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`EXHIBIT 2
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`Molecular Therapy
`Original Article
`
`NO) GENE«CELL
`
`wa THERAPY
`
`Preclinical and Clinical Demonstration
`of Immunogenicity by MRNA Vaccines
`against H1ON8 and H7N9Influenza Viruses
`
`Kapil Bahl,' Joe J. Senn,* Olga Yuzhakov,! Alex Bulychev,? Luis A. Brito,? Kimberly J. Hassett,! Michael E. Laska,?
`Mike Smith,? Orn Almarsson,? James Thompson,” Amilcar (Mick) Ribeiro,! Mike Watson,! Tal Zaks,”
`and Giuseppe Ciaramella'
`
`1Valera, A Moderna Venture, 500 Technology Square, Cambridge, MA 02139, USA; 7Moderna Therapeutics, 200 Technology Square, Cambridge, MA 02139, USA
`
`antigenic proteins (antigenic shift) and sustainable person to person
`transmission are hallmarks of pandemic influenza strains.’ 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.°* Thecase fatality rate in over 600 cases ofH7N9infections
`was ~30%.'” Mostrecently, the World Health Organization reported
`another 120 cases since September 2016 resulting in 37 deaths.'’ To
`date, H10N8infection in man has been limited; yet, of the three
`reported cases, two werefatal.''
`
`the World Health Organization confirmed 120
`Recently,
`new humancases 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 mRNApro-
`Thelimited efficacy ofexisting antiviral therapeutics (i.e., oseltamivir
`tected mice fromalethal challenge and reduced lung viral titers
`and zanamivir) makes vaccination the most effective meansofprotec
`tion againstinfluenza.'* Conventional influenza vaccines induce pro
`in ferrets. Interim results from a first-in-human,escalating-
`dose, phase 1 H10N8 study show very high seroconversion
`tection by generating HA specific neutralizing antibodies, the major
`correlate of protection, against the globular head domain.'*"'* Such
`rates, demonstrating robust prophylactic immunity in hu-
`mans. Adverse events (AEs) were mild or moderate with only
`vaccines utilize the HA protein, administered as a subunit, split
`a few severe and no serious events. These data show that
`virion, inactivated whole virus, or live attenuated virus. A majority
`LNP-formulated, modified mRNAvaccines can induce protec-
`of approved influenza vaccines are produced in embryonated chicken
`tive immunogenicity with acceptable tolerability profiles.
`eggs or cell substrates, This process takes several monthsand relies on
`the availability of sufficient supplies of pathogen free eggs and adap
`tation of the virus to grow within its substrate.'®'’ The 5 6 months
`required to produce enough vaccine to protect a substantial propor
`tion of the population consumes muchof the duration of the often
`devastating first wave of a pandemic.'® This mismatch between the
`speeds of vaccine production and epidemic spread drives the search
`for vaccine platforms that can respondfaster.'”
`
`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, mostof these strains are not
`able to sustain person to person transmission.' However, lessons
`learned from these outbreaks demonstrated that new approaches
`are neededto address potential future pandemic influenza outbreaks.”
`
`Two majorglycoproteins, crucial for influenza infection, are hemag
`glutinin (HA) and neuraminidase (NA);both are expressed on the sur
`faceofthe influenza A virion.’ HA mediates viral entry into hostcells
`by binding tosialic acid containing receptors on the cell mucosal sur
`face and the fusion ofviral and host endosomal membranes.*
`
`The segmented influenza A genome permits re assortment and ex
`change of HA (or NA) segments between different influenza strain
`subtypes during concomitanthost cell infection. Generation of novel
`
`Using mRNA complexed with protamine (RNActive, Curevac),
`Petsch et al.”’ demonstrated that intradermal (ID) vaccination of
`mice with RNActive encoding full length HA from influenza virus
`HINI (A/Puerto Rico/8/1934) inducedeffective 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 © 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/).
`
`@ con
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`virus neutralizing antibodiesin all vaccinated animals. Immunity was
`long lasting and protected both young and old animals from lethal
`challenge with the H1N1, H3N2, and H5N1strains of the influenza
`A virus.” Efficacy of these RNActive vaccines was also shown in fer
`rets and pigs.”!
`
`Theuseofa delivery system can dramatically reduce the doses needed
`to generate potent immuneresponses, 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 evaluatedin 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 Iand MDA5.”*”* The adjuvanteffect 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.”*”°
`This can be overcomebyreplacinguridine nucleosides with naturally
`occurring base modifications, such as pseudouridine and 5 methylcy
`tidine.””-?° Recently, we*’ and others*’ 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 HION8 HA mRNAin humans from an
`ongoing trial. In the animal studies, we show that both vaccines
`generated potentneutralizing antibodytiters in mice,ferrets, and cyn
`omolgus monkeys (cynos) after a single dose. Additionally, a single
`dose of H7N9 HA mRNAprotected mice from an autologous lethal
`challenge and reduced lungviral 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.
`
`by day 21 (Figure 1A). Unlike HAI, both anti H10 and anti H7
`IgG1 and IgG2atiters were detected on day 7 (Figures 1B and 1C).
`For H10, IgG1 and IgG2atiters continued to increase until day 21
`and were maintained at day 84. For H7, both IgG1 and IgG2aanti
`bodytiters increased 10 fold between day 21 and day 84 (Figure 1C).
`IgG2atiters were greater than IgG]titers at all time points following
`formulated H10 or H7 mRNA immunization, suggesting a TH1
`skewed immuneresponse. 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 pg H10 mRNAboosting immunization (21 days
`post prime) resulted in a2 to5 fold increase in HAItiters, compared
`to a single doseatall 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,” the ID route of administration has the
`potential to be dose sparing. Therefore, to examinetheeffect ofadmin
`istration route on immunogenicity, BALB/c mice were immunized ID
`or IM with formulated H10 or H7 mRNAatfour different doselevels.
`All animals received a boosting immunization on day 21, and serum
`wascollected 28 days post boost (day 49). Immune responses were
`observed for both vaccinesatall dose levels tested (Figures S2A and
`$2B). Titers were slightly higher following IM administration at
`2 and 0.4 ug for H10, but this difference was only significant at the
`2 wg dose (p = 0.0038) (Figure S2A). The differences in H10 HAItiters
`were significant between some ofthe doselevels following IM admin
`istration: 10 versus 0.4 wg, p = 0.0247; 10 versus 0.08 jig, p = 0.0002;
`2 versus0.08 pg, p = 0.0013; and0.4 versus 0.08 pg, p = 0.0279. HAI
`titers following H7 immunizationtrended higheras the dose increased
`although nosignificance was detected. In addition,there was nosignif
`icantdifference between IM and ID immunization (Figure $2B). T cell
`responses, as measured by IFNy ELISpot, were observed for both
`H10 and H7at all doses tested (Figures S2C and $2D). Similar to
`H7 HAItiters, T cell responses trended higher following IM adminis
`tration, especially for H7. However, significance could not be estab
`lished due to poolingofthe samplesby group. Overall, after two doses,
`immunization with either H10 or H7 mRNAelicited an immune
`response atall doses tested with both ID and IM administration.
`
`RESULTS
`H10N8 and H7N9S 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 $1), consistent with previous reports for other HAs.” Due
`to a lack of glycosylation, both H10 HA and H7 HAprotein controls
`had a molecular weight of 62 kDa.
`
`Hemagglutination inhibition (HAI), IgG1, and IgG2a titers were
`measured after a single 10 jg dose of either formulated H10 or H7
`mRNAin BALB/c mice immunized ID. HAI titers were below the
`limit of detection (<10) at day 7 but increased well above baseline
`
`Given this innovative vaccine platform, we examined the bio
`distribution of the mRNAvaccines for both routes of administration.
`Male CD 1 mice received 6 pg formulated H10 mRNAeither IM or ID.
`Following IM administration, the maximum concentration (Cynax) of
`the injection site muscle was 5,680 ng/mL, and the level declined
`with an estimated ti. 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 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 Cyax of
`86.9 ng/mL (area under the curve [AUC]o_264 of 2,270 ng.hr/mL)
`and 47.2 ng/mL (AUCp_264 of 276 ng.hr/mL), respectively. In the
`remaining tissues and plasma, H10 mRNA was found at 100 to
`1,000 fold lowerlevels.
`
`Molecular Therapy Vol. 25 No6 June 2017
`
`391317
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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 pg dose of formulated H10 or H7 mRNA.{A) H10 and H7indicate mean HAItiters (limit of detection is 1:10). Dotted line
`indicates the correlate of protection in humans (1:40). (6B and C) IgG1 and IgG2atiters were measured for both H10 (B) and H7(C) via ELISA (n = 5/group). *p = 0.0070,
`®5 = 0.0017,and °p = 0.0185 versus IgG2aat the sametimepoint. (D) BALB/c mice were immunized ID with a single 10 ug doseof formulated H10 mRNA.A subset of these
`mice received a 10 pg boost on day 21. Serum wascollected 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 sametimepoint. Error bars indicate standard mean error.
`
`Following ID administration, C,,.x within the skin at the injectionsite
`was 18.2 pg/mL. Levels declined by 24 hr with an estimated ty. of
`23.4 hr, suggesting that the H10 mRNAlikely 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_9¢ of 114.25 ng-hr/mL), had the highest levels
`amongdistal tissues. Only trace amounts of H10 mRNAwere 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
`wasobserved followed by drainingto the local lymph nodes and sub
`sequentcirculation in the lymphatic system (Table 1; Table $1).
`
`To understand the expression profile ofmRNAafter IM and ID admin
`istration, BALB/c mice were injected on day 0 with formulated lucif
`erase mRNAat four different dose levels (10, 2, 0.4, and 0.08 pg).
`Expression was found to be dose dependent. As the dose increased,
`expression was foundin distal tissues, with peak expression observed
`6 hrafter dosing. Therewere nosignificant 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 $3B and $3C). 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 $1).**
`
`H7 mRNAVaccine Provides Protection against Lethal Influenza
`H7NQ, A/Anhui/1/2013, in Mice and Ferrets
`To determinethe timeto onset and duration ofimmunity to influenza
`H7N9 (A/Anhui/1/2013) lethal challenge, BALB/c mice were immu
`nized ID with 10, 2, or 0.4 pg formulated H7 mRNA.For negative
`controls, placebo and 10 pg formulated H7 mRNA deficient in
`expression, due to the removal of a methyl group on the 2’ O position
`of the first nucleotide adjacent to the cap 1 structure at the 5’ end of
`the mRNA (_
`15 Dacap), were included. Serum wascollected on days
`6, 20, and 83, and mice were challenged via intranasal (IN)instillation
`with a target dose of 2.5 x 10° tissue culture infectious dose (TCIDs9)
`on days 7, 21, and 84. Changes in body weight andclinical signs of
`disease were monitored for 14 days post challenge. A single vaccina
`tion was found to be protective against H7N9 challenge (2.5 x 10°
`
`1818
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`Table 1. Biodistribution of H10 mRNAin PlasmaandTissueafter IM
`Administration in Mice
`
`back titer calculation (6.2 x 10° TCIDso versus 3.8 x 10° and 6.1 x
`10°, respectively), which was only ~3 fold higher than the LDso of
`1.88 x 10° (95% confidence interval [CI] = 8.02 x 107 5.51 x 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 confirming the significant protection
`observedin the positive vaccine groups. Additionally,it is not possible
`to rule out a lowlevel of protein expression from the de methylated
`cap ofthe negative mRNAcontrol.”°
`
`AUC) 264 b
`(ng-hr/mL)
`Chex (ng/mL)
`ty (h)
`Mean
`SE
`tmax (hr) Mean
`SE
`Matrix
`
`NC
`NA
`2.0
`335 1.87
`Bone marrow
`
`8.0
`0429 0.0447 39 161 NR
`Brain
`
`80
`0.886 0464 111 5120 NC
`Cecum
`
`80 Lil 0501 135 551 NC
`Colon
`
`8.0
`17.0 170.0 “4050 2,060 28.0
`Distal lymph nodes
`Unlike mice, ferrets are naturally susceptible to human influenza
`
`20
`0.799 0225 676 198 350
`Heart
`virus isolates. Human and avian influenza viruses both replicate effi
`
`ciently in the respiratory tract of ferrets, and numerous clinical signs
`
`
`Tleum ©3554 2.60 226=«108 54220
`found in humans following seasonal or avian influenza virus infection
`
`Jejunum
`20
`0.330 0.120 524 0931 824
`are also presentin the ferrets.°°*” Ferrets (n = 8/group) were vacci
`
`Kidney
`20
`131 0273 972 144 114
`nated ID on day 0 with 200 , 50, or 10 pg doses of formulated
`
`
`Liver 472—«BS6 276=37A NC20.
`
`H7 mRNA.Formulated H7 mRNA witha
`15 Da cap and placebo
`Lung
`20
`«1820555 127 «292 «160
`
`were included as negative controls. A subset of ferrets received a sec
`
`Muscle (injection site)
`2.0
`5,680 2,870 "95,100 "20,000 18.8
`ond ID vaccination on day 21. All groups were exposed to influenza
`
`Plasma
`20.
`547 0829 355 541 967
`H7N9via IN challenge (1 x 10° TCIDso). The primary endpointfor
`
`Proximal lymph nodes
`8.0
`"2,120 1,970 “38,600 "22,000 25.4
`this study was viral burden determined by TCIDsp in the lung at
`3 days post challenge, which is when the peak viral load is seen in
`
`Rectum
`20
`103 0493 147 367 NR
`control animals (data not shown). A reduction in lungviral titers
`
`Spleen
`20
`«869 291 2270 585 254
`wasobserved when ferrets were challenged 7 days post immunization
`
`Stomach
`20
`0626 «~O12 «116 «132 «127
`at all doses tested (Figures S6A S6C). Ferrets immunized with 200 pg
`Testes
`8.0
`237 103 36.6 18 NR
`and challenged on day 49 hadviral loads below the level of detection
`Male CD-1 mice received 300 pg/kg (6 jg) formulated H10 mRNAvia IM immuniza-
`(Figure S6C). Antibodytiters, as measured by HAI, increased signif
`tion. Tworeplicates ofbone marrow,lung, liver, heart, right kidney, inguinal- and popli-
`icantly by day 21 forall dose groups (p < 0.05); as measured by micro
`teal-draining lymph nodes, axillary distal lymph nodes, spleen, brain, stomach,ileum,
`neutralization (MN),significant increases were observedby day49 for
`jejunum, cecum, colon, rectum,testes (bilateral), and injection site muscle were
`collected for bDNAanalysis at 0, 2, 8, 24, 48, 72, 120, 168, and 264 hr after dosing
`all dose groups(p < 0.05) (Figures S7A and $7B). A second immuni
`(n=3mice/time point). NA, not applicable AUC with less than three quantifiable con-
`zation increased titers but showed nostatistical benefit compared to a
`centrations; NC, not calculated; NR, not reported because extrapolation exceeds 20% or
`single immunization, likely due to the two to four log reduction in
`R-squared is less than 0.80.
`viral lung titers seen in both the single and double immunization
`groups (Figures S7A S7D). Two immunizations with 50 pg doses
`significantly increased HAI and MNtiters compared to placebo
`(p < 0.05), and two immunizations with 200 yg doses generated
`significant HAI and MNtiters versus placebo and all other doses
`(p < 0.0001) (Figures $7C and $7D).
`
`TCIDs9; 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
`In the absence of an H10N8 (A/Jiangxi Donghu/346/2013) chal
`loss (incidence and duration) was moreprevalent for animals in the
`
`control groups and seen toalesser extent in the low dose vaccine lenge model, the onset and duration of immunity to formulated
`group (Figures 2D 2F). HAItiters were below the limit of detection
`H10 mRNAin ferrets was tested by HAI. Groups of ferrets were
`immunized ID once, twice, or three times with 50 or 100 pg H10
`until day 20 for both the 10 and 2 jg dose groups (Figure S5). There
`
`was a5 to7fold increase in HAItiters from day 20 to day 83atall mRNA. Immunization with a single dose of 50 or 100 pg resulted
`doses tested (p < 0.0001). Day 83 titers were dose dependent with
`in significant and comparable increases in HAItiters at days 21, 35,
`and 49 (p < 0.0001; Figure 3). Immunization with a 100 pg dose re
`mean titers of 224, 112, and 53 for the 10 pg dose, 2 ig dose, and
`0.4 ug dose groups, respectively (p < 0.0001). Interestingly, despite
`sulted in only slightly elevated antibody responses on day 7 compared
`complete protection to challenge at the 0.4 1g dose at day 21 (Fig
`to day 0 (p < 0.0001), with minimal differences observed with the
`ure 2B), a protective HAItiter (> 40) was not detected until day 83
`50 pg dose on day 7 compared to day 0 (p < 0.3251). Subsequent
`at this dose, suggesting additional mechanism(s)of protection.
`boosts with either a 50 or 100 pg 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 pg dose yielded significant increases in
`HAIantibody titers as compared with prevaccination baseline values
`
`The negative mRNAcontrol 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
`
`Molecular Therapy Vol. 25 No6 June 2017==1319
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`Molecular Therapy
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`Challenged on Day7 Post-Immunization
`
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`Days Post-Challenge
`
`Days Post-Challenge
`
`Figure 2. A Single Injection of an H7 mRNAVaccine Achieves Rapid and Sustained Protection in Mice
`BALB/c mice were vaccinated ID with 10, 2, or 0.4 wg formulated H7 mRNA.Placebo and 10 pg formulated H7 mRNAwith a reduced 5’ cap structure (_ 15 Da cap) were
`included as negative controls. On day7, 21, or 84 post immunization, mice were challenged via intranasal(IN) instillation with a target dose of 2.5 x 10° TCIDgp 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 day7 (A), day 21 (B), or day 84 (C) post
`immunization at the indicated doses. p < 0.0001 10 , 2 , and 0.4 yg 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 meanerror.
`
`and controls (p < 0.0001). A single booster vaccination provided a sig
`nificant increasein titers, but a second booster dose did notyield an
`additional increase (Figure 3).
`
`H10 HA and H7 HA mRNA Immunogenicity in Nonhuman
`Primates
`
`Oneof the majorlimitations with other nucleic acid based technolo
`gies, such as plasmid DNA,has beentranslation 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 doselevels (0.2 and
`0.4 mg) of formulated H7 mRNA administered IM andID (Figures
`4A and 4B). Formulated H10 mRNA wastested 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 mRNAvac
`cines generated HAItiters between 100 and 1,000after 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 injectionsite irritation, and, in somecases,
`decreased food consumption following either H10 or H7 immuniza
`tion. All symptomsresolved within 48 72 hr. Overall, both ID and IM
`administration elicited similar HAItiters regardless of dose, suggest
`ing that lower doses may generate a similar HAItiter.
`
`H10 mRNA Immunogenicity and Safety in Humans
`To evaluate the safety and immunogenicity ofH10 mRNAin humans, a
`randomized,double blind, placebo controlled, dose escalating phase 1
`trial is ongoing (Clinical Trials Identifier NCT03076385). We report
`hereinterim results, obtained 43 days post vaccination of 31 subjects
`(23 of whom received active H10 at 100 pg IM andeight 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 > 40 and MN = 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) whoreceived the H10 vaccine had an HAIbaseline <10
`andpost vaccination HAI > 40 or HAIfour or more timesbaseline,
`respectively, compared to 0% for placebo (Figures 5A and 5B). HAI
`geometric mean antibody titers of subjects given the H10 vaccine
`were 68.8 comparedto 6.5 for placebo, and the MN geometric mean ti
`ters were 38.3 versus 5.0, respectively (Figures 5C and 5D).
`
`The majority ofadverse 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
`influenza vaccines.’ Twenty three subjects whoreceived 100 pg 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/commoncold like symptoms
`
`1820
`
`Molecular Therapy Vol. 25 No6 June 2017
`
`

`

`Case 1:22-cv-00252-MSG Document 193-2 Filed 01/16/24 Page 7 of 24 PageID #: 12531
`Filed 01/16/24 Page 7 of 24 PagelD #: 12531
`Case 1:22-cv-00252-MSG Document 193-2
`
`www.moleculartherapy.org
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`Other mRNAvaccine approaches have previously been reported for
`influenza.””**” Unmodified, sequence optimized mRNA wasused
`to generate H1 specific responses in mice, ferrets, and pigs at dose
`levels ~4 to 8 fold higher than tested by us.”° Brazzoli et al.*° 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 yg dose (two of six re
`sponders) and at the 45 yg dose(three of six responders)after a single
`immunization. Following a boost,
`titers were measurable in all
`animals and provided protection to a homologouschallengestrain.*°
`In another study, mice singly immunized against H1N1 (A/WSN/33),
`receiving a self amplifying mRNA, showed no IgG responsesafter
`7 days. After a second immunization, responses were boosted and
`animals were protected against a homologous challenge.*° Immuniza
`tion in mice against either H1 or H7, with a self amplifying mRNA,
`induced HAIandIgG titers that were comparable to those achieved
`in our study at similar doses (Figure 1).*” Our platform, therefore,
`is surprisingly efficacious when comparedto 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 accommodatethe self replicating portions of
`the vectors (typically 7 9 kb).
`
`Modified mRNAhas been shown to express moreefficiently than un
`modified mRNA,likely due to its reduced indiscriminate activation of
`innate immunity.”? When included in a vaccine formulation, our
`modified mRNA technology balances immunestimulation and anti
`gen expression, leading to very potent immuneresponses 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 duringa viral infection. Indeed, the biodistribution
`weobserved (Table 1; Table $1) is similar to an influenza virus, where
`virus could be measured outside the primary site of inoculation after
`5 days.** Importantly, there was no wayfor our vaccineto revert to a
`virulent form because key parts of the virus were missing, including
`any nonstructural elements or capsid structures.
`
`Weselected LNPs for delivery of the mRNAas they have been vali
`datedin the clinic for siRNA and are well tolerated compared to other
`nonviral delivery systems.’”*° Other groupshaverelied on either exog
`enous RNAas an adjuvant or on the adjuvant properties generated
`duringself amplification of the mRNA. Using an LNP, we generate
`very high levels oftransient 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 immuneresponses in mice, ferrets, and cynomolgus mon
`keys. In animals, we showed that a range of doses of formulated
`mRNAencoding the HA protein ofeither H7N9 or H10N8is able
`to stimulate rapid, robust, and long lasting, immune responses, as
`measured by HAI, MNassay, andprotection from viral challenge. A
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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 pg formulated H10 mRNA. p < 0.0001,
`days 21, 35, and 49 versus day 0 with single doses of 50 or 100 pg; p < 0.0001
`100 yg single dose, day 7 versus day 0. A subsetof 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 yg boosting dose(s), days 35 and 49 versus day 0.
`
`(Table $2). 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
`moncold (0.6%) (Table 2; Table $2). No serious AE occurred andall
`events were expected and reversible. Overall, this reactogenicity profile
`is similar to that ofa monovalent ASO3 adjuvanted H1N]1vaccine, and
`it is comparable to that ofmeningococcal conjugate vaccine in healthy
`adults (19 55 years).*°*!
`
`DISCUSSION
`Nucleic acid vaccines (NAVs) offer the potential to accurately ex
`press any protein antigen, whetherintracellular, membrane bound,
`or secreted, Although first identified in the early 1990s, mRNA vac
`cines were not advancedintotheclinic until recently due to concerns
`around