Sterilizing Immunity against SARS-CoV-2 Infection in Mice by a Single-Shot and Lipid Amphiphile Imidazoquinoline TLR7/8 Agonist-Adjuvanted Recombinant Spike Protein Vaccine**

Article abstract of DOI:10.1002/anie.202015362

The search for vaccines that protect from severe morbidity and mortality because of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019 (COVID-19) is a race against the clock and the virus. Here we describe an amphiphilic imidazoquinoline (IMDQ-PEG-CHOL) TLR7/8 adjuvant, consisting of an imidazoquinoline conjugated to the chain end of a cholesterol-poly(ethylene glycol) macromolecular amphiphile. It is water-soluble and exhibits massive translocation to lymph nodes upon local administration through binding to albumin, affording localized innate immune activation and reduction in systemic inflammation. The adjuvanticity of IMDQ-PEG-CHOL was validated in a licensed vaccine setting (quadrivalent influenza vaccine) and an experimental trimeric recombinant SARS-CoV-2 spike protein vaccine, showing robust IgG2a and IgG1 antibody titers in mice that could neutralize viral infection in vitro and in vivo in a mouse model.

Full text of DOI:10.1002/anie.202015362

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How to cite: Angew. Chem. Int. Ed. 2021, 60, 9467–9473
International Edition: doi.org/10.1002/anie.202015362
German Edition: doi.org/10.1002/ange.202015362
Vaccines
Sterilizing Immunity against SARS-CoV-2 Infection in Mice by
a Single-Shot and Lipid Amphiphile Imidazoquinoline TLR7/8
Agonist-Adjuvanted Recombinant Spike Protein Vaccine**
+
+
+
Sonia Jangra , Jana De Vrieze , Angela Choi , Raveen Rathnasinghe, Gabriel Laghlali,
Annemiek Uvyn, Simon Van Herck, Lutz Nuhn, Kim Deswarte, Zifu Zhong, Niek N. Sanders,
Stefan Lienenklaus, Sunil A. David, Shirin Strohmeier, Fatima Amanat, Florian Krammer,
Hamida Hammad, Bart N. Lambrecht, Lynda Coughlan, Adolfo García-Sastre,
Bruno G. De Geest,* and Michael Schotsaert*
Abstract: The search for vaccines that protect from severe Introduction
morbidity and mortality because of infection with severe acute
respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus
that causes coronavirus disease 2019 (COVID-19) is a race
against the clock and the virus. Here we describe an amphi-
philic imidazoquinoline (IMDQ-PEG-CHOL) TLR7/8 adju-
vant, consisting of an imidazoquinoline conjugated to the
chain end of a cholesterol-poly(ethylene glycol) macromolec-
ular amphiphile. It is water-soluble and exhibits massive
translocation to lymph nodes upon local administration
through binding to albumin, affording localized innate im-
mune activation and reduction in systemic inflammation. The
adjuvanticity of IMDQ-PEG-CHOL was validated in a li-
censed vaccine setting (quadrivalent influenza vaccine) and an
experimental trimeric recombinant SARS-CoV-2 spike protein
vaccine, showing robust IgG2a and IgG1 antibody titers in
mice that could neutralize viral infection in vitro and in vivo in
a mouse model.
Severe acute respiratory syndrome coronavirus 2 (SARS-
CoV-2), as the causative agent of coronavirus disease 2019,
also known as COVID-19, is a beta-coronavirus which
belongs to the family of Coronaviridae and is currently
responsible for the third human coronavirus outbreak in the
[1–3]
past 20 years.
Therapeutic drugs are of limited use in the
clinic and effective mass immunization campaigns that induce
long lasting immunity are generally considered crucial to
mitigate the ongoing pandemic. More than hundred candi-
date vaccines, consisting of multiple vaccine types such as
recombinant viral epitopes, adenovirus-based vectors, puri-
fied inactivated or live-attenuated virus, virus like particles
and DNA or RNA based vaccine formulations, are currently
[
4,5]
being investigated.
At present mRNA-based vaccines
formulated in lipid nanoparticles, recombinant protein-based
and inactivated virus-based vaccines as well as viral vector-
[
+]
[+]
[
*] Dr. S. Jangra, A. Choi, R. Rathnasinghe, G. Laghlali,
S. Strohmeier, F. Amanat, Prof. Dr. F. Krammer,
Prof. Dr. L. Coughlan, Prof. Dr. A. García-Sastre,
Prof. Dr. M. Schotsaert
Dr. S. A. David
Virovax, Lawrence, KS (USA)
Prof. Dr. B. N. Lambrecht
Department of Pulmonary Medicine, Erasmus Medical Center
Rotterdam (The Netherlands)
Department of Microbiology
Icahn School of Medicine at Mount Sinai
New York, NY (USA)
Prof. Dr. A. García-Sastre, Prof. Dr. M. Schotsaert
Global Health and Emerging Pathogen Institute
Icahn School of Medicine at Mount Sinai, New York, NY (USA)
E-mail: michael.schotsaert@mssm.edu
[
+]
J. De Vrieze, A. Uvyn, Dr. S. Van Herck, Dr. Z. Zhong,
Prof. Dr. B. G. De Geest
Prof. Dr. A. García-Sastre
Department of Medicine, Division of Infectious Diseases
Icahn School of Medicine at Mount Sinai, New York, NY (USA)
and
The Tisch Cancer Institute
Icahn School of Medicine at Mount Sinai, New York, NY (USA)
Department of Pharmaceutics, Ghent University
Ghent (Belgium)
E-mail: br.degeest@ugent.be
[
+]
A. Choi, R. Rathnasinghe, S. Strohmeier, F. Amanat
Graduate School of Biomedical Sciences
Icahn School of Medicine at Mount Sinai, New York, NY (USA)
Dr. L. Nuhn
Max Planck Institute for Polymer Research, Ackermannweg 10,
K. Deswarte, Prof. Dr. H. Hammad, Prof. Dr. B. N. Lambrecht
Department of Internal Medicine and Pediatrics, Ghent University
and VIB Center for Inflammation Research
5
5128 Mainz (Germany)
+
[
] These authors contributed equally to this work.
[
**] A previous version of this manuscript has been deposited on
a preprint server (https://www.biorxiv.org/content/10.1101/2020.
Zwijnaarde (Belgium)
Prof. Dr. N. N. Sanders
Laboratory for Gene Therapy, Ghent University
Merelbeke (Belgium)
1
0.23.344085v1).
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.202015362.
Dr. S. Lienenklaus
Institute for Laboratory Animal Science, Institute of Immunology
Hannover Medical School, Hannover (Germany)
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[
17]
based vaccines have reached late stage of clinical develop-
ment and are entering the market. For these vaccines, pre-
clinical data in animal models and phase 3 clinical trials
tissues, which is currently a strong limitation for applying
imidazoquinoline TLR7/8 agonists in mass immunization
campaigns. We and others have reported on strategies to alter
the bio-distribution of imidazoquinolines through chemical
conjugation to a synthetic carrier that limits systemic circu-
lation but confers robust translocation to immune-inducing
[
6,7]
also support the hypothesis that these vaccines can effectively
prevent severe illness upon viral infection. However, little is
known about whether recombinant protein vaccines are
capable of conferring protective immunity. In contrast to
the aforementioned mRNA and viral vector-based vaccines,
recombinant protein vaccines are simpler as they consist of
a single entity antigen and do not require antigen expression
in the vaccinees. mRNA vaccines show promising results.
However, the need for formulation into lipid nanoparticles,
required to overcome the barrier of the endosomal membrane
before the mRNA reaches its molecular target the cytoplasm,
poses considerable challenges in the context of manufacturing
[
17–23]
sites in sentinel lymph nodes.
In the present work, we report on a novel amphiphilic
carrier for imidazoquinoline (IMDQ) TLR7/8 agonists with
high translational potential, based on conjugation of a single
imidazoquinoline to the chain end of a cholesteryl-polyeth-
ylene glycol macromolecular amphiphile (IMDQ-PEG-
CHOL; Figure 1A). This design mediates binding to serum
[
17,21–23]
proteins such as albumin
pure lipidation, the conjugate is well water-soluble.
(Figure 1B) and in contrast to
[
8]
and storage. Hence, exploring the viability of a recombinant
protein COVID-19 vaccine might be of considerable rele-
vance.
We demonstrate that IMDQ-PEG-CHOL is a potent
adjuvant which enhances vaccine efficiency and induces
robust Th1 skewed antibody responses in mice when deliv-
ered as a single shot with either admixed S protein (for SARS-
CoV-2) or seasonal quadrivalent inactivated influenza virus
vaccine (QIV, for influenza). Moreover, IMDQ-PEG-CHOL
was able to infer protection in SARS-CoV-2 or influenza virus
(H1N1)-infected mice. In this context, it is noteworthy that in
contrast to human ACE-2, murine ACE-2 is not targeted by
wild type SARS-CoV-2 virus because of species-specific
variations in ACE-2 receptors between mouse and human.
Here we made use of a mouse model where hACE-2 was
introduced through an adenoviral vector, which allows for
subsequent replication of SARS-CoV-2 upon infection in the
SARS-CoV-2 encodes four major structural proteins,
spike (S), membrane (M), nucleocapsid (N), and envelope
(
E). The spike protein comprises a homotrimeric structure
which is present on the surface of the virus and facilitates the
viral attachment and entry into the host via human angio-
tensin-converting enzyme 2 (hACE-2) receptors via its
[9–11]
receptor-binding domain (RBD).
Owing to its involvement in viral entry, the S protein is
a major target for current vaccine development against
[5]
SARS-CoV-2. Therefore, in this study we explored the
recombinant SARS-CoV-2 S protein as a potential vaccine
candidate. As recombinant protein antigens are poorly
immunogenic and are incapable of mounting antigen-specific
immunity of sufficient quality, amplitude and duration, co-
administration of adjuvants that shape B cell and T cell
responses are indispensable. Adjuvants like alum and oil-in-
water emulsions can act through a multitude of mechanisms.
More defined small molecule adjuvants that potently activate
innate immune cells by triggering specific innate immune
receptors might be more relevant for anti-viral vaccine
design. The Toll-like receptors 7 and 8 (TLR7/8) are widely
distributed amongst innate immune cell subsets over a broad
[
24]
airways of transduced mice.
[12]
range of species.
Akin to be an endosomal pattern
recognition receptor for viral RNA, triggering of these
receptors provokes robust type I interferon production that
can skew a Th1-type adaptive immune response against co-
Figure 1. A) Molecular structure of (A1) IMDQ-PEG-CHOL and (A2)
IMDQ-PEG. Conjugation was performed by amide bond formation
between, respectively cholesterylamine and PEG and PEG and IMDQ.
B) Representation of albumin hitchhiking-mediated lymphatic trans-
portation.
[13]
administered antigen.
The latter are characterized by
robust antibody titers capable of inducing viral neutralization
through a variety of mechanisms, including Fc-mediated
innate immune killing as well as inducing CD4 + and CD8 +
T-cell based immunological memory. Moreover, vaccines Results and Discussion
adjuvanted with TLR7/8 ligands have been shown to confer
enhanced protective immunity in both mouse and non-human
The imidazoquinoline 1-(4-(aminomethyl)benzyl)-2-bu-
[
14,15]
[25]
primate models.
Being well-defined small molecules, imidazoquinolines
tyl-1H-imidazo[4,5-c]quinolin-4-amine (IMDQ)
was con-
jugated to cholesteryl-poly(ethylene glycol) (PEG-CHOL),
yielding IMDQ-PEG-CHOL. As a control, non-amphiphilic
IMDQ-PEG was synthesized. (Figure 1A) PEG with a mo-
lecular weight of 3 kDa was chosen as an optimal compromise
between water solubility and drug load. Characterization of
the conjugate was performed by matrix assisted laser
desorption/ionization–time of flight (MALDI-ToF) (Support-
[16]
are a class of TLR7/8 agonists
that hold a massive
technological advantage in terms of production and phys-
icochemical stability. However, their pharmacokinetic profile
is characterized by rapid systemic dissemination upon local
(
subcutaneous or intramuscular) administration, thereby
causing unwanted innate immune activation at multiple distal
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ing Information, Figure S1) analysis whereas high pressure
line (Figure 2D), while being non-toxic within the tested
liquid chromatography (HPLC) analysis (Supporting Infor-
mation, Figure S2) proved absence of free soluble non-
conjugated IMDQ. Both IMDQ-PEG-CHOL and IMDQ-
PEG were water-soluble, but only IMDQ-PEG-CHOL
showed affinity towards albumin as measured by biolayer
interferometry (Figure 2A). On the in vitro level, the
presence of the CHOL motif dramatically improved cellular
uptake by DC2.4 (Figure 2B,C; note that for imaging
purpose, IMDQ was replaced by the fluorescent probe
Cynanine5), a murine model mouse dendritic cell line.
Furthermore, compared to IMDQ-PEG, IMDQ-PEG was
more potent in inducing NF-kB activation in a reporter cell
experimental window (Figure 2E). We attribute this to the
ability of the cholesterol motif to interact with the phospho-
lipid cell membrane. Note that the drop in activity of
conjugated IMDQ relative to native IMDQ is in accordance
[17–19]
to our earlier findings.
On the in vivo level, using a transgenic luciferase-reporter
[26]
mouse model for IFNb-production,
we found that local
administration (subcutaneous injection into the footpad) of
IMDQ-PEG-CHOL, in contrast to unformulated IMDQ,
dramatically reduced systemic innate immune activation,
while focusing its activity to the site of injection and the
draining (popliteal) lymph node (Figure 3A). For quantifica-
Figure 2. A) Biolayer interferometry (BLI) sensorgrams of non-amphiphilic IMDQ-PEG and amphiphilic IMDQ-PEG-CHOL binding to albumin-
À1
coated sensors. A dilution series of 50, 10, and 5 mgmL (dark to light color code, as marked by the black arrow) was measured. Sensors were
dipped into a PEG-or lipid-PEG solution at the 500 s time point, which marks the onset of adsorption. At the 1125 s time point, sensors were
dipped into PBS, which marks the onset of desorption. B) Flow cytometry analysis of association between DC2.4 cells and Cyanine5-PEG,
Cyanine5-PEG-CHOL, respectively. (n=3; Student t-test, ****:p<0.0001, ***:p<0.001) C) Confocal microscopy images of DC2.4 cells incubated
for 24 h at 378C with Cyanine5-PEG and Cyanine5-PEG-CHOL. Left panel represents the Cyanine5 channel, right panel represents the overlay of
the Cyanine5 and transmitted light channels. Scale bar: 15 mm. D) TLR agonistic activity of IMDQ-PEG-CHOL, IMDQ-PEG and native IMDQ
measured as NF-kB activation using the RAW-Blue reporter cell assay. (n=6, mean + SD). E) Cytotoxicity of IMDQ-PEG-CHOL, IMDQ-IMDQ and
native IMDQ, measured by MTT assay (n=6, mean + SD).
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Figure 3. A) Bioluminescence images of luciferase reporter mice (IFNb+/Db-luc); images taken 4, 24 and 48 h post footpad injection of IMDQ-
PEG-CHOL, IMDQ-PEG and native IMDQ. B1) Confocal microscopy images of lymph node tissue sections 48 h post subcutaneous injection of
Cyanine5-PEG-CHOL, respectively Cyanine5-PEG, into the footpad of mice. Scale bar: 100 mm. B2) Flow cytometry analysis of the draining
popliteal lymph node 48 h post subcutaneous injection of Cyanine5-PEG-CHOL, respectively Cyanine5-PEG into the footpad of mice. (n=3, mean
+
SD; Student’s t-test: ****: p<0.0001) C) Translocation of Cyanine5-PEG-CHOL to the draining popliteal lymph node analyzed 24 h post
injection into the footpad, measured by flow cytometry. (n=6, mean + SD) D) Flow cytometry analysis of the innate immune response in the
draining popliteal lymph node 24 h post injection of IMDQ-PEG-CHOL into the footpad (D1) Relative increase in innate immune cell subsets, B
and T cell numbers relative to a naïve control and (D2) maturation/activation of innate immune cell subsets, B and T cells (n=6, mean + SD;
Student’s t-test: ****: p<0.0001).
tion of the luminescence imaging data, we refer to the
Supporting Information, Figure S3. Interestingly, the CHOL
motif appeared crucial for mediating lymphatic translocation
as the IMDQ-PEG control induced very limited activity in the
draining lymph node. To further support this, we performed
microscopic (Figure 3B1) and flow cytometry (Figure 3B2)
analysis of popliteal lymph nodes of mice that received
fluorescent Cyanine5-PEG-CHOL or Cyanine-PEG, respec-
tively. These experiments revealed a dramatic increase in
fluorescence when the conjugates contained the CHOL motif.
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A more detailed analysis of immune cells subsets in the
assay (ELISA) plates. The total IgG antibody titers in mice
draining lymph node revealed that vast percentages of
lymphocytes, notably over 50% of dendritic cells (DCs) and
macrophages (Mf), as well as 40% of B cells, were targeted by
Cyanine5-PEG-CHOL (Figure 3C). In a similar experimen-
tal setting, IMDQ-PEG-CHOL induced recruitment (Fig-
ure 3D1) and robust activation (Figure 3D2) of immune cells.
Taken together, these data support our hypothesis that
IMDQ-PEG-CHOL is a potent adjuvant that focuses its
activity to draining lymphoid tissue, combined with a promis-
ing safety profile.
We evaluated the potential of IMDQ-PEG-CHOL to
adjuvant a licensed vaccine, that is, the quadrivalent influenza
vaccine (QIV), in a well-established preclinical vaccination-
infection model. Hereto we vaccinated BALB/c mice with
each of 1.5 mg of QIV with or without 100 mg of IMDQ-PEG-
CHOL or PEG-CHOL as a control. The study protocol is
outlined in Figure 4A. Six mice in each group received a total
of 100 mL vaccine-adjuvant mixture, intramuscularly, divided
over both hind legs and the blood was collected 3 weeks post
vaccination, followed by serological assays. No adverse effects
as a result of vaccination were observed. For detection of
influenza-specific antibodies induced by the QIV vaccines, we
used vaccine antigen to coat enzyme-linked immunosorbent
which received QIV only or QIV+ PEG-CHOL were very
low as compared to the mice vaccinated with QIV+ IMDQ-
PEG-CHOL (also shown as area under curve (AUC) in the
Supporting Information, Figure S4). Unadjuvanted QIV and
PEG-CHOL admixed QIV resulted mainly in vaccine-
specific IgG1 antibodies, whereas IMDQ-PEG-CHOL re-
sulted in a balanced IgG1/IgG2a response as shown in
Figure 4B. Interestingly, QIV + PEG-CHOL resulted in
even lower antibody responses than QIV alone, an observa-
tion we confirmed in an independent vaccination experiment
(data not shown). The sera from four out of six mice that
received QIV + IMDQ-PEG-CHOL could efficiently inhibit
hemagglutination of chicken red blood cells (RBC) in vitro,
by A/Singapore/gp1908/2015 IVR-180, the H1N1 virus com-
ponent in QIV, with hemagglutination inhibition (HI) titers
outperforming those of the other immunized groups (Fig-
ure 4C).
Next, the immunized mice were challenged with a hun-
dred-fold half-lethal dose (LD ) of IVR-180 (H1N1) virus to
50
examine the magnitude of protection against viral infection in
vivo. The lungs were harvested from three mice in each group
5 days post challenge to determine lung virus titers. Consis-
tent with the ELISA and HI data, mice immunized with
Figure 4. IMDQ-PEG-CHOL induces a balanced neutralizing antibody response to IVR-180 [Influenza A/Singapore/gp1908/2015 (H1N1)]
infection. A) Outline of the QIV immunization and influenza virus challenge study. B) Vaccine-specific ELISA titers (titers are expressed on the X
axis as the reciprocal of the dilution factor) for total IgG, IgG1 and IgG2a and IgG2a/IgG1 ratio (based on the AUC (OD at 450 nm) curve of the
individual serum samples) in mice sera collected 3 weeks post-vaccination. C) Control versus immunized sera analyzed for HI titers by
hemagglutination inhibition assay, using 4 haemagglutination units of IVR-180 virus. D) Body weight loss of mice reported as percentage of initial
body weight after challenge with 100 LD50 (18000 PFU) of IVR-180 virus. E) Viral lung titers after challenge with 100LD50 (18000 PFU) of IVR-180
À1
virus. Data are represented as plaque-forming-unit (PFU)mL (geometric mean + SD). Lungs were harvested on day-5 post infection with IVR-
1
80 virus.
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QIV + IMDQ-PEG-CHOL exhibited best reduction in viral
lung titers as evidenced by an almost negligible number of
plaques when compared to other groups (Figure 4E). This
was also reflected in the optimal protection from body weight
loss of QIV + IMDQ-PEG-CHOL mice after viral challenge
tion assay (Figure 5C1-2). By contrast, serum of mice immu-
nized with spike protein + IMDQ-PEG-CHOL was able to
neutralize > 50% of SARS-CoV-2 virus infection in this
assay, which was also significantly higher than the serum of
mice immunized with spike protein + AddaVax. Therefore, as
has been reported for human convalescent sera, higher anti-
spike ELISA titers correlate with microneutralization titers
(
Figure 4D). In conclusion, our novel adjuvant IMDQ-PEG-
CHOL was able to offer excellent control of viral infection
and therefore, in combination with the right antigen, might
also hold promise to confer protective immunity against other
respiratory viruses such as SARS-CoV-2.
We next investigated the potential of IMDQ-PEG-CHOL
to adjuvant recombinant SARS-CoV-2 S protein. For this
purpose, BALB/c mice were immunized intramuscularly with
[27]
(reference).
Finally, we investigated to what extent S protein +
IMDQ-PEG-CHOL immunization is able to confer protec-
tion against a SARS-CoV-2 viral challenge. As mice do not
express the hACE-2 receptor that is needed for the virus to
infect the host, we first transduced immunized mice with an
adenoviral vector encoding for hACE-2 by intranasal instal-
lation. Four days later, mice were challenged with SARS-
CoV-2 virus and again 4 days later, lungs were harvested, and
the residual viral infection was quantified by a plaque assay.
Interestingly, whereas non-adjuvanted Spike protein could
not confer any protection, relative to the non-immunized
group, Spike protein + IMDQ-PEG-CHOL vaccination
conferred sterilizing immunity against the SARS-CoV-2
infection with plaque numbers below the detection limit
(Figure 5D), and performed significantly better than spike
protein + AddaVax immunization, which correlates with the
higher microneutralization titers observed in the spike
protein + IMDQ-PEG-CHOL group (Figure 5C1-2).
6
mg of recombinant trimeric spike protein either unadju-
vanted or adjuvanted with IMDQ-PEG-CHOL or with
equivalent amounts of MF59-like water-in-oil vaccine Adda-
Vax as a control established vaccine adjuvant. The recombi-
nant vaccine consisted of the ectodomain of the SARS-CoV-2
spike protein from which the polybasic cleavage site was
removed. Stabilizing prolines were added at positions 986 and
9
87 and trimerization was promoted by fusion to a T4
trimerization domain (see the Supporting Information, Meth-
ods section for more details). The study protocol is outlined in
Figure 5A. Serum was collected after 21 days post immuni-
zation and analyzed for Spike protein specific IgG titers.
Whereas non-immunized mice evidently did not show any
detectable spike protein-specific titers in their sera, immuni-
zation with S protein induced Spike protein-specific titers in
all groups (Figure 5B), also shown as area under the curve Conclusion
AUC) titers in Supplementary Figure 54. The total S protein-
(
specific IgG titers were found to be the highest in the IMDQ-
PEG-CHOL adjuvanted group. Additionally, Spike protein +
IMDQ-PEG-CHOL immunization resulted in a higher Ig-
G2a/IgG1 ratio, suggesting a more potent Th1 immune
response and more efficient class switching towards IgG2a
as compared to spike protein only or spike protein +
AddaVax immunization.
Next, the sera from vaccinated mice were used to test the
ability to inhibit SARS-CoV-2 infection in vitro. Although
immunization with non-adjuvanted S protein was able to
induce some IgG titers, it was found ineffective in neutralizing
viral infection of Vero E6 cells in vitro in a microneutraliza-
We have shown in this work that IMDQ-PEG-CHOL is
a potent adjuvant with enhanced safety profile that induced
innate immune activation in lymphoid tissue upon local
administration. Whereas IMDQ in soluble, unformulated
form, rapidly enters systemic circulation, conjugation to
a lipid-polymer amphiphile prevents the latter while promot-
ing translocation to the draining lymph node, likely through
binding to albumin in the interstitial flow. In mouse models,
we have demonstrated that a single immunization with QIV
or S protein adjuvanted with IMDQ-PEG-CHOL induced
robust Th1 skewed antibody responses. Importantly, vacci-
nation with QIV and S protein adjuvanted with IMDQ-PEG-
Figure 5. IMDQ-PEG-CHOL induces a balanced neutralizing antibody response to SARS-CoV-2 infection. A) Outline of the Spike protein
vaccination and SARS-CoV-2 challenge. B) ELISA titers (titers are expressed on the X axis as the reciprocal of the dilution factor) for total IgG,
IgG1, and IgG2a and IgG2a/IgG1 ratio (based on the AUC (OD at 450 nm) curve of the individual serum samples) in mice sera collected 3 weeks
post-vaccination. C) Control versus vaccinated sera examined for presence of virus-neutralizing antibodies by microneutralization assay, using 100
tissue culture infectious dose 50 (TCID50) of SARS-CoV-2 virus. The outcome is represented as a percentage inhibition of viral growth in (C1) and
as the half maximal inhibitory concentration IC50 calculated by a non-linear regression analysis of percentage inhibition curve in (C2). D) Viral
À1
lung titers represented as Plaque-forming-unit (PFU)mL (geometric mean with geometric SD). The Ad5-hACE2 transduced mice were challenged
with 5”104 PFU of SARS-CoV-2 and the lungs were harvested on day-4 post infection.
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CHOL resulted in virus-specific neutralizing antibodies and
[
[
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control of viral infection after challenge with influenza and
SARS-CoV-2 viruses, respectively. Whereas we are aware of
the limitations of the present studies, we do believe that the
overarching message that single vaccination with a properly
adjuvanted recombinant S protein-based vaccine is able to
induce protective immunity in a mouse model, is of great
relevance with regard to broadening the arsenal of emerging
COVID-19 vaccines.
Furthermore, the concept of PEG-CHOL conjugation
could be applied to other small molecule drugs that have the
lymph node as a target tissue, including innate receptor
agonists, immune checkpoints blockers and chemotherapeu-
tics.
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Excellence for Influenza Research and Surveillance (CEIRS,
contract no. HHSN272201400008C), by SEM-CIVIC,
a NIAID funded Collaborative Influenza Vaccine Innovation
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lence for Influenza Research and Surveillance (CEIRS)
contract HHSN272201400008C (F.K., for reagent genera-
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Conflict of interest
A.G.-S. is inventor in patents on influenza and COVID-19
vaccines owned by the Icahn Shool of Medicine at Mount
Sinai. The laboratory of A.G.-S. has research agreements on
the study of viral vaccines and prophylaxis with Avimex,
Pfizer, and 7Hills Pharma. A.G.-S. is a consultant for Avimex
and Esperovax.
Manuscript received: November 17, 2020
Revised manuscript received: December 24, 2020
Accepted manuscript online: January 19, 2021
Version of record online: March 11, 2021
Keywords: amphiphiles · COVID-19 · innate immunity ·
SARS-CoV-2 · vaccines
Angew. Chem. Int. Ed. 2021, 60, 9467 – 9473
ꢀ 2021 Wiley-VCH GmbH
www.angewandte.org
9473