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Preclinical and Clinical Demonstration of Immunogenicity by mRNA Vaccines against H10N8 and H7N9 Influenza Viruses


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Recently, the World Health Organization confirmed 120 new human cases of avian H7N9 influenza in China resulting in 37 deaths, highlighting the concern for a potential pandemic and the need for an effective, safe, and high-speed vaccine production platform. Production speed and scale of mRNA based vaccines make them ideally suited to impede potential pandemic threats. Here we show that lipid nanoparticle (LNP)-formulated, modified mRNA vaccines, encoding hemagglutinin (HA) proteins of H10N8 (A/Jiangxi-Donghu/346/2013) or H7N9 (A/Anhui/1/2013), generated rapid and robust immune responses in mice, ferrets, and nonhuman primates, as measured by hemagglutination inhibition (HAI) and microneutralization (MN) assays. A single dose of H7N9 mRNA protected mice from a lethal challenge and reduced lung viral titers in ferrets. Interim results from a first-in-human, escalating dose, phase 1 H10N8 study show very high seroconversion rates, demonstrating robust prophylactic immunity in humans. Adverse events (AEs) were mild or moderate with only a few severe and no serious events. These data show that LNP-formulated, modified mRNA vaccines can induce protective immunogenicity with acceptable tolerability profiles.
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Original Article
Preclinical and Clinical Demonstration
of Immunogenicity by mRNA Vaccines
against H10N8 and H7N9 Influenza Viruses
Kapil Bahl,
Joe J. Senn,
Olga Yuzhakov,
Alex Bulychev,
Luis A. Brito,
Kimberly J. Hassett,
Michael E. Laska,
Mike Smith,
Örn Almarsson,
James Thompson,
Amilcar (Mick) Ribeiro,
Mike Watson,
Tal Zaks,
and Giuseppe Ciaramella
Valera, A Moderna Venture, 500 Technology Square, Cambridge, MA 02139, USA;
Moderna Therapeutics, 200 Technology Square, Cambridge, MA 02139, USA
Recently, the World Health Organization conrmed 120
new human cases of avian H7N9 inuenza in China resulting
in 37 deaths, highlighting the concern for a potential pandemic
and the need for an effective, safe, and high-speed vaccine
production platform. Production speed and scale of mRNA-
based vaccines make them ideally suited to impede potential
pandemic threats. Here we show that lipid nanoparticle
(LNP)-formulated, modied mRNA vaccines, encoding hem-
agglutinin (HA) proteins of H10N8 (A/Jiangxi-Donghu/346/
2013) or H7N9 (A/Anhui/1/2013), generated rapid and robust
immune responses in mice, ferrets, and nonhuman primates,
as measured by hemagglutination inhibition (HAI) and micro-
neutralization (MN) assays. A single dose of H7N9 mRNA pro-
tected mice from a lethal challenge and reduced lung viral titers
in ferrets. Interim results from a rst-in-human, escalating-
dose, phase 1 H10N8 study show very high seroconversion
rates, demonstrating robust prophylactic immunity in hu-
mans. Adverse events (AEs) were mild or moderate with only
a few severe and no serious events. These data show that
LNP-formulated, modied mRNA vaccines can induce protec-
tive immunogenicity with acceptable tolerability proles.
Several avian inuenza A viruses (H5N1, H10N8, H7N9, and H1N1)
have crossed the species barrier, causing severe and often fatal respi-
ratory disease in humans. Fortunately, most of these strains are not
able to sustain person-to-person transmission.
However, lessons
learned from these outbreaks demonstrated that new approaches
are needed to address potential future pandemic inuenza outbreaks.
Two major glycoproteins, crucial for inuenza infection, are hemag-
glutinin (HA) and neuraminidase (NA); both are expressed on the sur-
face of the inuenza A virion.
HA mediates viral entry into host cells
by binding to sialic acid-containing receptors on the cell mucosal sur-
face and the fusion of viral and host endosomal membranes.
The segmented inuenza A genome permits re-assortment and ex-
change of HA (or NA) segments between different inuenza strain
subtypes during concomitant host-cell infection. Generation of novel
antigenic proteins (antigenic shift) and sustainable person-to-person
transmission are hallmarks of pandemic inuenza strains.
strains can spread quickly and cause widespread morbidity and
mortality in humans due to high pathogenicity and little to no pre-
existing immunity. Recent cases (2013) of avian-to-human transmis-
sion of avian inuenza A virus subtypes included H7N9, H6N1, and
The case-fatality rate in over 600 cases of H7N9 infections
was 30%.
Most recently, the World Health Organization reported
another 120 cases since September 2016 resulting in 37 deaths.
date, H10N8 infection in man has been limited; yet, of the three
reported cases, two were fatal.
The limited efcacy of existing antiviral therapeutics (i.e., oseltamivir
and zanamivir) makes vaccination the most effective means of protec-
tion against inuenza.
Conventional inuenza vaccines induce pro-
tection by generating HA-specic neutralizing antibodies, the major
correlate of protection, against the globular head domain.
vaccines utilize the HA protein, administered as a subunit, split
virion, inactivated whole virus, or live-attenuated virus. A majority
of approved inuenza vaccines are produced in embryonated chicken
eggs or cell substrates. This process takes several months and relies on
the availability of sufcient supplies of pathogen-free eggs and adap-
tation of the virus to grow within its substrate.
The 56 months
required to produce enough vaccine to protect a substantial propor-
tion of the population consumes much of the duration of the often-
devastating rst wave of a pandemic.
This mismatch between the
speeds of vaccine production and epidemic spread drives the search
for vaccine platforms that can respond faster.
Using mRNA complexed with protamine (RNActive, Curevac),
Petsch et al.
demonstrated that intradermal (ID) vaccination of
mice with RNActive encoding full-length HA from inuenza virus
H1N1 (A/Puerto Rico/8/1934) induced effective seroconversion and
Received 23 January 2017; accepted 24 March 2017;
Correspondence: Giuseppe Ciaramella, Valera, 500 Technology Square, Cam-
bridge, MA 02139, USA.
Molecular Therapy Vol. 25 No 6 June 2017 ª2017 The Authors. 1
This is an open access article under the CC BY-NC-ND license (
Please cite this article in press as: Bahl et al., Preclinical and Clinical Demonstration of Immunogenicity by mRNA Vaccines against H10N8 and H7N9
Influenza Viruses, Molecular Therapy (2017),
virus-neutralizing antibodies in all vaccinated animals. Immunity was
long lasting and protected both young and old animals from lethal
challenge with the H1N1, H3N2, and H5N1 strains of the inuenza
A virus.
Efcacy of these RNActive vaccines was also shown in fer-
rets and pigs.
The use of a delivery system can dramatically reduce the doses needed
to generate potent immune responses, without an additional conven-
tional adjuvant. Lipid nanoparticles (LNPs) have been used exten-
sively for the delivery of small interfering RNA (siRNA), and they
are currently being evaluated in late-stage clinical trials via intrave-
nous administration.
Exogenous mRNA can stimulate innate immunity through Toll-like
receptors (TLRs) 3, 7, and 8 and cytoplasmic signal-recognition pro-
teins RIG-I and MDA5.
The adjuvant effect of stimulating innate
immunity may be advantageous for puried protein vaccines, but
indiscriminate immune activation can inhibit mRNA translation,
reducing antigen expression and subsequent immunogenicity.
This can be overcome by replacing uridine nucleosides with naturally
occurring base modications, such as pseudouridine and 5-methylcy-
Recently, we
and others
have shown how LNP-encap-
sulated modied mRNA vaccines can induce extraordinary levels of
neutralizing immune responses against the Zika virus in mice and
nonhuman primates, respectively.
In this study, we evaluated the immunogenicity of two LNP-formu-
lated, modied mRNA-based inuenza A vaccines encoding the
HA of H10N8 (A/Jiangxi-Donghu/346/2013) and H7N9 (A/Anhui/
1/2013) in animals and H10N8 HA mRNA in humans from an
ongoing trial. In the animal studies, we show that both vaccines
generated potent neutralizing antibody titers in mice, ferrets, and cyn-
omolgus monkeys (cynos) after a single dose. Additionally, a single
dose of H7N9 HA mRNA protected mice from an autologous lethal
challenge and reduced lung viral titers in ferrets. Encouraged by these
ndings, a rst-in-human, dose-escalating, phase 1 trial is ongoing,
with interim results reported here that conrm the observed, preclin-
ical immunogenicity data with a safety prole consistent with other
non-live vaccines.
H10N8 and H7N9 HA mRNA Immunogenicity in Mice
In vitro protein expression for both H10N8 HA (H10) and H7N9 HA
(H7) mRNA vaccines were conrmed by transfection of HeLa cells.
Western blot of resulting cell lysates demonstrated a 75-kDa band
for both constructs using the corresponding HA-specic antibodies
(Figure S1), consistent with previous reports for other HAs.
to a lack of glycosylation, both H10 HA and H7 HA protein controls
had a molecular weight of 62 kDa.
Hemagglutination inhibition (HAI), IgG1, and IgG2a titers were
measured after a single 10-mg dose of either formulated H10 or H7
mRNA in BALB/c mice immunized ID. HAI titers were below the
limit of detection (<10) at day 7 but increased well above baseline
by day 21 (Figure 1A). Unlike HAI, both anti-H10 and anti-H7
IgG1 and IgG2a titers were detected on day 7 (Figures 1B and 1C).
For H10, IgG1 and IgG2a titers continued to increase until day 21
and were maintained at day 84. For H7, both IgG1 and IgG2a anti-
body titers increased 10-fold between day 21 and day 84 (Figure 1C).
IgG2a titers were greater than IgG1 titers at all time points following
formulated H10 or H7 mRNA immunization, suggesting a TH1-
skewed immune response. For H10, these differences were signicant
at day 84 (p = 0.0070) and for H7 at day 7 (p = 0.0017) and day 21
(p = 0.0185). A 10-mg H10 mRNA-boosting immunization (21 days
post-prime) resulted in a 2- to 5-fold increase in HAI titers, compared
to a single dose at all time points tested (p < 0.05) (Figure 1D). Titers
remained stable for more than a year, regardless of the number of
While most vaccines are delivered via an intramuscular (IM) or sub-
cutaneous administration,
the ID route of administration has the
potential to be dose sparing. Therefore, to examine the effect of admin-
istration route on immunogenicity, BALB/c mice were immunized ID
or IM with formulated H10 or H7 mRNA at four different dose levels.
All animals received a boosting immunization on day 21, and serum
was collected 28 days post-boost (day 49). Immune responses were
observed for both vaccines at all dose levels tested (Figures S2A and
S2B). Titers were slightly higher following IM administration at
2 and 0.4 mg for H10, but this difference was only signicant at the
2-mg dose (p = 0.0038) (Figure S2A). The differences in H10 HAI titers
were signicant between some of the dose levels following IM admin-
istration: 10 versus 0.4 mg, p = 0.0247; 10 versus 0.08 mg, p = 0.0002;
2 versus 0.08 mg, p = 0.0013; and 0.4 versus 0.08 mg, p = 0.0279. HAI
titers following H7 immunization trended higher as the dose increased
although no signicance was detected. In addition, there was no signif-
icant difference between IM and ID immunization (Figure S2B). T cell
responses, as measured by IFNɣELISpot, were observed for both
H10 and H7 at all doses tested (Figures S2C and S2D). Similar to
H7 HAI titers, T cell responses trended higher following IM adminis-
tration, especially for H7. However, signicance could not be estab-
lished due to pooling of the samples by group. Overall, after two doses,
immunization with either H10 or H7 mRNA elicited an immune
response at all doses tested with both ID and IM administration.
Given this innovative vaccine platform, we examined the bio-
distribution of the mRNA vaccines for both routes of administration.
Male CD-1 mice received 6 mg formulated H10 mRNA either IM or ID.
Following IM administration, the maximum concentration (C
the injection site muscle was 5,680 ng/mL, and the level declined
with an estimated t
of 18.8 hr (Table 1). Proximal lymph nodes
had the second highest concentration at 2,120 ng/mL (t
of 8 hr
with a relatively long t
of 25.4 hr), suggesting that H10 mRNA
distributes from the injection site to systemic circulation through
the lymphatic system. The spleen and liver had a mean C
86.9 ng/mL (area under the curve [AUC]
of 2,270
and 47.2 ng/mL (AUC
of 276, respectively. In the
remaining tissues and plasma, H10 mRNA was found at 100- to
1,000-fold lower levels.
Molecular Therapy
2 Molecular Therapy Vol. 25 No 6 June 2017
Please cite this article in press as: Bahl et al., Preclinical and Clinical Demonstration of Immunogenicity by mRNA Vaccines against H10N8 and H7N9
Influenza Viruses, Molecular Therapy (2017),
Following ID administration, C
within the skin at the injection site
was 18.2 mg/mL. Levels declined by 24 hr with an estimated t
23.4 hr, suggesting that the H10 mRNA likely dissipated to systemic
circulation via the proximal draining lymph node, as seen for the IM
dosing. Consistent with this, the spleen, with a C
of 1.66 ng/mL
(1,663.52 pg/mL; AUC
of 114.25, had the highest levels
among distal tissues. Only trace amounts of H10 mRNA were found
in the heart, kidney, liver, and lung. Overall, whether administered
ID or IM, the biodistribution of this vaccine was consistent with
that observed for other vaccines,
where a local deposition effect
was observed followed by draining to the local lymph nodes and sub-
sequent circulation in the lymphatic system (Table 1;Table S1).
To understand the expression prole of mRNA after IM and ID admin-
istration, BALB/c mice were injected on day 0 with formulated lucif-
erase mRNA at four different dose levels (10, 2, 0.4, and 0.08 mg).
Expression was found to be dose dependent. As the dose increased,
expression was found in distal tissues, with peak expression observed
6 hr after dosing. There were no signicantdifferences when comparing
maximum expression and time of maximal expression across IM and
ID routes (Figure S3A). The time course of expression was also similar
with both routes (Figures S3B and S3C). However, the distribution of
expression changed slightly when the two routes were compared.
Expression outside of the site of administration was observed across
all dose levels, but it was more pronounced following IM administra-
tion, which is consistent with the biodistribution data (Figures S4A
S4E; Table 1;Table S1).
H7 mRNA Vaccine Provides Protection against Lethal Influenza
H7N9, A/Anhui/1/2013, in Mice and Ferrets
To determine the time to onset and duration of immunity to inuenza
H7N9 (A/Anhui/1/2013) lethal challenge, BALB/c mice were immu-
nized ID with 10, 2, or 0.4 mg formulated H7 mRNA. For negative
controls, placebo and 10 mg formulated H7 mRNA decient in
expression, due to the removal of a methyl group on the 20-O position
of the rst nucleotide adjacent to the cap 1 structure at the 50end of
the mRNA (15 Da cap), were included. Serum was collected on days
6, 20, and 83, and mice were challenged via intranasal (IN) instillation
with a target dose of 2.5 10
tissue culture infectious dose (TCID
on days 7, 21, and 84. Changes in body weight and clinical signs of
disease were monitored for 14 days post-challenge. A single vaccina-
tion was found to be protective against H7N9 challenge (2.5 10
Figure 1. Mice Immunized with H10 or H7 mRNA Generate Robust and Stable Antibody Responses Consistent with a TH1 Profile
BALB/c mice were vaccinated ID with a single 10-mg dose of formulated H10 or H7 mRNA. (A) H10 and H7 indicate mean HAI titers (limit of detection is 1:10). Dotted line
indicates the correlate of protection in humans (1:40). (B and C) IgG1 and IgG2a titers were measured for both H10 (B) and H7 (C) via ELISA (n = 5/group).
p = 0.0070,
p = 0.0017, and
p = 0.0185 versus IgG2a at the same time point. (D) BALB/c mice were immunized ID with a single 10-mg dose of formulated H10 mRNA. A subset of these
mice received a 10-mg boost on day 21. Serum was collected at the indicated time points, and neutralizing antibody titers were determined by HAI (n = 15/group). Placebo
controls were also included.
p < 0.05 single dose versus boosting dose at the same time point. Error bars indicate standard mean error.
Molecular Therapy Vol. 25 No 6 June 2017 3
Please cite this article in press as: Bahl et al., Preclinical and Clinical Demonstration of Immunogenicity by mRNA Vaccines against H10N8 and H7N9
Influenza Viruses, Molecular Therapy (2017),
;Figures 2A2C). There was a signicant increase in sur-
vival for animals in the three vaccine dose groups compared to the an-
imals from the two control groups (p < 0.0001). Clinical observations
in inuenza-infected mice included rough coat, hunched posture,
orbital tightening, and, in some cases, labored breathing. Weight
loss (incidence and duration) was more prevalent for animals in the
control groups and seen to a lesser extent in the low-dose vaccine
group (Figures 2D2F). HAI titers were below the limit of detection
until day 20 for both the 10- and 2-mg dose groups (Figure S5). There
was a 5- to 7-fold increase in HAI titers from day 20 to day 83 at all
doses tested (p < 0.0001). Day-83 titers were dose dependent with
mean titers of 224, 112, and 53 for the 10-mg dose, 2-mg dose, and
0.4-mg dose groups, respectively (p < 0.0001). Interestingly, despite
complete protection to challenge at the 0.4-mg dose at day 21 (Fig-
ure 2B), a protective HAI titer (R40) was not detected until day 83
at this dose, suggesting additional mechanism(s) of protection.
The negative mRNA control unexpectedly showed some delayed ef-
cacy by day 21. However, this group of animals appeared to have
received a dose lower than the day 7 and day 84 groups, based on
back titer calculation (6.2 10
versus 3.8 10
and 6.1
, respectively
), which was only 3-fold higher than the LD
1.88 10
(95% condence interval [CI] = 8.02 10
5.51 10
Nonetheless, this group had comparable weight loss to the placebo
group, and it was just above the threshold for euthanasia (30%) for
some of the animals, thus conrming the signicant protection
observed in the positive vaccine groups. Additionally, it is not possible
to rule out a low level of protein expression from the de-methylated
cap of the negative mRNA control.
Unlike mice, ferrets are naturally susceptible to human inuenza
virus isolates. Human and avian inuenza viruses both replicate ef-
ciently in the respiratory tract of ferrets, and numerous clinical signs
found in humans following seasonal or avian inuenza virus infection
are also present in the ferrets.
Ferrets (n = 8/group) were vacci-
nated ID on day 0 with 200-, 50-, or 10-mg doses of formulated
H7 mRNA. Formulated H7 mRNA with a 15 Da cap and placebo
were included as negative controls. A subset of ferrets received a sec-
ond ID vaccination on day 21. All groups were exposed to inuenza
H7N9 via IN challenge (1 10
). The primary endpoint for
this study was viral burden determined by TCID
in the lung at
3 days post-challenge, which is when the peak viral load is seen in
control animals (data not shown). A reduction in lung viral titers
was observed when ferrets were challenged 7 days post-immunization
at all doses tested (Figures S6AS6C). Ferrets immunized with 200 mg
and challenged on day 49 had viral loads below the level of detection
(Figure S6C). Antibody titers, as measured by HAI, increased signif-
icantly by day 21 for all dose groups (p < 0.05); as measured by micro-
neutralization (MN), signicant increases were observed by day 49 for
all dose groups (p < 0.05) (Figures S7A and S7B). A second immuni-
zation increased titers but showed no statistical benet compared to a
single immunization, likely due to the two to four log reduction in
viral lung titers seen in both the single- and double-immunization
groups (Figures S7AS7D). Two immunizations with 50-mg doses
signicantly increased HAI and MN titers compared to placebo
(p < 0.05), and two immunizations with 200-mg doses generated
signicant HAI and MN titers versus placebo and all other doses
(p < 0.0001) (Figures S7C and S7D).
In the absence of an H10N8 (A/Jiangxi-Donghu/346/2013) chal-
lenge model, the onset and duration of immunity to formulated
H10 mRNA in ferrets was tested by HAI. Groups of ferrets were
immunized ID once, twice, or three times with 50 or 100 mg H10
mRNA. Immunization with a single dose of 50 or 100 mg resulted
in signicant and comparable increases in HAI titers at days 21, 35,
and 49 (p < 0.0001; Figure 3). Immunization with a 100-mg dose re-
sulted in only slightly elevated antibody responses on day 7 compared
to day 0 (p < 0.0001), with minimal differences observed with the
50-mg dose on day 7 compared to day 0 (p < 0.3251). Subsequent
boosts with either a 50- or 100-mg dose (delivered on day 21 or on
both days 21 and 35) resulted in signicant and comparable increases
in HAI titers on days 35 and 49 (p < 0.0001). Overall, the H10 mRNA
administered at a 50- or 100-mg dose yielded signicant increases in
HAI antibody titers as compared with prevaccination baseline values
Table 1. Biodistribution of H10 mRNA in Plasma and Tissue after IM
Administration in Mice
Matrix t
0264 h
(h)Mean SE Mean SE
Bone marrow 2.0 3.35 1.87 NA NC
Brain 8.0 0.429 0.0447 13.9 1.61 NR
Cecum 8.0 0.886 0.464 11.1 5.120 NC
Colon 8.0 1.11 0.501 13.5 5.51 NC
Distal lymph nodes 8.0 177.0 170.0 4,050 2,060 28.0
Heart 2.0 0.799 0.225 6.76 1.98 3.50
Ileum 2.0 3.54 2.60 22.6 10.8 5.42
Jejunum 2.0 0.330 0.120 5.24 0.931 8.24
Kidney 2.0 1.31 0.273 9.72 1.44 11.4
Liver 2.0 47.2 8.56 276 37.4 NC
Lung 2.0 1.82 0.555 12.7 2.92 16.0
Muscle (injection site) 2.0 5,680 2,870 95,100 20,000 18.8
Plasma 2.0 5.47 0.829 35.5 5.41 9.67
Proximal lymph nodes 8.0 2,120 1,970 38,600 22,000 25.4
Rectum 2.0 1.03 0.423 14.7 3.67 NR
Spleen 2.0 86.9 29.1 2,270 585 25.4
Stomach 2.0 0.626 0.121 11.6 1.32 12.7
Testes 8.0 2.37 1.03 36.6 11.8 NR
Male CD-1 mice received 300 mg/kg (6 mg) formulated H10 mRNA via IM immuniza-
tion. Two replicates of bone marrow, lung, liver, heart, right kidney, inguinal- and popli-
teal-draining lymph nodes, axillary distal lymph nodes, spleen, brain, stomach, ileum,
jejunum, cecum, colon, rectum, testes (bilateral), and injection site muscle were
collected for bDNA analysis at 0, 2, 8, 24, 48, 72, 120, 168, and 264 hr after dosing
(n = 3 mice/time point). NA, not applicable AUC with less than three quantiable con-
centrations; NC, not calculated; NR, not reported because extrapolation exceeds 20% or
R-squared is less than 0.80.
Molecular Therapy
4 Molecular Therapy Vol. 25 No 6 June 2017
Please cite this article in press as: Bahl et al., Preclinical and Clinical Demonstration of Immunogenicity by mRNA Vaccines against H10N8 and H7N9
Influenza Viruses, Molecular Therapy (2017),
and controls (p < 0.0001). A single booster vaccination provided a sig-
nicant increase in titers, but a second booster dose did not yield an
additional increase (Figure 3).
H10 HA and H7 HA mRNA Immunogenicity in Nonhuman
One of the major limitations with other nucleic acid-based technolo-
gies, such as plasmid DNA, has been translation to higher-order spe-
cies, such as nonhuman primates. To evaluate the immune responses
elicited in nonhuman primates, HAI titers were measured in cynos af-
ter two immunizations (days 1 and 22) at two dose levels (0.2 and
0.4 mg) of formulated H7 mRNA administered IM and ID (Figures
4A and 4B). Formulated H10 mRNA was tested with only the
0.4-mg dose delivered ID and IM with the same immunization
schedule (days 1 and 22) (Figure 4C). Both H10 and H7 mRNA vac-
cines generated HAI titers between 100 and 1,000 after a single immu-
nization (day 15). HAI titers of 10,000 were generated for both H10
and H7 at 3 weeks following the second immunization (day 43),
regardless of dose or route of administration. At 0.4 mg, the cynos
experienced some systemic symptoms, such as warm to touch pain
at the injection site, minor injection site irritation, and, in some cases,
decreased food consumption following either H10 or H7 immuniza-
tion. All symptoms resolved within 4872 hr. Overall, both ID and IM
administration elicited similar HAI titers regardless of dose, suggest-
ing that lower doses may generate a similar HAI titer.
H10 mRNA Immunogenicity and Safety in Humans
To evaluate the safety and immunogenicityof H10 mRNA in humans, a
randomized, double-blind, placebo-controlled, dose-escalating phase 1
trial is ongoing (Clinical Trials Identier NCT03076385). We report
here interim results, obtained 43 days post-vaccination of 31 subjects
(23 of whom received active H10 at 100 mg IM and eight of whom
received placebo). Immunogenicity data show that 100% (n = 23)
and 87% (n = 20) of subjects who received the H10 vaccine had an
HAI R40 and MN R20 at day 43, respectively, compared to 0% of
placebo subjects (Figures 5A and 5B). A total of 78% (n = 18) and
87% (n = 20) who received the H10 vaccine had an HAI baseline <10
and post-vaccination HAI R40 or HAI four or more times baseline,
respectively, compared to 0% for placebo (Figures 5A and 5B). HAI
geometric mean antibody titers of subjects given the H10 vaccine
were 68.8 compared to 6.5 for placebo, and the MN geometric mean ti-
ters were 38.3 versus 5.0, respectively (Figures 5C and 5D).
The majority of adverse events (AEs) were mild (107/163 events; 66%)
or moderate (52/163 events; 32%), using the Center for Biologics Eval-
uation and Research (CBER) severity scale.
AEs were comparable in
frequency, nature, and severity to unadjuvanted and adjuvanted H1N1
inuenza vaccines.
Twenty-three subjects who received 100 mg H10
IM reported 163 reactogenicity events with no idiosyncratic or persis-
tent AEs observed. The majority of events were injection site pain,
myalgia, headache, fatigue, and chills/common-cold-like symptoms
Figure 2. A Single Injection of an H7 mRNA Vaccine Achieves Rapid and Sustained Protection in Mice
BALB/c mice were vaccinated ID with 10, 2, or 0.4 mg formulated H7 mRNA. Placebo and 10 mg formulated H7 mRNA with a reduced 50cap structure (15 Da cap) were
included as negative controls. On day 7, 21, or 84 post-immunization , mice were challenged via intranasal (IN) instillation with a target dose of 2.5 10
of influenza A/
Anhui/1/2013 (H7N9). Serum was collected prior to challenge (days 6, 20, and 83). (A–C) Survival curves of mice challenged on day 7 (A), day 21 (B), or day 84 (C) post-
immunization at the indicated doses. p < 0.0001 10-, 2-, and 0.4-mg dose groups versus placebo or 15 Da cap at days 7, 21, and 84 post-immunization. (D–F) Weight
curves of mice challenged on day 7 (D), day 21 (E), or day 84 (F) post-immunization at the indicated doses (n = 15/group). Error bars indicate standard mean error.
Molecular Therapy Vol. 25 No 6 June 2017 5
Please cite this article in press as: Bahl et al., Preclinical and Clinical Demonstration of Immunogenicity by mRNA Vaccines against H10N8 and H7N9
Influenza Viruses, Molecular Therapy (2017),
(Table S2). Only four events (2.5%), reported by three subjects (13% of
exposed subjects), were categorized as severe and included injection
site erythema (1.2%), injection site induration (0.6%), and chills/com-
mon cold (0.6%) (Table 2;Table S2). No serious AE occurred and all
events were expected and reversible. Overall, this reactogenicity prole
is similar to that of a monovalent AS03-adjuvanted H1N1 vaccine, and
it is comparable to that of meningococcal conjugate vaccine in healthy
adults (1955 years).
Nucleic acid vaccines (NAVs) offer the potential to accurately ex-
press any protein antigen, whether intracellular, membrane bound,
or secreted. Although rst identied in the early 1990s, mRNA vac-
cines were not advanced into the clinic until recently due to concerns
around stability and production.
The mRNA vaccines are pro-
duced by a well-controlled, enzymatic, and well-characterized scal-
able process that is agnostic to the antigen being produced. Addi-
tionally, host cell production and presentation of the antigen more
closely resemble viral antigen expression and presentation than
compared to an exogenously produced, puried, and formulated
protein antigen. They offer advantages in speed, precision, adapt-
ability of antigen design and production control that cannot be repli-
cated with conventional platforms. This may be especially valuable
for emerging infections, such as potential pandemic inuenza.
The mRNA vaccine platform described here allows for rapid
mRNA production and formulation, within a few weeks, at suf-
cient quantities to support typical-sized clinical trials. Moreover,
this mRNA-based vaccine technology overcomes the challenges
other nucleotide approaches pose, such as pre-existing antivector
immunity for viral vectors, and concern for genome integration,
or the high doses and devices needed (e.g., electroporation), for
DNA-based vaccines.
Other mRNA vaccine approaches have previously been reported for
Unmodied, sequence-optimized mRNA was used
to generate H1-specic responses in mice, ferrets, and pigs at dose
levels 4- to 8-fold higher than tested by us.
Brazzoli et al.
ated a self-amplifying mRNA that expressed H1 HA from the 2009
pandemic formulated with a cationic nanoemulsion in ferrets. HAI
titers were low but measurable for the 15-mg dose (two of six re-
sponders) and at the 45-mg dose (three of six responders) after a single
immunization. Following a boost, titers were measurable in all
animals and provided protection to a homologous challenge strain.
In another study, mice singly immunized against H1N1 (A/WSN/33),
receiving a self-amplifying mRNA, showed no IgG responses after
7 days. After a second immunization, responses were boosted and
animals were protected against a homologous challenge.
tion in mice against either H1 or H7, with a self-amplifying mRNA,
induced HAI and IgG titers that were comparable to those achieved
in our study at similar doses (Figure 1).
Our platform, therefore,
is surprisingly efcacious when compared to existing self-replicating
RNA approaches. It also offers potential additional advantages in
terms of rapid onset of immunity, as shown by the protection from
challenge achieved after one immunization at low doses (Figure 2),
and manufacturability, since it obviates the need to produce very
large-sized mRNAs to accommodate the self-replicating portions of
the vectors (typically 79 kb).
Modied mRNA has been shown to express more efciently than un-
modied mRNA, likely due to its reduced indiscriminate activation of
innate immunity.
When included in a vaccine formulation, our
modied-mRNA technology balances immune stimulation and anti-
gen expression, leading to very potent immune responses that are su-
perior to unmodied mRNA approaches. The very high, transient
levels of protein, expressed shortly after administration, are similar
to what is seen during a viral infection. Indeed, the biodistribution
we observed (Table 1;Table S1) is similar to an inuenza virus, where
virus could be measured outside the primary site of inoculation after
5 days.
Importantly, there was no way for our vaccine to revert to a
virulent form because key parts of the virus were missing, including
any nonstructural elements or capsid structures.
We selected LNPs for delivery of the mRNA as they have been vali-
dated in the clinic for siRNA and are well tolerated compared to other
nonviral delivery systems.
Other groups have relied on either exog-
enous RNA as an adjuvant or on the adjuvant properties generated
during self-amplication of the mRNA. Using an LNP, we generate
very high levels of transient expression without the need for additional
immunostimulatory compounds.
In the studies summarized here, we demonstrated that the LNP-based,
modied-mRNA vaccine technology is able to generate robust and
protective immune responses in mice, ferrets, and cynomolgus mon-
keys. In animals, we showed that a range of doses of formulated
mRNA encoding the HA protein of either H7N9 or H10N8 is able
to stimulate rapid, robust, and long-lasting, immune responses, as
measured by HAI, MN assay, and protection from viral challenge. A
Figure 3. A Single Dose of H10 mRNA in Ferrets Generates Robust HAI
Titers, Which Are Significant and Comparable at All Time Points
Ferrets were vaccinated ID with 50 or 100 mg formulated H10 mRNA. p < 0.0001,
days 21, 35, and 49 versus day 0 with single doses of 50 or 100 mg; p < 0.0001
100-mg single dose, day 7 versus day 0. A subset of immunized ferrets received a
boost on day 21 and an additional subset received a second boost on day 35. HAI
titers were measured on days 0, 7, 21, 35, and 49 (n = 8/group). p < 0.0001 50 or
100 mg boosting dose(s), days 35 and 49 versus day 0.
Molecular Therapy
6 Molecular Therapy Vol. 25 No 6 June 2017
Please cite this article in press as: Bahl et al., Preclinical and Clinical Demonstration of Immunogenicity by mRNA Vaccines against H10N8 and H7N9
Influenza Viruses, Molecular Therapy (2017),
single vaccination on day 0 with as little as 0.4 mg was shown to protect
mice against challenge with H7N9 on days 7, 21, and 84 (Figure 2),
despite the fact that H7 HA has demonstrated relatively poor immuno-
Increased survival of mice vaccinated with H7 HA and
challenged with H7N9 (A/Anhui/1/2013) at early time points (Fig-
ure 2) suggests additional mechanism(s) of protection, since HAI titers
were below the level of detection (Figure S5). T cells have been shown to
elicit protection against pandemic inuenza strains.
We detected
T cell responses to both H10 and H7 vaccines at multiple doses (Figures
S2C and S2D). Additional follow-up studies are ongoing, to determine
whether T cell responses alone offer protective benets, to lend insight
into the specic mechanism of vaccine protection.
These interim results of H10 mRNA vaccination in humans are the
rst published example of a nucleic acid vaccine against an infectious
disease working in man without the use of electroporation. Although
strategies, such as electroporation, have been developed to increase
the efcacy of DNA-based vaccines, they continue to have relatively
poor immunogenicity compared to protein vaccines.
Initial data
from the rst-in-human trial appear to conrm a robust immune
response with a safe and well-tolerated prole. However, the full data-
set from the trial will need to be evaluated in order to conrm this
interim analysis. Nonetheless, these results are encouraging in that
microgram-dose levels provided immunogenicity with a safety prole
comparable to traditional vaccines.
The completion of these and additional clinical trials is needed to
conrm whether mRNA vaccines will become an effective vaccine
platform that can overcome many of the shortcomings of conven-
tional vaccines. Our initial ndings are nonetheless encouraging
and provide support for further clinical exploration.
mRNA Synthesis and Formulation
Our mRNA was synthesized in vitro by T7 polymerase-mediated
transcription from a linearized DNA template, which incorporates
50and 30UTRs, including a poly-A tail.
The mRNA is puried
and resuspended in a citrate buffer at the desired concentration.
A donor methyl group S-adenosylmethionine (SAM) is added to
methylated capped RNA (cap-0), resulting in a cap-1 to increase
mRNA translation efciency.
LNP formulations were prepared using a modied procedure of a
method previously described for siRNA.
Briey, lipids were dissolved
in ethanol at molar ratios of 50:10:38.5:1.5 (ionizable lipid: 1,2-dis-
tearoyl-sn-glycero-3-phosphocholine (DSPC): cholesterol: PEG-lipid).
The lipid mixture was combined with a 50 mM citrate buffer (pH 4.0)
containing mRNA at a ratio of 3:1 (aqueous:ethanol) using a microui-
dic mixer (Precision Nanosystems).Formulations were dialyzedagainst
PBS (pH 7.4) in dialysis cassettes for at least 18 hr. Formulations were
concentrated using Amicon ultra centrifugal lters (EMD Millipore),
Figure 4. Vaccination with Either H10 or H7 mRNA Generates Strong HAI Titers in Nonhuman Primates following ID and IM Immunizations
(A and B) Male or female cynomolgus monkeys (cynos) were immunized on day 1 with 0.2 or 0.4 mg formulated H7 mRNA, both IM and ID, and received a boosting
immunization on day 22. Serum was collected on days 1, 8, 15, 22, 29, 36, and 43 to determine HAI titers. (C) Male and female cynos were immunized with 0.4 mg
formulated H10 mRNA via an IM or ID route and received a boosting immunization on day 22. Serum was collected on days 1, 8, 15, 22, 29, 36, and 43 to determine HAI titers
(n = 1/group).
Molecular Therapy Vol. 25 No 6 June 2017 7
Please cite this article in press as: Bahl et al., Preclinical and Clinical Demonstration of Immunogenicity by mRNA Vaccines against H10N8 and H7N9
Influenza Viruses, Molecular Therapy (2017),
passed through a 0.22-mmlter, and stored at 4Cuntil use. All formu-
lations were tested for particle size, RNA encapsulation, and endotoxin,
and they were found to be between 80 and 100 nm in size, with >90%
encapsulation and <1 EU/mL endotoxin.
In Vitro Expression
The day before transfection, 400,000 HeLa cells (ATCC) were seeded
in a six-well cell culture plate, and 2.5 mg of either H10 or H7 HA
mRNA was transfected using the Transit mRNA transfection kit (Mi-
rus Bio). Recovered protein lysate, 30 mg, was resolved on a NuPage
Novex 4%12% Bis-Tris Protein Gel and transferred onto nitrocellu-
lose using an iBlot 2 (7-min transfer). Blots were incubated with either
anti-H10 HA polyclonal antibody (rabbit, 11693; Sino Biological) or
anti-H7 HA monoclonal antibody (mouse, 11082-MM04; Sino Bio-
logical) overnight at 4C. Included as positive controls were 0.5 mg
recombinant H10 HA protein (1505-001; IBT) and recombinant
H7 HA protein (1502-001; IBT). A polyclonal antibody against actin
was also included as a loading control (rabbit, A2066; Sigma-Aldrich).
Blots were scanned and analyzed on an Odyssey CLx (LI-COR
Animal Studies
Female BALB/c mice 58 weeks old were purchased from Charles
River Laboratories and housed at the study site (Noble Life Sciences
or Moderna Therapeutics,). For mouse H7N9 challenge studies, fe-
male BALB/c mice 78 weeks old were purchased from Harlan Lab-
oratories and housed at MRIGlobals ABSL-3 facility.
Male ferrets 1315 weeks old (Triple F Farms) with a baseline HAI titer
of %20 to inuenza virus, A/California/07/2009 (H1N1), A/Wiscon-
sin/15/2009 (H3N2), and B/Massachusetts/2/2012, were used for
studies at MRIGlobals ABSL-3 facility.
Nonhuman primate studies were conducted at Charles River Labora-
tories using naive cynomolgus monkeys (cynos), 24 years old, weigh-
ing 26 kg. Animals were housed in stainless steel, perforated-oor
cages, in a temperature- and humidity-controlled environment (21
26C and 30%70%, respectively), with an automatic 12-hr dark/light
cycle. Animals were fed PMI Nutrition Certied Primate Chow No.
5048 twice daily. Tuberculin tests were carried out on arrival at
the test facility. The study plan and procedures were approved by
Figure 5. H10 mRNA Immunogenicity in Humans
(A and B) A greater percentage of subjects who received active vaccine had an HAI R40 (A) and MN R20 (B) compared to placebo. (C and D) HAI (C) and MN (D) titers
of individual subjects were substantially more pronounced in those who received active vaccine compared to plac ebo. Error bars indicate SEM (100 mg IM, n = 23; placebo,
n = 8).
Molecular Therapy
8 Molecular Therapy Vol. 25 No 6 June 2017
Please cite this article in press as: Bahl et al., Preclinical and Clinical Demonstration of Immunogenicity by mRNA Vaccines against H10N8 and H7N9
Influenza Viruses, Molecular Therapy (2017),
PCS-SHB Institutional Animal Care and Use Committee (IACUC).
Animal experiments and husbandry followed the NIH (NIH Publica-
tions No. 8023, eighth edition)and the USA National Research Council
and the Canadian Council on Animal Care (CCAC) guidelines. No
treatment randomization or blinding methods were used for any of
the animal studies. Sample sizes were determined by the resource equa-
tion method.
First-in-Human Phase 1 Study
A single-center, randomized, double-blind, placebo-controlled, dose-
ranging study is ongoing to evaluate the safety and immunogenicity
of H10N8 antigen mRNA in humans between the ages of 18 and 64
(Clinical Trials Identier NCT03076385). Subjects are being followed
for up to 1 year post-vaccination for safety and immunogenicity. Only
interim analysis (day 43) of one dose-group cohort (100 mg IM) in
healthy adults is reported (all other analyses are ongoing).
Briey, males and females were eligible for this study if they had a body
mass index between 18.0 and 30.0 kg/m
, were considered in general
good health with no ongoing acute or chronic illness, did not have
any asymptomatic (e.g., mild hypertension) or any suspected immu-
nosuppressive condition, or and did not have a history of serious re-
actions to inuenza vaccinations or Guillain-Barre Syndrome. Eligible
adults were randomized at a ratio of 3:1 to receive either H10N8
mRNA 100 mg IM or placebo. All study personnel who conducted
assessments were blinded to treatment. Immunogenicity was deter-
mined by HAI and MN assays.
Safety was assessed from solicited (local and systemic reactogenicity
events) and unsolicited AEs via scheduled clinic visit (vital signs,
laboratory assessments, and physical examinations), subject diaries,
and follow-up telephone calls at specic intervals. AEs were dened
as any problematic medical occurrence even if seemingly unrelated
to treatment and graded by the Toxicity Grading Scale and dened
as mild (transient with no normal daily activity limitations), moder-
ate (some normal daily limitations), and severe (unable to perform
normal daily activities).
Serious AEs were dened as any occurrence
of death, a life-threatening situation, hospitalization, persistent or sig-
nicant disability/incapacity, congenital anomaly/birth defect, or any
medical event that jeopardizes the subject or requires medical inter-
vention. A safety review committee reviewed safety data at key inter-
vals throughout the study before allowing dose expansion or dose
escalation. Prior to study enrollment, all subjects completed a written
informed consent in accordance with all applicable local- and coun-
try-specic regulations. This study was conducted by PAREXEL In-
ternational and was reviewed and approved by an Independent Ethics
Committee. This study was conducted in compliance with the Inter-
national Conference on Harmonization Good Clinical Practice guide-
lines and the ethical principles of the Declaration of Helsinki.
For mouse IM immunizations, 50 mL was injected in either the left or
right quadriceps. For ferret and mouse ID immunizations, the needle
was inserted bevel up with the point visualized through the skin. The
vaccine (50 mL) was administered slowly, creating a blister-like
For ID delivery to cynos, the material was injected into the lumbar re-
gion in a 100 mL vol for the 0.2-mg dose and delivered at two sites for
the 0.4-mg dose (0.2 mg in 100 mL per site). For IM delivery to cynos,
the material was injected into the left thigh in a 100 mL vol for the
0.2-mg dose or a 200 mL vol for the 0.4-mg dose.
In the human study, each subject in the 100-mg IM cohort received two
treatment doses on day 1 and day 22. Each subject received their vac-
cine via IM administration according to standard procedures in their
deltoid muscle, with the second dose administered in the same arm.
Viral Challenges
Inuenza strain A/Anhui/1/2013 (H7N9) was grown and characterized
at MRIGlobal to a concentration of 3.3 10
/mL. BALB/c
mice were challenged via IN instillation (2.5 10
in 50 ml
Dulbeccos phosphate-buffered saline [DPBS]). Anesthetized ferrets
were inoculated IN with 1 10
with 2 250 mL per nostril.
Serum Collection
Approximately 200 mL blood was collected from mice via tail vein or
retro-orbital bleed (1 mL for terminal bleeds) and centrifuged at
1,200 gfor serum isolation (10 min at 4C). Collected blood
Table 2. Number and Percentage of Subjects Who Experienced a Solicited
Reactogenicity Event after Receiving 100 mg H10N8 mRNA IM or Placebo
100 mg IM H10N8
mRNA n (%) Placebo n (%)
Total number of subjects 23 (100) 8 (100)
Any reactogenicity event 23 (100) 5 (62.5)
Mild 23 (100) 3 (37.5)
Moderate 12 (52.2) 1 (12.5)
Severe 3 (13.0) 1 (12.5)
Any local reactogenicity event 12 (91.3) 2 (25.0)
Mild 20 (87.0) 2 (25.0)
Moderate 9 (39.1) 0
Severe 2 (8.7) 0
Any systemic reactogenicity event 21 (91.3) 5 (62.5)
Mild 21 (91.3) 3 (37.5)
Moderate 11 (47.8) 1 (12.5)
Severe 1 (4.3) 1 (12.5)
Reactogenicity was dened as selected AE signs and symptoms occurring after dose
administration that were reported by the subject using diary cards during the day of
and 6 days after each dose administration. Events were categorized according to the
toxicity grading scale for heathy adult and adolescent volunteers enrolled in preventative
vaccine clinical trials (CBER 2007). AEs were dened as any unfavorable and unintended
medical occurrence. Mild AEs were dened as those having no limitations in normal daily
activities, moderate AEs as causing some limitations, and severe AEs were dened as
events causing inability to perform normal daily activities. The total number of patients
are those who received at least one dose of treatment. Percentages are based on the num-
ber of patients who reported at least one solicited reactogenicity event after treatment.
Molecular Therapy Vol. 25 No 6 June 2017 9
Please cite this article in press as: Bahl et al., Preclinical and Clinical Demonstration of Immunogenicity by mRNA Vaccines against H10N8 and H7N9
Influenza Viruses, Molecular Therapy (2017),
(13 mL) from the ferretscranial vena cava was processed to serum us-
ing a serum separator tube (SST). Blood collected from the peripheral
vein of cynos (0.5 mL) was centrifuged at 1,200 g(10 min at 4C). All
serum was frozen immediately and stored at 80C.
In the human study, blood samples for immunogenicity analysis
were collected via intravenous cannula or by direct venipuncture of
the forearm. Serum samples were stored and transported under
controlled conditions to Synexa Life Sciences for HAI analysis and
to Southern Research Institute for MN testing.
Lung Homogenate
Ferrets were then euthanized by intraperitoneal injection of Euthasol,
and lungs (1 cm
of the lower part of each of the three right lung
lobes), nasal turbinates, and a portion of the trachea were collected.
The lung portions were weighed and immediately homogenized
and tested in the TCID
Inuenza virus levels in nasal washes and lung homogenates were
determined by TCID
assay. Madin-Darby canine kidney (MDCK)
cells were seeded in 96-well plates in serum-free media and incubated
at 37C with 5% CO
. Nasal washes and lung homogenates (four to
eight replicates) were serially diluted in serum-free media and added
to plates that were R95% conuent after a single wash. Cytopathic
effects (CPEs) were determined after 35 days at 37C with 5%
. The TCID
/mL was calculated using the lowest dilution at
which CPE was observed. Lung homogenate results were reported
/g lung tissue.
Biodistribution Studies
Male CD-1 mice received 300 mg/kg (6 mg) H10 HA mRNA (50 uL
vol) via ID or IM (left side) administration. Blood, heart, lung, spleen,
kidney, liver, and skin injection sites were collected pre-dose and 2,
4, 8, 24, 48, 72, and 96 hr post-ID dosing (n = 4 mice/time point).
Two replicates each of bone marrow (left and right femur), lung,
liver, heart, right kidney, inguinal- and popliteal-draining lymph
nodes, axillary distal lymph nodes, spleen, brain, stomach, ileum,
jejunum, cecum, colon, rectum, testes (bilateral), and injection site
muscle were collected pre-dose and 2, 8, 24, 48, 72, 120, 168, and
264 hr post-IM dosing (n = 3 mice/time point). Blood samples
were collected from jugular venipuncture at study termination.
H10 HA mRNA quantication for both serum and tissues was
performed by AxoLabs using the Quantigene 2.0 branched DNA
(bDNA) Assay (Panomics/Affymetrix).
A standard curve on each
plate of known amounts of mRNA (added to untreated tissue sam-
ples) was used to quantitate the mRNA in treated tissues. The calcu-
lated amount in picograms (pg) was normalized to the amount of
weighed tissue in the lysate applied to the plate.
Luciferase Studies
Female BALB/c mice 68 weeks old were dosed with formulated lucif-
erase mRNA via IM or ID administration at four dose levels as follows:
10, 2, 0.4, and 0.08 mg (n = 6 per group). At 6, 24, 48, 72, and 96 hr post-
dosing, animals were injected with 3 mg luciferin and imaged on an
in vivo imaging system (IVIS Spectrum, PerkinElmer). At 6 hr post-
dosing, three animals were sacriced and dissected, and the muscle,
skin, draining lymph nodes, liver, and spleen were imaged ex vivo.
MN Assay
Heat-inactivatedserum was serially diluted on 96-well plates, and 2
/mL H7N9 (A/Anhui/1/2013) was added to each dilution.
Following a 1-hr incubation at room temperature, the serum/
virus mixtures from each well were transferred to plates containing
MDCK cells and incubated at 37C(5%CO
). After 35 days, the
CPE titer was determined based on the most dilute sample at which
no CPE was observed. Each sample was tested three times, and the geo-
metric mean of the three replicates was reported as the overall titer.
HAI Assay
The HAI titers of serum samples in both the animal and human
studies were determined using a protocol adapted from the World
Health Organization protocol.
Sera were rst treated with recep-
tor-destroying enzyme (RDE) to inactivate nonspecic inhibitors.
RDE was inactivated by incubation at 56C for 30 min. Treated
sera were serially diluted in 96-well plates, mixed with a standardized
amount of recombinant HA (eight HA units of H10N8 or H7N9 rHA;
Medigen), and incubated for 30 min at room temperature. Turkey red
blood cells (RBCs) (Lampire Biological Laboratories) were then added
to the wells of the 96-well plates, mixed, and incubated at room tem-
perature for 45 min. The most dilute serum sample that completely
inhibited hemagglutination was the reported titer for that replicate.
Each serum sample was analyzed in triplicate and the results are re-
ported as the geometric mean of the three results.
Mouse IFNgELISpot assays were performed using the IFNgpre-
coated ELISpot kit catalog 3321-4APW (MabTech), according to
the manufacturers protocol. Briey, the plates were blocked using
complete RPMI (R10) and incubated for 30 min prior to plating cells.
Peptide libraries for H7 or H10 were diluted to a nal concentration
of 10 mg/mL. Mouse splenocytes were pooled by group and plated at
600,000 cells/well, with peptide, phorbol myristate acetate (PMA) +
Ionomycin or R10 media alone. Cells were stimulated in a total vol-
ume of 125 mL/well. Plates were then incubated at 37C, 5% CO
for 1824 hr. Assay plates were developed and counted using the
automated ELISpot reader CTL ImmunoSpot/FluoroSpot. Overlap-
ping peptide libraries (15mers with ten amino acid overlaps) for
H10 HA (A/Jiangxi-Donghu/346/2013) and H7 HA (A/Anhui/1/
2013) were ordered from Genscript.
Statistical Analysis and Data Collection
In general, two datasets were compared by two sample t test and more
than two groups were compared by ANOVA proc mixed model.
Two-way ANOVA was used to analyze titers in lung tissue. Survival
curves were compared via log-rank (Mantel-Cox) test. Statistical an-
alyses for the animal studies were performed with GraphPad Prism 6.
Molecular Therapy
10 Molecular Therapy Vol. 25 No 6 June 2017
Please cite this article in press as: Bahl et al., Preclinical and Clinical Demonstration of Immunogenicity by mRNA Vaccines against H10N8 and H7N9
Influenza Viruses, Molecular Therapy (2017),
The phase 1 human clinical trial is being conducted by PAREXEL
International, and data were collected utilizing their electronic re-
cords ClinBase system. All statistical analyses for the human trial
are performed using SAS (SAS Institute, version 9.1 or higher).
Supplemental Information includes seven gures and two tables and
can be found with this article online at
G.C. led and planned the studies. K.B. and G.C. contributed to the
experimental design and analysis of all in vivo studies. O.Y. contrib-
uted to the HAI analysis. J.J.S. and A.B. contributed to the toxicology
and biodistribution studies, respectively. L.A.B., K.J.H., and O.A.
contributed to formulation design and expression optimization.
M.E.L. and M.S. contributed to mRNA synthesis and process optimi-
zation. A.R., T.Z., and M.W. supervised the conduct of the human
study. All authors drafted and revised the manuscript for critical in-
tellectual content and have reviewed and approved the nal paper.
This study was funded by Valera Therapeutics, a Moderna Therapeu-
tics venture. Authors K.B., O.Y., K.J.H., A.R., M.W., and G.C. are em-
ployees of Valera Therapeutics. Authors J.J.S., A.B., L.A.B., M.E.L,
M.S., O.A., J.T., and T.Z. are employees of Moderna Therapeutics.
Statistical analyses were conducted by Georges Carlettis of Strategie-
stat and Katherine Kacena of BioBridges. Editorial support was pro-
vided by Stephanie Eide of BioBridges.
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Influenza Viruses, Molecular Therapy (2017),
In vitro-transcribed mRNAs (IVT-mRNAs) are easily and rapidly designed in vitro synthesized RNA molecules. After their intracellular delivery through an efficient delivery system, the host cell ribosomes will be recruited to translate and produce the corresponding desired proteins. Nowadays, about 20 years after the first report as for the use of IVT-mRNA, this technology has all the spotlight on it, due to the recently produced vaccines against SARS-CoV-2. All this enthusiasm around IVT-mRNA has pushed a wave of biotech companies to leverage this technology, raising significant investments annually. Thus, IVT-mRNA technology has gained an impressive dynamic, with multidimensional applications [as protein replacement therapy (PRT), cancer immunotherapy, vaccine production, production of antibodies, cytokines and growth factors, gene silencing, cellular reprogramming, and gene editing] with remarkable results. This chapter will emphasize the recent advances in IVT-mRNA delivery. A wide range of in vitro and in vivo transfection reagents have been shown to protect IVT-mRNA from degradation, to escape immunosurveillance and facilitate its intracellular delivery. IVT-mRNA delivery systems can be classified into two broad categories: (i) physical transfection methods, like electroporation, that temporarily disrupt cell membrane barrier function and (ii) chemically formulated nanocarriers, like polymer-based, lipid-based nanovectors, lipid–polymer hybrid nanoparticles, and peptide vectors. As the peptide-based delivery systems are gaining ground due to the flexibility that peptides can offer, this chapter will present this very interesting aspect that combines IVT-mRNA technology with protein transduction domain (PTD) technology. Compared to cationic polymers, the peptides are of low-molecular weight, with degradable amino acid sequences and distinct biological properties, such as cell permeability efficiency and cell and nuclear surface targeting. Either by non-covalent or covalent binding, peptide-based carriers and hybrids are suggested as interesting alternatives to the various existing non-viral vectors for IVT-mRNA delivery.
Self-amplifying RNA (saRNA) is a next-generation nucleic acid technology that is structurally similar to mRNA, but capable of replicating upon delivery into the cytosol. This amplification results in high protein expression from a relatively low dose of saRNA (~100-fold lower than mRNA). The rapid, cost-effective, cell-free manufacturing, low dosage requirement, and acceptable safety profile have drawn spotlight on saRNA, which has recently entered clinical trials. However, similar to mRNA, saRNA formulations need highly protective and robust delivery vehicles to achieve a therapeutic effect. The delivery systems have an integral role on the therapeutic efficacy of the saRNA including, biodistribution, cellular uptake, protein expression, and immunogenicity. In the last three decades, a broad range of non-viral delivery systems for RNA have been investigated. Herein, we discuss the cutting-edge advancements in saRNA delivery platforms including the variety of delivery approaches that have been used for saRNA formulations to date, and the resulting immunogenicity, biodistribution, cellular uptake, protein expression, and effect of route of administration.
Messenger RNA (mRNA) can be harnessed as vaccines and therapeutic drugs via transient in situ expression of protein antigens and therapeutic proteins, respectively. Currently, mRNA-based vaccines are used worldwide in mass vaccination programs to induce protective immunity against COVID-19, and a number of prophylactic vaccines, therapeutic vaccines, and therapeutic drugs based on mRNA are now tested in clinical trials. Although chemical modification of the mRNA components has considerably ameliorated mRNA stability and immunogenicity, further improvements in formulation and delivery systems, which are used to transport mRNA to the cytosol of target cells, are still required to enhance the efficacy and safety of mRNA therapeutics. However, our knowledge about the mechanisms by which mRNA therapeutics activate the immune system is still very limited, partly because the activation of immune cells by ionizable lipids commonly used in mRNA delivery systems is poorly understood. Lipid-mediated induction of innate immune pathways can be exploited in mRNA vaccines by providing an adjuvant effect, whereas innate immune activation is undesired for the therapeutic use of mRNA. Here, we review recent studies focusing on the hurdles that challenge in vivo delivery of mRNA. We subsequently discuss the state of the art in formulation design approaches, which are used to overcome these challenges, with focus on the marketed COVID-19 mRNA vaccines. Finally, we present research centered on how ionizable and cationic lipids used for delivery of mRNA therapeutics activate immune cells and engage innate immune pathways, including future challenges and opportunities in formulation and delivery to optimize the safe and efficacious use of mRNA therapeutics.KeywordsMessenger RNA (mRNA)Lipid nanoparticlesIonizable lipidsImmune activationToll-like receptors
Hospital-based programs democratize mRNA therapeutics by facilitating the processes to translate a novel RNA idea from the bench to the clinic. Because mRNA is essentially biological software, therapeutic RNA constructs can be rapidly developed. The generation of small batches of clinical-grade mRNA to support IND applications and first-in-man clinical trials, as well as personalized mRNA therapeutics delivered at the point-of-care, is feasible at a modest scale of cGMP manufacturing. Advances in mRNA manufacturing science and innovations in mRNA biology are increasing the scope of mRNA clinical applications.KeywordsMessenger RNAHospital-based mRNA therapeuticsCircular mRNASelf-amplifying mRNARNA-based CAR T cellRNA-based gene-editing tools
Remarkable advances in mRNA and ionizable lipid-based carrier innovations have allowed the unprecedented speed of development for these technologies as vaccines to prevent SARS-CoV2 disease. Their validation in the field of prophylaxis now paves the way for other infectious diseases indications and manufacturing advantages over certain traditional vaccine technologies. In this chapter, platform advances and critical quality attributes important for vaccination will be discussed and related to SARS-CoV2 vaccines for which field efficacy data are available.KeywordsmRNALNPCritical quality attributesVaccinesImmunogensFormulationsManufacturingInfectious diseaseSARS-COV-2COVID-19
Influenza A virus (IAV) infection is a contagious respiratory infection responsible for high morbidity and mortality rates across the planet. The human immune system contains a wide range of soluble activators, membrane-bound receptors, and regulators to eliminate IAVs. Despite these various immune mechanisms that neutralize IAV or restrict their replication, IAVs still have developed distinct strategies to evade the host immunity and establish a successful infection. Given the higher and continuous rate of mutations in IAVs, decades of research have focused on understanding the host's immune mechanisms against IAVs, and the evasion strategies employed by the virus to overcome the e host immune system. Future IAV pandemics or epidemics remain inevitable, and a greater understanding of the host-pathogen interaction involved is required to develop universal vaccines and treatment against IAV. Here, we review how the host immune system responds to IAV infection as well as the strategies employed by the IAV to evade the host immune surveillance. Furthermore, this review also focuses on the treatments and vaccines that have been developed to counter IAV infection.
Salmonella infections are continuously growing. Causative serovars have gained enhanced drug resistance and virulence. Current vaccines have fallen short of providing sufficient protection. mRNA vaccines have come up with huge success against SARS-CoV-2; Pfizer-BioNTech and Moderna vaccines have resulted in >90% efficacy with efficient translocation, expression, and presentation of antigen to the host immune system. Herein, based on the same approach a mRNA vaccine construct has been designed and analyzed against Salmonella by joining regions of genes of outer membrane proteins C and F of S. Typhi through a flexible linker. Construct was flanked by regulatory regions that have previously shown better expression and translocation of encoded protein. GC content of the construct was improved to attain structural and thermodynamic stability and smooth translation. Sites of strong binding miRNAs were removed through codon optimization. Protein encoded by this construct is structurally plausible, highly antigenic, non-allergen to humans, and does not cross-react to the human proteome. It is enriched in potent, highly antigenic, and conserved linear and conformational epitopes. Most conserved conformational epitopes of core protein lie on extended beta hairpins exposed to the cellular exterior. Stability and thermodynamic attributes of the final construct were found highly comparable to the Pfizer-BioNTech vaccine construct. Both contain a stable stem-loop structure downstream of the start codon and do not offer destabilizing secondary structures upstream of the start codon. Given structural and thermodynamic stability, effective immune response, and epitope composition the construct is expected to provide broad-spectrum protection against clinically important Salmonella serovars. Communicated by Ramaswamy H. Sarma
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With the rapid development of gene therapy technology and the outbreak of coronavirus disease 2019 (COVID-19), messenger RNA (mRNA) therapeutics have attracted more and more attention, and the COVID-19 mRNA vaccine has been approved by the Food and Drug Administration (FDA) for emergency authorization. To improve the delivery efficiency of mRNA in vitro and in vivo, researchers have developed a variety of mRNA carriers and explored different administration routes. This review will systematically introduce the types of mRNA vectors, routes of administration, storage methods, safety of mRNA therapeutics, and the type of diseases that mRNA drugs are applied for. Finally, some suggestions are supplied on the development direction of mRNA therapeutic agents in the future.
Modified mRNA (modRNA) is a safe and effective vector for gene-based therapies. Notably, the safety of modRNA has been validated through COVID-19 vaccines which incorporate modRNA technology to translate spike proteins. Alternative gene delivery methods using plasmids, lentiviruses, adenoviruses, and adeno-associated viruses have suffered from key challenges such as genome integration, delayed and uncontrolled expression, and immunogenic responses. However, modRNA poses no risk of genome integration, has transient and rapid expression, and lacks an immunogenic response. Our lab utilizes modRNA-based therapies to promote cardiac regeneration following myocardial infarction and heart failure. We have also developed and refined an optimized and economical method for synthesis of modRNA. Here, we provide an updated methodology with improved translational efficiency for in vitro and in vivo application.Key wordsModified mRNASynthetic mRNAIn vitro transcriptionmRNA translationGene therapyGene-based therapyGenetically based therapy Cardiac gene therapy
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Messenger ribonucleic acid (mRNA) vaccines made their successful public debut in the effort against the COVID-19 outbreak starting in late 2019, although the history of mRNA vaccines can be traced back decades. This review provides an overview to discuss the historical course and present situation of mRNA vaccine development in addition to some basic concepts that underly mRNA vaccines. We discuss the general preparation and manufacturing of mRNA vaccines and also discuss the scientific advances in the in vivo delivery system and evaluate popular approaches (i.e., lipid nanoparticle and protamine) in detail. Next, we highlight the clinical value of mRNA vaccines as potent candidates for therapeutic treatment and discuss clinical progress in the treatment of cancer and coronavirus disease 2019. Data suggest that mRNA vaccines, with several prominent advantages, have achieved encouraging results and increasing attention due to tremendous potential in disease management. Finally, we suggest some potential directions worthy of further investigation and optimization. In addition to basic research, studies that help to facilitate storage and transportation will be indispensable for practical applications.
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Zika virus (ZIKV) has recently emerged as an explosive pandemic associated with severe neuropathology in newborns and adults1. There are no ZIKV-specific treatments or preventatives; thus, development of a safe and effective vaccine is a high priority. Messenger RNA (mRNA) has emerged as a versatile and highly effective platform to deliver vaccine antigens and therapeutic proteins2,3. Here, we demonstrate that a single low-dose intradermal immunization with lipid nanoparticle-encapsulated nucleoside-modified mRNA (mRNA-LNP) encoding the pre-membrane and envelope (prM-E) glycoproteins of a 2013 ZIKV outbreak strain elicited potent and durable neutralizing antibody responses in mice and non-human primates. Immunization with 30 μg of nucleoside-modified ZIKV mRNA-LNPs protected mice from ZIKV challenges at 2 weeks or 5 months post-vaccination, and a single dose of 50 μg was sufficient to protect non-human primates from a challenge at 5 weeks post-vaccination. These data demonstrate that nucleoside-modified mRNA-LNPs elicit rapid and durable protective immunity and thus represent a new and promising vaccine candidate for the global fight against ZIKV.
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Vaccines have had broad medical impact, but existing vaccine technologies and production methods are limited in their ability to respond rapidly to evolving and emerging pathogens, or sudden outbreaks. Here, we develop a rapid-response, fully synthetic, single-dose, adjuvant-free dendrimer nanoparticle vaccine platform wherein antigens are encoded by encapsulated mRNA replicons. To our knowledge, this system is the first capable of generating protective immunity against a broad spectrum of lethal pathogen challenges, including H1N1 influenza, Toxoplasma gondii, and Ebola virus. The vaccine can be formed with multiple antigen-expressing replicons, and is capable of eliciting both CD8(+) T-cell and antibody responses. The ability to generate viable, contaminant-free vaccines within days, to single or multiple antigens, may have broad utility for a range of diseases.
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The stalk region of the influenza virus hemagglutinin is relatively well conserved compared with the globular head domain, which makes it a potential target for use as a universal vaccine against influenza. However, the role of CD4 T cells in the hemagglutinin stalk-specific immune response is not clear. Here we identified a mouse CD4 T-cell epitope that encompasses residues HA2113-131 from the hemagglutinin stalk domain after a sub-lethal infection of influenza. In response to stimulation with the identified epitope, splenocytes derived from the infected mice showed significant polyfunctionality as shown by IL-2, TNF-α and IFN-γ production as well as degranulation. Moreover, mice immunized with the peptide corresponding to this CD4 T-cell epitope exhibited interindividual sharing of the CD4 T-cell receptor β sequences, and they had a higher survival rate following a challenge with a lethal dose of pandemic H1N1 influenza virus. Thus, our data demonstrated a crucial role of hemagglutinin stalk-specific CD4 T cells in the host immune response against influenza virus infection.Cellular & Molecular Immunology advance online publication, 9 May 2016; doi:10.1038/cmi.2016.20.
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Since 2013, avian influenza A(H7N9) viruses have diversified into multiple lineages by dynamically reassorting with other viruses, especially H9N2, in Chinese poultry. Despite concerns about the pandemic threat posed by H7N9 viruses, little is known about the biological properties of H7N9 viruses that may recruit internal genes from genetically distinct H9N2 viruses circulating among wild birds. Here, we generated 63 H7N9 reassortants derived from an avian H7N9 and a wild-bird-origin H9N2 virus. Compared with the wild-type parent, 25/63 reassortants had increased pathogenicity in mice. A reassortant containing PB1 of the H9N2 virus was highly lethal to mice and chickens but was not transmissible to guinea pigs by airborne routes; however, three substitutions associated with adaptation to mammals conferred airborne transmission to the virus. The emergence of the H7N9-pandemic reassortant virus highlights that continuous monitoring of H7N9 viruses is needed, especially at the domestic poultry/wild bird interface.
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Vaccination is the most effective method to prevent influenza infection. However, current influenza vaccines have several limitations. Relatively long production times, limited vaccine capacity, moderate efficacy in certain populations and lack of cross-reactivity are important issues that need to be addressed. We give an overview of the current status and novel developments in the landscape of influenza vaccines from an interdisciplinary point of view. The feasibility of novel vaccine concepts not only depends on immunological or clinical outcomes, but also depends on biotechnological aspects, such as formulation and production methods, which are frequently overlooked. Furthermore, the next generation of influenza vaccines is addressed, which hopefully will bring cross-reactive influenza vaccines. These developments indicate that an exciting future lies ahead in the influenza vaccine field. Copyright © 2015. Published by Elsevier B.V.
The emergence of ZIKV infection has prompted a global effort to develop safe and effective vaccines. We engineered a lipid nanoparticle (LNP) encapsulated modified mRNA vaccine encoding wild-type or variant ZIKV structural genes and tested immunogenicity and protection in mice. Two doses of modified mRNA LNPs encoding prM-E genes that produced virus-like particles resulted in high neutralizing antibody titers (∼1/100,000) that protected against ZIKV infection and conferred sterilizing immunity. To offset a theoretical concern of ZIKV vaccines inducing antibodies that cross-react with the related dengue virus (DENV), we designed modified prM-E RNA encoding mutations destroying the conserved fusion-loop epitope in the E protein. This variant protected against ZIKV and diminished production of antibodies enhancing DENV infection in cells or mice. A modified mRNA vaccine can prevent ZIKV disease and be adapted to reduce the risk of sensitizing individuals to subsequent exposure to DENV, should this become a clinically relevant concern.
Influenza vaccines confer considerable but incomplete protection and are recommended for everyone. The Advisory Committee on Immunization Practices does not endorse a specific vaccine but recommends against the live attenuated vaccine during 2016–2017 in the United States.
Importance: In this paper, we describe protective immune responses in mice and ferrets after vaccination with a novel HA-based influenza. This novel type of vaccine elicits both humoral and cellular immune responses. While vaccine-specific antibodies are the key players in mediating protection from homologous influenza virus infections, vaccine-specific T cells contribute to the control of heterologous infections. The rapid production capacity and the synthetic origin of the vaccine antigen make this platform particularly exploitable in case of influenza pandemic.