Original Article
`
`Preclinical and Clinical Demonstration
`of Immunogenicity by mRNA Vaccines
`against H10N8 and H7N9 Influenza Viruses
`Kapil Bahl,1 Joe J. Senn,2 Olga Yuzhakov,1 Alex Bulychev,2 Luis A. Brito,2 Kimberly J. Hassett,1 Michael E. Laska,2
`Mike Smith,2 Örn Almarsson,2 James Thompson,2 Amilcar (Mick) Ribeiro,1 Mike Watson,1 Tal Zaks,2
`and Giuseppe Ciaramella1
`
`1Valera, A Moderna Venture, 500 Technology Square, Cambridge, MA 02139, USA; 2Moderna Therapeutics, 200 Technology Square, Cambridge, MA 02139, USA
`
`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 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 first-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, modified mRNA vaccines can induce protec-
`tive immunogenicity with acceptable tolerability profiles.
`
`INTRODUCTION
`Several avian influenza 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.1 However, lessons
`learned from these outbreaks demonstrated that new approaches
`are needed to address potential future pandemic influenza outbreaks.2
`
`Two major glycoproteins, crucial for influenza infection, are hemag-
`glutinin (HA) and neuraminidase (NA); both are expressed on the sur-
`face of the influenza A virion.3 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.4
`
`The segmented influenza A genome permits re-assortment and ex-
`change of HA (or NA) segments between different influenza strain
`subtypes during concomitant host-cell infection. Generation of novel
`
`antigenic proteins (antigenic shift) and sustainable person-to-person
`transmission are hallmarks of pandemic influenza strains.5 Such
`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 influenza A virus subtypes included H7N9, H6N1, and
`H10N8.6–8 The case-fatality rate in over 600 cases of H7N9 infections
`was 30%.1,9 Most recently, the World Health Organization reported
`another 120 cases since September 2016 resulting in 37 deaths.10 To
`date, H10N8 infection in man has been limited; yet, of the three
`reported cases, two were fatal.11
`
`The limited efficacy of existing antiviral therapeutics (i.e., oseltamivir
`and zanamivir) makes vaccination the most effective means of protec-
`tion against influenza.12 Conventional influenza vaccines induce pro-
`tection by generating HA-specific neutralizing antibodies, the major
`correlate of protection, against the globular head domain.13–15 Such
`vaccines utilize the HA protein, administered as a subunit, split
`virion, inactivated whole virus, or live-attenuated virus. A majority
`of approved influenza vaccines are produced in embryonated chicken
`eggs or cell substrates. This process takes several months and relies on
`the availability of sufficient supplies of pathogen-free eggs and adap-
`tation of the virus to grow within its substrate.16,17 The 5–6 months
`required to produce enough vaccine to protect a substantial propor-
`tion of the population consumes much of the duration of the often-
`devastating first wave of a pandemic.18 This mismatch between the
`speeds of vaccine production and epidemic spread drives the search
`for vaccine platforms that can respond faster.19
`
`Using mRNA complexed with protamine (RNActive, Curevac),
`Petsch et al.20 demonstrated that intradermal (ID) vaccination of
`mice with RNActive encoding full-length HA from influenza virus
`H1N1 (A/Puerto Rico/8/1934) induced effective seroconversion and
`
`Received 23 January 2017; accepted 24 March 2017;
`http://dx.doi.org/10.1016/j.ymthe.2017.03.035.
`Correspondence: Giuseppe Ciaramella, Valera, 500 Technology Square, Cam-
`bridge, MA 02139, USA.
`E-mail: giuseppe.ciaramella@valeratx.com
`
`1316
`
`Molecular Therapy Vol. 25 No 6 June 2017 ª 2017 The Authors.
`This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
`
`ARBUTUS - EXHIBIT 2027
`Moderna Therapeutics, Inc. v. Arbutus Biopharma Corporation - IPR2019-00554
`
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`

`www.moleculartherapy.org
`
`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 influenza
`A virus.20 Efficacy of these RNActive vaccines was also shown in fer-
`rets and pigs.21
`
`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.22
`
`Exogenous mRNA can stimulate innate immunity through Toll-like
`receptors (TLRs) 3, 7, and 8 and cytoplasmic signal-recognition pro-
`teins RIG-I and MDA5.23,24 The adjuvant effect of stimulating innate
`immunity may be advantageous for purified protein vaccines, but
`indiscriminate immune activation can inhibit mRNA translation,
`reducing antigen expression and subsequent immunogenicity.25,26
`This can be overcome by replacing uridine nucleosides with naturally
`occurring base modifications, such as pseudouridine and 5-methylcy-
`tidine.27–29 Recently, we30 and others31 have shown how LNP-encap-
`sulated modified 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, modified mRNA-based influenza 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
`findings, a first-in-human, dose-escalating, phase 1 trial is ongoing,
`with interim results reported here that confirm the observed, preclin-
`ical immunogenicity data with a safety profile consistent with other
`non-live vaccines.
`
`RESULTS
`H10N8 and H7N9 HA mRNA Immunogenicity in Mice
`In vitro protein expression for both H10N8 HA (H10) and H7N9 HA
`(H7) mRNA vaccines were confirmed by transfection of HeLa cells.
`Western blot of resulting cell lysates demonstrated a 75-kDa band
`for both constructs using the corresponding HA-specific antibodies
`(Figure S1), consistent with previous reports for other HAs.22 Due
`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 significant
`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
`doses.
`
`While most vaccines are delivered via an intramuscular (IM) or sub-
`cutaneous administration,32 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 significant at the
`2-mg dose (p = 0.0038) (Figure S2A). The differences in H10 HAI titers
`were significant 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 significance was detected. In addition, there was no signif-
`icant difference between IM and ID immunization (Figure S2B). T cell
`responses, as measured by IFNɣ ELISpot, 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, significance 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 (Cmax) of
`the injection site muscle was 5,680 ng/mL, and the level declined
`with an estimated t1/2 of 18.8 hr (Table 1). Proximal lymph nodes
`had the second highest concentration at 2,120 ng/mL (tmax of 8 hr
`with a relatively long t1/2 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 Cmax of
`86.9 ng/mL (area under the curve [AUC]0–264 of 2,270 ng.hr/mL)
`and 47.2 ng/mL (AUC0–264 of 276 ng.hr/mL), respectively. In the
`remaining tissues and plasma, H10 mRNA was found at 100- to
`1,000-fold lower levels.
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`Molecular Therapy
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`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). ap = 0.0070,
`bp = 0.0017, and cp = 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. dp < 0.05 single dose versus boosting dose at the same time point. Error bars indicate standard mean error.
`
`Following ID administration, Cmax within the skin at the injection site
`was 18.2 mg/mL. Levels declined by 24 hr with an estimated t1/2 of
`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 Cmax of 1.66 ng/mL
`(1,663.52 pg/mL; AUC0–96 of 114.25 ng.hr/mL), 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,33 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 profile 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 significant differences 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).34
`
`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 influenza
`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 deficient in
`0
`expression, due to the removal of a methyl group on the 2
`-O position
`0
`of the first nucleotide adjacent to the cap 1 structure at the 5
`end 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  105 tissue culture infectious dose (TCID50)
`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  105
`
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`www.moleculartherapy.org
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`Table 1. Biodistribution of H10 mRNA in Plasma and Tissue after IM
`Administration in Mice
`
`Matrix
`
`Bone marrow
`
`Brain
`
`Cecum
`
`Colon
`
`Distal lymph nodes
`
`Heart
`
`Ileum
`
`Jejunum
`
`Cmax (ng/mL)
`
`AUC0–264 h
`(ng.hr/mL)
`
`tmax (hr)
`
`Mean
`
`SE
`
`Mean
`
`SE
`
`t1/2 (h)
`
`2.0
`
`8.0
`
`8.0
`
`8.0
`
`8.0
`
`2.0
`
`2.0
`
`2.0
`
`3.35
`
`1.87
`
`0.429
`
`0.0447
`
`0.886
`
`0.464
`
`1.11
`
`0.501
`
`NA
`
`13.9
`
`11.1
`
`13.5
`
`1.61
`
`5.120
`
`5.51
`
`177.0
`
`170.0
`
`4,050
`
`2,060
`
`0.799
`
`0.225
`
`3.54
`
`2.60
`
`0.330
`
`0.120
`
`1.31
`
`0.273
`
`6.76
`
`22.6
`
`5.24
`
`9.72
`
`1.98
`
`10.8
`
`0.931
`
`1.44
`
`NC
`
`NR
`
`NC
`
`NC
`
`28.0
`
`3.50
`
`5.42
`
`8.24
`
`11.4
`
`back titer calculation (6.2  103 TCID50 versus 3.8  105 and 6.1 
`105, respectively.), which was only 3-fold higher than the LD50 of
`1.88  103 (95% confidence interval [CI] = 8.02  102–5.51  103).
`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 confirming the significant 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.35
`
`Kidney
`
`Liver
`
`Lung
`
`Muscle (injection site)
`
`Plasma
`
`Proximal lymph nodes
`
`Rectum
`
`Spleen
`
`Stomach
`
`Testes
`
`2.0
`
`2.0
`
`2.0
`
`2.0
`
`2.0
`
`8.0
`
`2.0
`
`2.0
`
`2.0
`
`8.0
`
`47.2
`
`1.82
`
`8.56
`
`0.555
`
`276
`
`12.7
`
`37.4
`
`2.92
`
`5,680
`
`2,870
`
`95,100
`
`20,000
`
`5.47
`
`0.829
`
`35.5
`
`5.41
`
`2,120
`
`1,970
`
`38,600
`
`22,000
`
`1.03
`
`86.9
`
`0.423
`
`14.7
`
`29.1
`
`2,270
`
`0.626
`
`0.121
`
`2.37
`
`1.03
`
`11.6
`
`36.6
`
`3.67
`
`585
`
`1.32
`
`11.8
`
`NC
`
`16.0
`
`18.8
`
`9.67
`
`25.4
`
`NR
`
`25.4
`
`12.7
`
`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 quantifiable con-
`centrations; NC, not calculated; NR, not reported because extrapolation exceeds 20% or
`R-squared is less than 0.80.
`
`TCID50; Figures 2A–2C). There was a significant 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 influenza-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 2D–2F). 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 effi-
`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
`
`Unlike mice, ferrets are naturally susceptible to human influenza
`virus isolates. Human and avian influenza viruses both replicate effi-
`ciently in the respiratory tract of ferrets, and numerous clinical signs
`found in humans following seasonal or avian influenza virus infection
`are also present in the ferrets.36,37 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 influenza
`H7N9 via IN challenge (1  106 TCID50). The primary endpoint for
`this study was viral burden determined by TCID50 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 S6A–S6C). 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), significant 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 benefit 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 S7A–S7D). Two immunizations with 50-mg doses
`significantly increased HAI and MN titers compared to placebo
`(p < 0.05), and two immunizations with 200-mg doses generated
`significant 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 significant 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 significant 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 significant increases in
`HAI antibody titers as compared with prevaccination baseline values
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`Molecular Therapy
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`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 5
`
`cap structure (15 Da cap) were
`0
`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  105 TCID50 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.
`
`and controls (p < 0.0001). A single booster vaccination provided a sig-
`nificant 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
`Primates
`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 48–72 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 immunogenicity of H10 mRNA in humans, a
`randomized, double-blind, placebo-controlled, dose-escalating phase 1
`trial is ongoing (Clinical Trials Identifier 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 R 40 and MN R 20 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 R 40 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.38 AEs were comparable in
`frequency, nature, and severity to unadjuvanted and adjuvanted H1N1
`influenza vaccines.39 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
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`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.
`
`(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 profile
`is similar to that of a monovalent AS03-adjuvanted H1N1 vaccine, and
`it is comparable to that of meningococcal conjugate vaccine in healthy
`adults (19–55 years).40,41
`
`DISCUSSION
`Nucleic acid vaccines (NAVs) offer the potential to accurately ex-
`press any protein antigen, whether intracellular, membrane bound,
`or secreted. Although first identified in the early 1990s, mRNA vac-
`cines were not advanced into the clinic until recently due to concerns
`around stability and production.42,43 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, purified, 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 influenza.44
`The mRNA vaccine platform described here allows for rapid
`mRNA production and formulation, within a few weeks, at suffi-
`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
`influenza.20,45–47 Unmodified, sequence-optimized mRNA was used
`to generate H1-specific responses in mice, ferrets, and pigs at dose
`levels 4- to 8-fold higher than tested by us.20 Brazzoli et al.45 evalu-
`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.45
`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.46 Immuniza-
`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).47 Our platform, therefore,
`is surprisingly efficacious 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 7–9 kb).
`
`Modified mRNA has been shown to express more efficiently than un-
`modified mRNA, likely due to its reduced indiscriminate activation of
`innate immunity.29 When included in a vaccine formulation, our
`modified-mRNA technology balances immune stimulation and anti-
`gen expression, leading to very potent immune responses that are su-
`perior to unmodified 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 influenza virus, where
`virus could be measured outside the primary site of inoculation after
`5 days.48 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.22,49 Other groups have relied on either exog-
`enous RNA as an adjuvant or on the adjuvant properties generated
`during self-amplification 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,
`modified-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
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`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).
`
`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-
`genicity.50,51 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
`w