulted in a negligible effect on SARS-CoV-2 specific IgG titers. Fur-
and MNA delivery is a viable approach for the clinical development ther, in our preliminary study, the immunogenicity of gamma irradia-
of subunit vaccines against emerging infectious diseases such as tion sterilized MNA vaccines was comparable to that of unirradiated
COVID-19. MNA vaccines (Data not shown), thereby supporting the feasibility of
gamma irradiation as a terminal sterilization approach for our clinical
MNA-SARS-CoV-2 subunit vaccines.
3.5. Immunogenicity of MNA delivered SARS-CoV-2-S1 subunit vaccines
Based on these MERS-S1 vaccine results and the urgency of the 3.6. Rapid development of clinically applicable MNA SARS-CoV-2 S1
public health threat by COVID-19, we focused our efforts on the subunit vaccines
development of SARS-CoV-2-S1 and SARS-CoV-2-S1fRS09 subunit
vaccines (shown in Fig. 5A). The size and trimerization product of the Relying on our experience with MERS-CoV and SARS-CoV
vaccine was confirmed by western blot (Fig. 5B). The aggregation in vaccines, and our experience with clinical production and human
Fig. 5B could be attributed to the trimerization of the S1’s segments applications of MNAs, we developed standard operating procedures
NTD, CTD1, and CDT2 domains or the ability of the histidine tag to oli- (SOPs) for the rapid development of clinic grade MNA SARS-CoV-2
gomerize the tagged proteins [47 49]. We used MNAs to vaccinate subunit vaccines. Our production strategy and the related timeline is
mice with SARS-CoV-2-S1 and SARS-CoV-2-S1fRS09 subunit vac- outlined in Table 1. First, we designed, optimized, and cloned the S1
cines. Sera was collected prior to immunization (week 0) and at subunits beginning when the sequence became publicly available.
weeks 1 2, 3, and 4, and evaluated for the presence of SARS-CoV-2-S1 Subsequently, we produced S1 subunit proteins, purified them, and
JID: EBIOM
Fig. 3. Induction of humoral immune response in mice vaccinated with recombinant MERS-S1 vaccines. BALB/c mice were immunized subcutaneously or intracutaneously
with 20 mg of each rMERS-S1 subunit vaccines s.c., with or without 20 mg of MPLA, or by MNA delivery of the same subunit proteins, or with 1011vp of Ad5.MERS-S1 as a positive
control. On day 14 mice were boosted using the same regimen as the prime immunization. On weeks 0, 2, 4, and 6 after treatment, immune sera from mice were collected, diluted
(200x), and tested for the presence of MERS-S1-specific antibodies by ELISA (A and B) or by MERS-CoV-neutralization assay without dilution (C and D). MERS-CoV virus-neutralizing
titers (VNTs) were measured every week after primary immunization using Vero cells by determining the highest dilution inhibiting MERS-CoV infection by 100%. Significance was
determined by one-way ANOVA followed by Tukey’s test. *p < 0.05.White and black circles in Fig. 3C and D represent week 4 and week 6, respectively.
Fig. 4. Longevity of immune response in mice vaccinated with subunit vaccines. BALB/c mice were immunized with subunit vaccines as described in the Fig. 3 legend. On week
23 and 55 after immunization sera were collected, diluted (200x) in PBS, and tested for the presence of MERS-S1-specific antibodies by ELISA. Significance was determined by one-
way ANOVA followed by Tukey’s test. *p < 0.05.
JID: EBIOM
Fig. 5. Induction of humoral immune response in mice immunized with MNA delivered SARS-CoV-2-S1 vaccines. (A) Schematic diagram of rSARS-CoV-2-S1s. The positions of the
RBD (small dots) and transmembrane domain (stripes) are indicated and S is divided into two subdomains, S1 and S2, at position 681. The SARS-CoV-2-S1 or SARS-CoV-2-S1fRS09
gene was inserted into pmax Cloning expression vector. (B) Western blot of the purified rSARS-CoV-2-S1 (lane 1 and 3) or rSARS-CoV-2-S1fRS09 (lane 2 and 4) proteins with anti-6His
monoclonal antibody. The purified proteins were resolved on 10% Tris/Glycine gel after being boiled in 2% SDS sample buffer with b-ME or in native sample buffer without b-ME. (C)
C57BL/6 mice were immunized intradermally with MNAs containing 20 mg of rSARS-CoV-2-S1 or rSARS-CoV-2-S1fRS09. Immune sera from mice were collected every week, diluted
(100x) in PBS, and tested for the presence of SARS-CoV-2-S1-specific antibodies by ELISA. Significance was determined by one-way ANOVA followed by Tukey’s test. *p < 0.05.
then fabricated dissolvable MNAs integrating the protein subunits for were initiated within 4 weeks of the SARS-CoV-2 spike sequence
pre-clinical testing in the animal model. Next, we vaccinated mice becoming publicly available. In parallel, to enable future regulatory
with MNA-rSARS-CoV-2 vaccines and began analysis of antibody approval and rapid clinical testing, we employed Good Manufactur-
responses induced in immunized animals. These preclinical studies ing Processes (GMP) using a certified master cell bank (MCB) to scale
JID: EBIOM
Table 1
Timeline for the development of SARS-CoV-2 subunit vaccines.
S1 subunit protein production, purification, and validation, and incor- alone (Fig. 3C,D). Thus, though the contributions of the integrated TLR
porated the subunits into dissolvable MNAs or syringes using pro- ligands to the strong immune responses induced by MNA vaccines
cesses developed through our clinical production and quality control appear to be minimal, they provide promising benefit for s.c. vaccine
of MNAs (ClinicalTrials.gov Identifier: NCT02192021). This was fol- administration, and their potential for contribution to immunogenicity
lowed by (1) gamma irradiation to assure sterilization of the vaccine in humans has yet to be determined. Importantly, we and others have
loaded MNAs and syringes that will be used as control groups in our shown effective protection against MERS-CoV challenge in hDPP4-
clinical trials, and (2) analysis of established release criteria, the latter expressing mice at comparable or lower neutralizing titers in previous
of which is now ongoing and will be used for an application for IND passive immunotherapy studies with MERS-CoV-specific antibodies or
approval that will enable a safety focused phase I clinical trial. neutralizing sera obtained from MERS-CoV immune humans or animals
[50 53]. Furthermore, we have previously demonstrated that antibod-
4. Discussion ies directed against S1 domain of the spike protein, as detected in our
study by ELISA, exert neutralizing activity against live coronaviruses
Here, we present the rapid design and translational development of [54].
MNA SARS-CoV-2 vaccines capable of generating potent antigen-specific Based on our results with MERS-CoV-S1 vaccines, we focused our
antibody responses by capitalizing on our substantial experience with efforts to develop MNA SARS-CoV-2 vaccines. Using the described
Coronavirus vaccines and MNA delivery platforms. approach, within three weeks of publication of the SARS-CoV-2- spike
First, we describe the design and production of trimeric recombinant glycoprotein sequence, we produced both rSARS-CoV-S1 and rSARS-
subunit vaccines against MERS-CoV-S1 with and without integrated CoV-S1fRS09 immunogens and fabricated dissolvable MNAs incorporat-
immunostimulatory TLR ligand sequences. We tested the immunoge- ing these vaccines in quantities sufficient for pre-clinical testing. MNA
nicity of these vaccine variants delivered either by traditional subcuta- delivery of either rSARS-CoV-2-S1 or rSARS-CoV-2-S1fRS09 induced
neous needle injection or using MNAs to more specifically target substantial and statistically significant increases in antigen-specific anti-
vaccine components to the immune fertile skin microenvironment. We bodies responses as soon as week 2 compared to pre-immunization and
found that MNA delivery of MERS-CoV-S1 vaccines induced stronger week 1 responses. The inclusion of RS09 did not have a significant effect
humoral responses than traditional needle injections regardless of the on antibody titer at the 2, 3, or 4-week time points. Furthermore, the
inclusion of TLR ligand binding sequences (Fig. 3A,B). On the other immunogenicity of MNA vaccines was maintained after gamma irradia-
hand, integration of the RS09 TLR4 binding sequence in the subunit tri- tion sterilization, suggesting a viable method to assure sterility of our
mer resulted in relatively stronger antibody responses than those with- MNA vaccines for clinical applications. The significant antibody titers
out RS09 when vaccines were delivered by subcutaneous injection we observed at the early time points before boosting strongly supports
(Fig. 3A). Interestingly, the inclusion of the flagellin TLR5 binding the feasibility of our MNA-SARS-CoV-2 vaccines, particularly in the
sequence had no effect on the immunogenicity of the s.c. delivered vac- context of similar results obtained with the analogous MERS-CoV-S1
cines (Fig. 3B). Importantly, significant neutralizing activity was constructs under the same conditions.
observed in vaccinated animals at week 6. Consistent with previous IgG Neutralization assays are important to ensure antibody function;
results, the presence of RS09 in the subunit trimer improved the neu- however, at this early timepoint, we do not have access to validated
tralization function of antibodies from s.c. immunized mice, and this assays for neutralizing antibodies against SARS-CoV-2. Further, at the
was the only immunogen that resulted in significant neutralization. time of submission of this manuscript, we are just over four weeks
Notably, MNA delivery was more effective in eliciting neutralizing anti- post-immunization and based on our MERS-CoV studies, the reliable
bodies for all vaccines. All MNA vaccines generated higher levels of neu- detection of neutralizing SARS-CoV-2 antibodies may require at least
tralizing antibody, even beyond those induced by an s.c. delivered six weeks post immunization. We speculate that this delay in the
MPLA adjuvanted vaccine, and similar to those seen following the deliv- neutralizing response is likely a result of the time needed for affinity
ery of the rMERS-S1f construct via recombinant adenoviral vector. maturation of the SARS-CoV-2 neutralizing IgG antibodies. However,
Though levels of neutralizing antibody were elevated in mice immu- we believe that MNA-SARS-CoV-2 vaccines will very likely induce
nized with trimers including either RS09 or flagellin, these increases neutralizing immunity as suggested by the vigorous early antibody
were not statistically significant when compared to the MNA vaccine responses, and commonalities between both the vaccines and the
JID: EBIOM
viruses. Accordingly, even though it is still early to predict whether Acknowledgments

humans immunized with these vaccine candidates will have similar
responses and be protected from SARS-CoV-2 or MERS-CoV infec- AG is funded by NIH NIAID (R21-AI114264) and LDF is funded by
tions, our studies demonstrate that development, production, and NIH NIAMS (R01-AR074285, R01-AR071277, and R01-AR068249 to
initial animal testing of clinically translatable MNA vaccine candi- LDF). SCB is supported by a fellowship from the NIH National Cancer
dates against SARS-CoV-2 and other emerging infections can be rap- Institute (T32-CA175294). These funding institutions had no role in
idly accomplished. Studies directly assessing important correlates of the study design, data collection, data analysis, and interpretation of
human efficacy would provide additional support for efficacy in this publication.
humans. In this regard it will be important to determine whether
antibodies from MNA-SARS-CoV-2 immunized animals will neutral- References
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TO HEIGHTEN THE COLOUR AND THE FLAVOUR OF GRAVIES.
This is best done by the directions given for making Espagnole. An

ounce or two of the lean of unboiled ham, cut into dice and coloured
slowly in a small stewpan, or smoothly-tinned iron saucepan, with
less than an ounce of butter, a blade of mace, two or three cloves, a
bay-leaf, a few small sprigs of savoury herbs, and an eschalot or
two, or about a teaspoonful of minced onion, and a little young
parsley root, when it can be had, will convert common shin of beef
stock, or even strong broth, into an excellent gravy, if it be gradually
added to them after they have stewed slowly for quite half an hour,
and then boiled with them for twenty minutes or more. The liquid
should not be mixed with the other ingredients until the side of the
stewpan is coloured of a reddish brown; and should any thickening
be required, a teaspoonful of flour should be stirred in well, and
simmered for three or four minutes before the stock is added; the
pan should be strongly shaken round afterwards, to detach the
browning from it, and this must be done often while the ham is
stewing.
Obs.—The cook who is not acquainted with this mode of preparing
or enriching gravies, will do well to make herself acquainted with it;
as it presents no difficulties, and is exceedingly convenient and
advantageous when they are wanted in small quantities, very highly
flavoured and well coloured. An unboiled ham, kept in cut, will be
found, as we have already said, a great economy for this, and other
purposes, saving much of the expense commonly incurred for gravy-
meats. As eschalots, when sparingly used, impart a much finer
savour than onions, though they are not commonly so much used in
England, we would recommend that a small store of them should
always be kept.
BARON LIEBEG’s BEEF GRAVY.
(Most excellent for hashes, minces, and other dishes made of cold
meat.)
For particulars of this most useful receipt, for extracting all its
juices from fresh meat of every kind in the best manner, the cook is
referred to the first part of the chapter on soups. The preparation, for
which minute directions are given there, if poured on a few bits of
lean ham lightly browned, with the other ingredients indicated above,
will be converted into gravy of fine flavour and superior quality.
With no addition, beyond that of a little thickening and spice, it will
serve admirably for dressing cold meat, in all the usual forms of
hashes, minces, blanquettes, &c., &c., and convert it into dishes as
nourishing as those of meat freshly cooked, and it may be
economically made in small quantities with any trimmings of
undressed beef, mutton, or veal, mixed together, which are free from
fat, and not sinewy: flavour may be given to it at once by chopping
up with them the lean part only of a slice or two of ham, or of highly-
cured beef.
SHIN OF BEEF STOCK FOR GRAVIES.
There is no better foundation for strong gravies than shin of beef

stewed down to a jelly (which it easily becomes), with the addition
only of some spice, a bunch of savoury herbs, and a moderate
proportion of salt; this, if kept in a cool larder, boiled softly for two or
three minutes every second or third day, and each time put into a
clean, well-scalded pan, will remain good for many days, and may
easily be converted into excellent soup or gravy. Let the bone be
broken in one or two places, take out the marrow, which, if not
wanted for immediate use, should be clarified, and stored for future
occasions; put a pint and a half of cold water to the pound of beef,
and stew it very gently indeed for six or seven hours, or even longer
should the meat not then be quite in fragments. The bones of calf’s
feet which have been boiled down for jelly, the liquor in which the
head has been cooked, and any remains of ham quite freed from the
smoky parts, from rust, and fat, will be serviceable additions to this
stock. A couple of pounds of the neck of beef may be added to six of
the shin with very good effect; but for white soup or sauces this is
better avoided.
Shin of beef, 6 lbs.; water, 9 pints; salt, 1 oz.; large bunch of
savoury herbs; peppercorns, 1 teaspoonful; mace, 2 blades.
RICH PALE VEAL GRAVY, OR CONSOMMÉ.
The French, who have always at hand their stock-pot of good

bouillon (beef soup or broth), make great use of it in preparing their
gravies. It is added instead of water to the fresh meat, and when this,
in somewhat larger proportions, is boiled down in it, with the addition
only of a bunch of parsley, a few green onions, and a moderate
seasoning of salt, a strong and very pure-flavoured pale gravy is
produced. When the best joints of fowls, or of partridges have been
taken for fricassees or cutlets, the remainder may be stewed with a
pound or two of veal into a consommé, which then takes the name of
chicken or of game gravy. For a large dinner it is always desirable to
have in readiness such stock as can easily and quickly be converted
into white and other sauces. To make this, arrange a slice or two of
lean ham in a stewpan or saucepan with three pounds of the neck of
veal once or twice divided (unless the thick fleshy part of the knuckle
can be had), and pour to them three full pints of strong beef or veal
broth; or, if this cannot conveniently be done, increase the proportion
of meat or diminish that of the liquid, substituting water for the broth;
throw in some salt after the boiling has commenced, and the gravy
has been well skimmed, with one mild onion, a bunch of savoury
herbs, a little celery, a carrot, a blade of mace, and a half-
saltspoonful of peppercorns; stew those very gently for four hours;
then, should the meat be quite in fragments, strain off the gravy, and
let it become sufficiently cold to allow the fat to be entirely cleared
from it. A handful of nicely prepared mushroom-buttons will much
improve its flavour; and the bones of boiled calf’s feet, or the fresh
ones of fowls, will be found excellent additions to it. A better method
of making it, when time and trouble are not regarded, is to heat the
meat, which ought to be free of bones, quite through, with from a
quarter to half a pint of broth only, and when on probing it with the
point of a knife no blood issues from it, and it has been turned and
equally done, to moisten it with the remainder of the broth, which
should be boiling.
Lean of ham, 6 to 8 oz.; neck or knuckle of veal, 3 lbs.; strong
broth, 3 pints (or veal, 4 lbs., and water, 3 pints); salt; bunch of
savoury herbs; mild onion, 1; carrot, 1 large or 2 small; celery 1/2
small head; mace, 1 large blade; peppercorns, 1/2 saltspoonful; 4
hours or more. Or: ham, 1/2 lb.; veal, 4 lbs.; broth, third of a pint;
nearly 1 hour. Additional broth, 3 pints: 3-1/2 to 4-1/2 hours.
RICH DEEP-COLOURED VEAL GRAVY.
Lay into a large thick stewpan or saucepan, from half to three

quarters of a pound of undressed ham, freed entirely from fat, and
from the smoked edges, and sliced half an inch thick; on this place
about four pounds of lean veal, cut from the best part of the knuckle
or from the neck (part of the fillet, which in France is often used for it
instead, not being generally purchasable here, the butchers seldom
dividing the joint); pour to them about half a pint of good broth,[54]
and place the pan over a brisk fire until it is well reduced; then thrust
a knife into the meat, and continue the stewing more gently until a
glaze is formed as we have described at page 10. The latter part of
the process must be very slow; the stewpan must be frequently
shaken, and the gravy closely watched that it may not burn: when it
is of a fine deep amber colour, pour in sufficient boiling broth to cover
the meat, add a bunch of parsley, and a few mushrooms and green
onions. A blade or two of mace, a few white peppercorns, and a
head of celery, would, we think, be very admissible additions to this
gravy, but it is extremely good without. Half the quantity can be
made, but it will then be rather more troublesome to manage.
54. When there is no provision of this in the house, the quantity may be made
with a small proportion of beef, and the trimmings of the veal, by the
directions for Bouillon, Chapter I.
Undressed ham, 8 to 12 oz.; lean veal, 4 lbs.; broth, 1/2 pint; 1 to 2

hours. Broth, 3 to 4 pints: bunch of parsley and green onions, or 1
Portugal onion; mushrooms, 1/4 to 1/2 pint: 1-1/2 to 2 hours.
GOOD BEEF OR VEAL GRAVY. (ENGLISH RECEIPT.)
Flour and fry lightly in a bit of good butter a couple of pounds of

either beef or veal; drain the meat well from the fat, and lay it into a
small thick stewpan or iron saucepan; pour to it a quart of boiling
water; add, after it has been well skimmed and salted, a large mild
onion sliced, very delicately fried, and laid on a sieve to drain, a
carrot also sliced, a small bunch of thyme and parsley, a blade of
mace, and a few peppercorns; stew these gently for three hours or
more, pass the gravy through a sieve into a clean pan, and when it is
quite cold clear it entirely from fat, heat as much as is wanted for
table, and if not sufficiently thick stir into it from half to a whole
teaspoonful of arrow-root mixed with a little mushroom catsup. Beef
or veal, 2 lbs.; water, 2 pints; fried onion, 1 large; carrot, 1; small
bunch of herbs; salt, 1 small teaspoonful or more; mace, 1 blade;
peppercorns, 20: 3 to 3-1/2 hours.
A RICH ENGLISH BROWN GRAVY.
Brown lightly and carefully from four to six ounces of lean ham,
thickly sliced and cut into large dice; lift these out, and put them into
the pan in which the gravy is to be made; next, fry lightly also, a
couple of pounds of neck of beef dredged moderately with flour, and
slightly with pepper; put this, when it is done, over the ham; and then
brown gently and add to them two or three eschalots, or a Portugal
onion; should neither of these be at hand, one not large common
onion must be used instead. Pour over these ingredients a quart of
boiling water, or of weak but well-flavoured broth; bring the whole
slowly to a boil, clear off the scum with great care, throw in a
saltspoonful of salt, four cloves, a blade of mace, twenty corns of
pepper, a bunch of savoury herbs, a carrot, and a few slices of
celery: these last two may be fried or not as is most convenient. Boil
the gravy very softly until it is reduced to little more than a pint;
strain, and set it by until the fat can be taken from it. Heat it anew,
add more salt if needed and a little mushroom catsup, cayenne-
vinegar, or whatever flavouring it may require for the dish with which
it is to be served; it will seldom require any thickening. A dozen small
mushrooms prepared as for pickling, or two or three morels,
previously well washed and soaked, may be added to it at first with
advantage. Half this quantity of gravy will be sufficient for a single
tureen, and the economist can diminish a little the proportion of meat
when it is thought too much.
PLAIN GRAVY FOR VENISON.
Trim away the fat from some cutlets, and lay them into a stewpan;
set them over a clear fire, and let them brown a little in their own
gravy; then add a pint of boiling water to each pound of meat. Take
off the scum, throw in a little salt, and boil the gravy until reduced
one half. Some cooks broil the cutlets lightly, boil the gravy one hour,
and reduce it after it is strained. For appropriate gravy to serve with
venison, see “Haunch of Venison,” Chapter XV.
A RICH GRAVY FOR VENISON.
There are few eaters to whom this would be acceptable, the

generality of them preferring infinitely the flavour of the venison itself
to any which the richest gravy made of other meats can afford; but
when the flavour of a well-made Espagnole is likely to be relished,
prepare it by the receipt of the following page, substituting plain
strong mutton stock for the veal gravy.
SWEET SAUCE, OR GRAVY FOR VENISON.
Add to a quarter-pint of common venison gravy a couple of

glasses of port wine or claret, and half an ounce of sugar in lumps.
Christopher North’s sauce, mixed with three times its measure of
gravy, would be an excellent substitute for this.
ESPAGNOLE (SPANISH SAUCE).
A highly-flavoured Gravy.
Dissolve a couple of ounces of good butter in a thick stewpan or
saucepan, throw in from four to six sliced eschalots, four ounces of
the lean of an undressed ham, three ounces of carrot, cut in small
dice, one bay leaf, two or three branches of parsley, and one or two
of thyme, but these last must be small; three cloves, a blade of
mace, and a dozen corns of pepper; add part of a root of parsley, if it
be at hand, and keep the whole stirred or shaken over a moderate
fire for twenty minutes, then add by degrees one pint of very strong
veal stock or gravy, and stew the whole gently from thirty to forty
minutes; strain it, skim off the fat, and it will be ready to serve.
Butter, 2 oz.; eschalots, 4 to 6; lean of undressed ham, 4 oz.;
carrots, 3 oz.; bay leaf, 1; little thyme and parsley, in branches;
cloves, 3; mace, 1 blade; peppercorns, 12; little parsley root: fried
gently, 20 minutes. Strong veal stock, or gravy, 1 pint: stewed very
softly, 30 to 40 minutes.
ESPAGNOLE, WITH WINE.
Take the same proportions of ingredients as for the preceding

Espagnole, with the addition, if they should be at hand, of a dozen
small mushrooms prepared as for stewing; when these have fried
gently in the stewpan until it appears of a reddish colour all round,
stir in a tablespoonful of flour, and when it is lightly browned, add in
small portions, letting each one boil up before the next is poured in,
and shaking the pan well round, three quarters of a pint of hot and
good veal gravy, and nearly half a pint of Madeira or sherry. When
the sauce has boiled gently for half an hour, add to it a small quantity
of cayenne and some salt, if this last be needed; then strain it, skim
off the fat entirely should any appear upon the surface, and serve it
very hot. A smaller proportion of wine added a few minutes before
the sauce is ready for table, would perhaps better suit with English
taste, as with longer boiling its flavour passes off almost entirely.
Either of these Espagnoles, poured over the well bruised remains of
pheasants, partridges, or moor fowl, and boiled with them for an
hour, will become most admirable game gravy, and would generally
be considered a superlative addition to other roast birds of their kind,
as well as to the hash or salmi, for which see Chapter XV.
Ingredients as in preceding receipt, with mushrooms 12 to 18;
Madeira, or good sherry, 1/4 to 1/2 pint.
JUS DES ROGNONS, OR, KIDNEY GRAVY.
Strip the skin and take the fat from three fresh mutton kidneys,
slice and flour them; melt two ounces of butter in a deep saucepan,
and put in the kidneys, with an onion cut small, and a teaspoonful of
fine herbs stripped from the stalks. Keep these well shaken over a
clear fire until nearly all the moisture is dried up; then pour in a pint
of boiling water, add half a teaspoonful of salt, and a little cayenne or
common pepper, and let the gravy boil gently for an hour and a half,
or longer, if it be not thick and rich. Strain it through a fine sieve, and
take off the fat. Spice or catsup may be added at pleasure.
Mutton kidneys, 3; butter, 2 oz.; onion, 1; fine herbs, 1 teaspoonful:
1/2 hour. Water, 1 pint; salt, 1/2 teaspoonful; little cayenne, or black
pepper: 1-1/2 hour.
Obs.—This is an excellent cheap gravy for haricots, curries, or
hashes of mutton; it may be much improved by the addition of two or
three eschalots, and a small bit or two of lean meat.
GRAVY IN HASTE.
Chop fine a few bits of lean meat, a small onion, a few slices of
carrot and turnip, and a little thyme and parsley; put these with half
an ounce of butter into a thick saucepan, and keep them stirred until
they are slightly browned; add a little spice, and water in the
proportion of a pint to a pound of meat; clear the gravy from scum,
let it boil half an hour, then strain it for use.
Meat, 1 lb.; 1 small onion; little carrot, turnip, thyme, and parsley;
butter, 1/2 oz.; cloves, 6; corns of pepper, 12; water, 1 pint: 1/2 hour.
CHEAP GRAVY FOR A ROAST FOWL.
When there is neither broth nor gravy to be had, nor meat of which
either can be made, boil the neck of the fowl after having cut it small,
in half a pint of water, with any slight seasonings of spice or herbs, or
with a little salt and pepper only; it should stew very softly for an hour
or more, or the quantity will be too much reduced. When the bird is
just ready for table, take the gravy from the dripping-pan, and drain
off the fat from it as closely as possible; strain the liquor from the
neck to it, mixing them smoothly, pass the gravy again through the
strainer, heat it, add salt and pepper or cayenne, if needed, and
serve it extremely hot. When this is done, the fowl should be basted
with good butter only, and well floured when it is first laid to the fire.
Many cooks always mix the gravy from the pan when game is
roasted, with that which they send to table with it, as they think that it
enriches the flavour; but to many persons it is peculiarly distasteful.
Neck of fowl; water, 1/2 pint; pepper, salt (little vegetable and spice
at choice): stewed gently, 1 hour; strained, stirred to the gravy of the
roast, well cleared from fat.
ANOTHER CHEAP GRAVY FOR A FOWL.
A little good broth added to half a dozen dice of lean ham, lightly
browned in a morsel of butter, with half a dozen corns of pepper and
a small branch or two of parsley, and stewed for half an hour, will
make excellent gravy of a common kind. When there is no broth, the
neck of the chicken must be stewed down to supply its place.
GRAVY OR SAUCE FOR A GOOSE.
Mince, and brown in a small saucepan, with a slice of butter, two

ounces of mild onion,. When it begins to brown, stir to it a
teaspoonful of flour, and in five or six minutes afterwards, pour in by
degrees the third of a pint of good brown gravy; let this simmer
fifteen minutes; strain it, bring it again to the point of boiling, and add
to it a teaspoonful of made mustard mixed well with a glass of port
wine. Season it with cayenne and pepper and salt, if this last be
needed. Do not let the sauce boil after the wine is added, but serve it
very hot.
Onions, 2 oz.; butter, 1-1/2 oz.: 10 to 15 minutes. Flour, 1
teaspoonful: 5 to 6 minutes. Gravy, 1/3 pint: 15 minutes. Mustard, 1
teaspoonful; port wine, 1 glassful; cayenne pepper; salt. See also
Christopher North’s own sauce, page 119.
ORANGE GRAVY FOR WILD FOWL.
Boil for about ten minutes, in half a pint of rich and highly-
flavoured brown gravy, or Espagnole, half the rind of a Seville
orange, pared as thin as possible, and a small strip of lemon-rind,
with a bit of sugar the size of a hazel-nut. Strain it off, add to it a
quarter pint of port or claret, the juice of half a lemon, and a
tablespoonful of Seville orange-juice: season it with cayenne, and
serve it as hot as possible.
Gravy, 1/2 pint; 1/2 the rind of a Seville orange; lemon-peel, 1
small strip; sugar, size of hazel-nut: 10 minutes. Juice of 1/2 a
lemon; Seville orange-juice, 1 tablespoonful; cayenne. See also
Christopher North’s own sauce, page 119.
MEAT JELLIES FOR PIES AND SAUCES.
A very firm meat jelly is easily made by stewing slowly down equal
parts of shin of beef, and knuckle or neck of veal, with a pint of cold
water to each pound of meat; but to give it flavour, some thick slices
of lean unboiled ham should be added to it, two or three carrots,
some spice, a bunch of parsley, one mild onion, or more, and a
moderate quantity of salt; or part of the meat may be omitted, and a
calf’s head, or the scalp of one, very advantageously substituted for
it, though the flavouring must then be heightened, because, though
very gelatinous, these are in themselves exceedingly insipid to the
taste. If rapidly boiled, the jelly will not be clear, and it will be difficult
to render it so without clarifying it with the whites of eggs, which it
ought never to require; if very gently stewed, on the contrary, it will
only need to be passed through a fine sieve, or cloth. The fat must
be carefully removed, after it is quite cold. The shin of beef
recommended for this and other receipts, should be from the middle
of the leg of young heifer beef, not of that which is large and coarse.
Middle of small shin of beef, 3 lbs.; knuckle or neck of veal, 3 lbs.;
lean of ham, 1/2 lb.; water, 3 quarts; carrots, 2 large, or 3 small;
bunch of parsley; 1 mild onion, stuck with 8 cloves; 2 small bay-
leaves; 1 large blade of mace; small saltspoonful of peppercorns;
salt, 3/4 oz. (more if needed): 5 to 6 hours’ very gentle stewing.
Obs.—A finer jelly may be made by using a larger proportion of
veal than of beef, and by adding clear beef or veal broth to it instead
of water, in a small proportion at first, as directed in the receipt for
consommé, see page 98, and by pouring in the remainder when the
meat is heated through. The necks of poultry, any inferior joints of
them omitted from a fricassee or other dish, or an old fowl, will
further improve it much; an eschalot or two may at choice be boiled
down in it, instead of the onion, but the flavour should be scarcely
perceptible.

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Microneedle array delivered

recombinant coronavirus vaccines:

EBioMedicine 000 (2020) 102743

Contents lists available at ScienceDirect

Microneedle array delivered recombinant coronavirus vaccines:

2 E. Kim et al. / EBioMedicine 00 (2020) 102743

E. Kim et al. / EBioMedicine 00 (2020) 102743 3

4 E. Kim et al. / EBioMedicine 00 (2020) 102743

2.6. FACS analysis 3. Results

E. Kim et al. / EBioMedicine 00 (2020) 102743 5

6 E. Kim et al. / EBioMedicine 00 (2020) 102743

E. Kim et al. / EBioMedicine 00 (2020) 102743 7

8 E. Kim et al. / EBioMedicine 00 (2020) 102743

E. Kim et al. / EBioMedicine 00 (2020) 102743 9

10 E. Kim et al. / EBioMedicine 00 (2020) 102743

E. Kim et al. / EBioMedicine 00 (2020) 102743 11

viruses. Accordingly, even though it is still early to predict whether Acknowledgments

12 E. Kim et al. / EBioMedicine 00 (2020) 102743

This is best done by the directions given for making Espagnole. An

There is no better foundation for strong gravies than shin of beef

The French, who have always at hand their stock-pot of good

Lay into a large thick stewpan or saucepan, from half to three

Undressed ham, 8 to 12 oz.; lean veal, 4 lbs.; broth, 1/2 pint; 1 to 2

Flour and fry lightly in a bit of good butter a couple of pounds of

There are few eaters to whom this would be acceptable, the

Add to a quarter-pint of common venison gravy a couple of

Take the same proportions of ingredients as for the preceding

Mince, and brown in a small saucepan, with a slice of butter, two

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