Case 1:22-cv-00252-MSG Document 193-5 Filed 01/16/24 Page 1 of 22 PageID #: 12578
`Case 1:22-cv-00252-MSG Document 193-5 Filed 01/16/24 Page 1 of 22 PagelD #: 12578
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`EXHIBIT 5
`EXHIBIT 5
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`Case 1:22-cv-00252-MSG Document 193-5 Filed 01/16/24 Page 2 of 22 PageID #: 12579
`Article
`
`SARS-CoV-2 mRNAvaccine design enabled
`by prototype pathogen preparedness
`
`
`
`https://doi.org/10.1038/s41586-020-2622-0
`Received: 10 June 2020
`
`
`Accepted: 29 July 2020
`
`Published online: 5 August 2020
`
`|® Check for updates
`
` Case 1:22-cv-00252-MSG Document 193-5 Filed 01/16/24 Page 2 of 22 PagelD #: 12579
`
`Since its emergence in December 2019, SARS-CoV-2 has accounted for
`more than 30 million cases of coronavirus disease 2019 (COVID-19)
`worldwide in 9 months?. SARS-CoV-2is the third novel Betacoronavirus
`in the past 20 yearsto cause substantial human disease; however, unlike
`its predecessors SARS-CoVand Middle East respiratory syndrome
`coronavirus (MERS-CoV), SARS-CoV-2 is transmitted efficiently from
`person to person.In the absenceof a vaccine, public health measures
`such as quarantine of newly diagnosed cases, contacttracing, use of
`face masks and physical distancing have been putinto place toreduce
`transmission‘. It is estimated that until 60-70% of the population have
`immunity, COVID-19 is unlikely to be sufficiently well-controlled for
`normal human activities to resume.Ifimmunity remains solely depend-
`ent oninfection, even at acasefatality rate of1%, more than4O million
`people could succumb to COVID-19 globally*. Therefore, rapid develop-
`mentof vaccines against SARS-CoV-2will be critical for changing the
`global dynamicsofthis virus.
`The spike (S) protein, a class I fusion glycoprotein analogous to
`influenza haemagglutinin, respiratory syncytial virus (RSV) fusion
`glycoprotein (F) and human immunodeficiency virus gp160 (Env),
`
`Kizzmekia S. Corbett", Darin K. Edwards”, Sarah R. Leist*"°, Olubukola M. Abiona’,
`Seyhan Boyoglu-Barnum', RebeccaA.Gillespie’, Sunny Himansu?, Alexandra Schafer’,
`Cynthia T. Ziwawo', AnthonyT. DiPiazza', Kenneth H. Dinnon®, Sayda M.Elbashir’,
`Christine A. Shaw’, Angela Woods’, EthanJ. Fritch*, David R. Martinez®, Kevin W. Bock°,
`Mahnaz Minai’, Bianca M. Nagata’, Geoffrey B. Hutchinson’, Kai Wu’, Carole Henry’, Kapil Bahl’,
`Dario Garcia-Dominguez’, LingZhi Ma’, Isabella Renzi”, Wing-Pui Kong’, Stephen D. Schmidt',
`Lingshu Wang, YiZhang', Emily Phung", Lauren A. Chang’, RebeccaJ. Loomis’,
`Nedim Emil Altaras?, Elisabeth Narayanan’,Mihir Metkar?, Vlad Presnyak?, Cuiping Liu’,
`MarkK. Louder’, Wei Shi’, Kwanyee Leung’, Eun Sung Yang’, Ande West®, Kendra L. Gully’,
`Laura J. Stevens’, Nianshuang Wang*, Daniel Wrapp*,Nicole A. Doria-Rose’,
`Guillaume Stewart-Jones?, Hamilton Bennett’, Gabriela S. Alvarado’, Martha C. Nason’,
`Tracy J. Ruckwardt', Jason S. McLellan®, Mark R. Denison’, James D. Chappell’, lan N. Moore®,
`Kaitlyn M. Morabito’, John R. Mascola’, Ralph S. Baric**, Andrea Carfi?™ & Barney S. Graham'™
`
`Avaccinefor severe acute respiratory syndrome coronavirus2 (SARS-CoV-2)is
`needed to control the coronavirus disease 2019 (COVID-19) global pandemic.
`Structural studies have led to the developmentof mutationsthatstabilize
`Betacoronavirus spike proteinsin the prefusionstate, improving their expression and
`increasing immunogenicity’. This principle has been applied to design mRNA-1273, an
`mRNAvaccine that encodesa SARS-CoV-2 spike protein thatis stabilized in the
`prefusion conformation. Here we show that mRNA-1273 inducespotentneutralizing
`antibody responsesto both wild-type (D614) and D614G mutant? SARS-CoV-2 as well
`as CD8* T cell responses, and protects against SARS-CoV-2 infection in the lungs and
`nosesof mice without evidence of immunopathology. mRNA-1273is currently ina
`phaseIll trial to evaluateitsefficacy.
`
`is the major surface protein on the coronavirus virion and thepri-
`marytarget for neutralizing antibodies. S proteins undergo marked
`structural rearrangementto fuse virus and host cell membranes,
`enabling delivery of the viral genome into targetcells. We previously
`showedthat prefusion-stabilized protein immunogens that preserve
`neutralization-sensitive epitopesare an effective vaccine strategy
`for enveloped viruses such as RSV°”. Subsequently,we identified 2
`proline substitutions (2P) at the apex of the central helix and heptad
`repeat 1 that effectively stabilized MERS-CoV, SARS-CoV and human
`coronavirus HKU1S proteinsinthe prefusion conformation“ Similar
`to other prefusion-stabilized fusion proteins, MERS-CoV S(2P) protein
`was more immunogenic at lower doses than wild-type S protein’. The
`2P mutationhas similar effects on the stability ofS proteins from other
`betacoronaviruses, suggesting a generalizable approachfordesigning
`stabilized-prefusion BetacoronavirusS protein antigensfor vaccina-
`tion. Such generalizability is fundamental to the prototype pathogen
`approachfor pandemic preparedness®*.
`Coronaviruses have long been predicted to havea high probability of
`causing zoonotic disease and pandemics’.As part ofour pandemic
`
`‘Vaccine Research Center, NationalInstitute of Allergy and Infectious Diseases, NationalInstitutes of Health, Bethesda, MD, USA. Moderna Inc, Cambridge, MA, USA. "Department of
`Epidemiology, Gillings Schoolof Global Public Health, University of North Carolina at ChapelHill, Chapel Hill, NC, USA. ‘Departmentof Microbiology and Immunology, School of Medicine,
`University of North Carolina at ChapelHill, ChapelHill, NC, USA. °NationalInstitute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA.“institute for Biomedical
`Sciences, George Washington University, Washington, DC, USA. Departmentof Pediatrics, Vanderbilt University Medical Center, Nashville, TN, USA. "Departmentof Molecular Biosciences,
`University of Texas at Austin, Austin, TX, USA. °Biostatistics Research Branch,Division ofClinical Research, NationalInstitute of Allergy and Infectious Diseases, NationalInstitutes of Health,
`Bethesda, MD, USA. These authorscontributed equally: Kizzmekia S. Corbett, Darin K. Edwards, Sarah R. Leist. e-mail: andrea.carfi@modernatx.com; bgraham@nih.gov
`
`Nature | Vol586 | 22 October 2020 | 567
`
`

`

`andcorrelates of protection. Achieving an effective and rapid vac-
`@=~Neutralizing antibodies 9oa
`cine response to a newly emerging virus requires both the precision
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`mRNAvaccinesare potently immunogenic and elicit both humoral
`& 901234567 8 9101112
`and cellular immunity’. We therefore evaluated mRNA formulated
`Time after challenge (d)
`inlipid nanoparticles (mRNA-LNP)asa delivery vehicle for MERS-CoV
`S(2P), and found that transmembrane-anchored MERS-CoVS(2P) mRNA
`elicited more potent pseudovirus-neutralizing antibody responses
`than secreted MERS-CoVS(2P) (Extended DataFig. 1a). Additionally,
`consistent with protein immunogens, MERS-CoV S(2P) mRNA was
`more immunogenic than wild-type MERS-CoVS mRNA(Extended Data
`Fig. 1b). Immunization with MERS-CoV S(2P) mRNA-LNP elicited potent
`pseudovirus-neutralizing activity with a dose as lowas0.1 pg and pro-
`tected transgenic mice expressing human DPP4(288/330*")” against
`lethal MERS-CoV challenge ina dose-dependentmanner,establishing
`that mRNA encoding S(2P)proteinis protective. Notably, a subprotec-
`tive 0.01,1g dose of MERS-CoV S(2P) mRNAdid not cause exaggerated
`disease following MERS-CoVinfection, but instead resultedin par-
`tial protection against weight loss followed byfull recovery without
`Fig.1| MERS-CoVS-2P mRNAprotectsmice from lethal challenge.
`evidenceof enhancedillness (Fig. 1).
`a-d, 288/330" mice were immunized at weeks 0 and3with0.01 (green), 0.1
`SARS-CoV-2 wasfirst identified as the cause of an outbreak of res-
`(blue) or 1 pg (red) MERS-CoV S(2P) mRNA.Control mice wereadministered
`piratory disease in Wuhan, Chinain earlyJanuary 2020. Within 24 h of
`phosphate-bufferedsaline (PBS) (grey).a, Two weekspost-boost, sera were
`the release of genomic sequences of SARS-CoV-2isolates on 10Janu-
`collected fromthree mice per group and assessed for neutralizing antibodies
`ary 2020, the 2P mutations were substitutedinto S protein residues
`against MERS m35c4 pseudovirus. b-d, Four weeks post-boost, 12 mice per
`986 and 987 to produce prefusion-stabilized SARS-CoV-2 S(2P) pro-
`groupwerechallenged witha lethal dose ofmouse-adapted MERS-CoV
`tein for structural analysis” and serological assay development”**>
`(m35c4). b, Following challenge, mice were monitoredfor weight loss. c,d, Two
`dayspost-challenge,at peak viral load,lungviral titres (c) and haemorrhage
`in silico, without additional experimentalvalidation. Within 5 days
`(scored as: 0,no haemorrhage, 4, severe haemorrhageinall lobes) (d) were
`ofthe release of the sequence, current good manufacturing practice
`assessed from five mice per group.Inc,d, alldoselevels were compared by
`(cGMP) production of mRNA-LNPencoding the SARS-CoV-2 S(2P) as
`Kruskal-Wallis analysis ofvariance (ANOVA) with Dunn’s multiple comparisons
`a transmembrane-anchored protein with the native furin cleavage
`test. Inb, for weightloss, all comparisonsare with PBS control mice at the same
`site (mRNA-1273)was initiatedin parallel with preclinical evaluation.
`time point by two-sided Mann-WhitneyU-test. **P< 0.01,****P< 0.0001. Data
`This led to a first-in-human phaseIclinical trial starting on 16 March
`are GMT+geometrics.d.(a,c) ormean+s.d.(b, d). Inc, thedottedline
`2020, 66 days after the viral sequence wasreleased, and a phase lltrial
`representsassaylimit ofdetection.
`74 days later on 29 May 2020 (Extended DataFig. 2). Expression and
`antigenicity of the S(2P) antigen delivered by mRNAwasconfirmed
`in vitro before vaccinationofthe first human participant (Extended
`Data Fig. 3), and immunogenicity of mMRNA-1273 was documentedin
`several mousestrains. The results of those studies are detailed here.
`
` Case 1:22-cv-00252-MSG Document 193-5 Filed 01/16/24 Page 3 of 22 PagelD #: 12580
`
`Fig. 2| mRNA-1273 elicits robust binding and
`pseudovirus-neutralizing antibody responsesin
`multiple mousestrains. a-f, BALB/c) (a,d), CS7BL/6)
`(b, e) or B6C3F1/J (c, f) mice (nr =10 per group) were
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`post-boost(filled circles) and assessed for SARS-CoV-2
`S-specific|gG byenzyme-linked immunosorbentassay
`(ELISA)(a-c),and forpost-boostsera, neutralizing
`antibodies against homotypicSARS-CoV-2
`pseudovirus(d-f). In a—c,timepointswerecompared
`signed-ranktest, and doses were compared post-boost
`by Kruskal-Wallis ANOVA with Dunn’s multiple
`comparisonstest.In d-f, Vaccine groups were
`compared by two-sided Mann-Whitney U-test.
`*P< 0.05, **P< 0.01,***P< 0.001, ****P< 0.0001. Data
`are presented as GMT+ geometric s.d. Dotted lines
`represent assay limits ofdetection.
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`568 | Nature | Vol586 | 22 October 2020
`
`Case 1:22-cv-00252-MSG Document 193-5 Filed 01/16/24 Page 3 of 22 PageID #: 12580
`Article
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`preparednessefforts, we have studied MERS-CoVas a prototype
`Betacoronavirus pathogento optimize vaccine design, dissect the
`humoral immuneresponseto vaccination, and identify mechanisms
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`Case 1:22-cv-00252-MSG Document 193-5 Filed 01/16/24 Page 4 of 22 PageID #: 12581
`
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` Case 1:22-cv-00252-MSG Document 193-5 Filed 01/16/24 Page 4 of 22 PagelD #: 12581
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`Fig. 3| Immunizations with mRNA-1273 and S(2P) protein, delivered with
`TLR4agonist,elicit S-specific T,1-biased T cell responses. B6C3F1/J mice
`(n=10 per group) were immunizedat weeks0 and 3 with0O.01, 0.1or1pg of
`mRNA-1273 or SARS-CoV-2S(2P)protein with SAS adjuvant. a—c, Sera were
`collected two weekspost-boostand assessed by ELISA for SARS-CoV-2
`S-specific lgG1, and1gG2a and IgG2c. End-pointtitres (a, b) and end-pointtitre
`ratios oflgG2a plus lgG2c tolgG1(c) werecalculated. Ratios were not
`calculated for mice for which end-pointtitres didnot reach the lowerlimit of
`detection (dotted line; N/A). d-g, Seven weekspost-boost, splenocytes were
`isolated from five mice per groupand restimulated with vehicle or pools of
`
`overlapping peptides from SARS-CoV-2S protein in the presence of aprotein
`transport inhibitor cocktail. After 6h,intracellular cytokine staining was
`performed to quantify CD4* and CD8* Tcell responses. Cytokineexpressionin
`the presenceofvehicle only was considered as background and subtracted
`from the responses measured from the S1 and S2 peptide pools for each
`individual mouse.d, e, PercentageofCD4"Tcellsexpressing IFN-y, TNF,IL-2,
`IL-4 and IL-5 inresponseto the S1 (d) and S2(e) peptide pools. f, g, Percentage of
`CD8*T cells expressing IFN-y, TNF and IL-2inresponsetotheS1(f) and S2(g)
`peptide pools.
`
`Immunogenicity was assessed in six-week-old female BALB/c],
`C57BL/6] and B6C3F1/J mice by two intramuscular immunizations
`with 0.01, 0.1 or 1 pg MRNA-1273, separated by a 3-weekinterval.
`mRNA-1273 induced dose-dependent specific S-binding antibod-
`ies after prime and boostin all mousestrains (Fig. 2a—c). Potent
`pseudovirus-neutralizing activity waselicited by 1 pg MRNA-1273,
`reaching reciprocal half-maximal inhibitory concentration (IC,,)
`geometric mean titres (GMTs) of 819 (BALB/cJ), 89 (CS7BL/6]) and
`1,115 (B6C3F1/J) (Fig. 2d-f). Additionally, mice immunized with 1 pg
`mRNA-1273 had robust neutralizing antibodies against pseudovi-
`rusesthat express S protein with the D614G substitution; SARS-CoV-2
`expressing the D614G variantof the S protein has recently become
`dominant around the world? (ExtendedData Fig.4). To further gauge
`immunogenicity across a wide dose range, BALB/c mice were immu-
`nized with 0.0025-20 pg mMRNA-1273, revealing a strong positive cor-
`relation between dose-dependent mRNA-1273-elicited binding and
`pseudovirus-neutralizing antibody responses (ExtendedDataFig.5).
`BALB/c] mice that received a single dose ofmRNA-1273 were evaluated
`to ascertain theutility of a single-dose vaccine. S-binding antibod-
`ies were induced in mice immunizedwith one 1 pg or 10 pg dose of
`mRNA-1273. The 10 pg dose elicited pseudovirus-neutralizing antibody
`
`activity thatincreased between week 2and week4,reaching 315 recip-
`rocal IC.¢g GMT (Extended Data Fig.6a, b). These datademonstrate that
`mRNAexpressing SARS-CoV-2 S(2P) is a potent immunogenand that
`pseudovirus-neutralizing activity can be elicited with a single dose.
`Vaccine-associated enhancedrespiratory disease (VAERD)has been
`associated with T helper2 cell (T,,2)-biased immune responsesin chil-
`dren immunized with whole-inactivated-virus vaccines against RSV
`and measles virus”. A similar phenomenonhasalso been reported in
`some animal models with whole-inactivated vaccines and other types
`of experimental SARS-CoVvaccines”**°. We therefore evaluated the
`balanceof T,,1 and T,,2 cells in immunized mice. Wefirst compared
`levels of S-specific immunoglobulins, IgG2a and IgG2c, and IgG1—
`which are surrogatesofT,,1 and T,,2 responses, respectively—elicited
`by mRNA-1273 with thoseelicited by immunization with SARS-CoV-2
`S(2P) protein using the TLR4 agonist Sigma Adjuvant System (SAS).
`Both immunogenselicited S-binding antibodies in the lgG2a and
`IgG1 subclasses, indicating a balanced T,,1-T,,2 response(Fig. 3a—c,
`Extended DataFig. 7). The S-specific IgG-subclassprofile following a
`single dose of mRNA-1273 (ExtendedData Fig. 6c) was similar to that
`observed following two doses. By contrast, T,,2-biased responses,
`with lower IgG2a/IgG1 ratios, were observed in mice immunized with
`
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` Case 1:22-cv-00252-MSG Document 193-5 Filed 01/16/24 Page 5 of 22 PagelD #: 12582
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`Wealso directly measured cytokine patterns in vaccine-induced mem-
`oryT cells by intracellular cytokine staining seven weeksafter the boost
`injection; MRNA-1273-elicited CD4'T cells re-stimulated with S1 or
`S2 peptide pools exhibited a T,,1-dominant response,particularly at
`higher immunogendoses (Fig. 3d, e). Furthermore, 1 pg mMRNA-1273
`induced arobust CD8'T cell responseto the S1 peptide pool(Fig. 3f, g).
`Together, the IgG subclass and T cell cytokine data demonstrate that
`immunization with MRNA-1273elicits balanced T,,1 and T,2 responses,
`in contrast to the T,,2-biased response seen whenusingS protein with
`alum adjuvant, suggesting that mRNAvaccination avoids T,,2-biased
`immune responses, which have been linked to VAERD.
`Protective immunity was assessed in young adult BALB/c] mice
`challenged with mouse-adapted (MA) SARS-CoV-2. SARS-CoV-2 MA
`contains the substitutions Q498Y/P499T in the receptor-binding
`domain*. The substitutions enable the virus to bind to the mouse
`angiotensin-converting enzyme 2 (ACE2) receptor and infect and
`replicate in the upper and lowerrespiratory tract”. BALB/c] mice that
`received two 1 pg doses of mRNA-1273 were completely protected from
`viral replication inlungs after challenge 5 or 13 weeks after boostinjec-
`tion (Fig.4a, Extended DataFig. 9a). mRNA-1273-induced immunity also
`resulted in undetectable viral replication in nasal turbinates in 6 out of
`7 mice (Fig. 4b, Extended DataFig. 9b). The efficacy of mRNA-1273 was
`dose-dependent; two 0.1 pg doses of mRNA-1273 reduced lungviral
`load by about 100-fold, whereas two 0.01 pg doses reduced lungviral
`load by about 3-fold (Fig. 4a). Of note, mice challenged 7 weeks after
`asingle dose of1or 10 pg MRNA-1273 were alsocompletely protected
`againstlungviral replication (Fig. 4c). Challenging animals immunized
`with subprotective dosesprovides an orthogonal assessmentofsafety
`signals such asincreased clinicalillness or pathology. Similar observa-
`tions with MERS-CoVS(2P) mRNA,mice immunized with subprotective
`0.1 or 0.01 1g doses of MRNA-1273 showed noevidence of enhanced
`lung pathologyor excessive mucus production(Fig.4d). Insummary,
`mRNA-1273is immunogenic,efficacious and does not produce evidence
`ofVAERD whengivenat subprotective doses in mice.
`Here we have shownthat 1 pg of MRNA-1273is sufficient to induce
`robust pseudovirus-neutralizing activity and CD8T cell responses,
`balanced T,,1-T,,2 antibody isotype responses,and protection from
`viral replication for more than three monthsfollowing a prime-
`boost regimen similar to the one being tested in humans.Thelevel of
`pseudovirus-neutralizing activity induced by 14g MRNA-1273in miceis
`similar in magnitudeto that induced by 100 pg mRNA-1273 inhumans*,
`whichis the dose selected for mRNA-1273 to advanceinto phaseIII
`clinical trials. The inclusion oflower subprotective doses demonstrates
`the dose-dependenceofantibody,T,,1 CD4T cell responses andprotec-
`tion, suggesting that immune correlates of protection can be further
`elucidated. Animal studies supporting candidate SARS-CoV-2 vaccines
`throughclinicaltrials aim to demonstrateelicitation of potent protec-
`tive immune responsesas well as to show that subprotective responses
`do not cause VAERD*. Subprotective doses ofmRNA-1273did not prime
`mice for enhancedimmunopathology following challenge. Moreover,
`the induction of protective immunity following a single dose suggests
`single-dose administration of this vaccine could be considered in the
`outbreak setting. These data, combined with immunogenicity data
`from non-humanprimates and human participants of early phase I
`clinical trials, have been used to inform the dose and regimen ofmRNA-
`1273 in advanced clinical efficacytrials.
`The COVID-19 pandemic of2020is the widely predicted ‘pathogen X
`event’?“*, Here we providea paradigmfor rapid vaccine development.
`Combining structure-guided stabilization ofthe MERS-CoVS protein
`with a fast, scalable and safe mRNA-LNPvaccine platform has led toa
`generalizable vaccine solution for Betacoronavirus and acommercial
`mRNAvaccine delivery platform; these developments enabled a rapid
`response to the COVID-19 outbreak. This response demonstrates how
`new technology-driven concepts such as synthetic vaccinology can
`facilitate a vaccine development programmeinitiated on the basis of
`
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`Fig. 4|mRNA-1273 protects mice from upper- and lower-airway SARS-CoV-2
`infection. a,b, BALB/c) mice (n=10 per group) immunized at weeks 0 and3
`with 0.01 pg (green), 0.1 pg (blue) or1 pg (red) mRNA-1273 or PBS were
`challenged with SARS-CoV-2 MAfive weekspost-boost. c, Other groups were
`immunizedwith single dosesof0.1,1g (blue), 1g (red) or 10 pg (purple)
`mRNA-1273 and challenged 7 weeksafter immunization. Two daysafter
`challenge, at peakviral load, mouse lungs(a, c) and nasal turbinates (b) were
`collected from five mice per groupto measureviral titres. a—c, Dataare
`presented as GMT + geometrics.d. and dotted lines represent assay limits of
`detection. Group comparisons were made by Kruskal-Wallis ANOVA with
`Dunn’s multiple comparisonstest. **P< 0.01,***P< 0.001. d, At days 2and4
`after challenge, lung sections from 5 mice pergroup werestained with
`haematoxylin and eosin, and representative photomicrographs(original
`magnification x4 (scale bars, 600 pm) and 10 (scale bars, 300 um)as
`indicated) from each groupwith detectablevirusin lung are shown. Day 2lungs
`from PBS control mice demonstrated moderate-to-severe, predominantly
`neutrophilic inflammation present within and surrounding small bronchioles
`(arrowheads); alveolar capillaries were markedly expandedbyinfiltrating
`inflammatorycells. In the 0.01 pg two-dose group,inflammation was minimal
`to absent. Inthe 0.1,1g two-dose group,occasional areasofinflammation
`intimately associated with small airways (bronchioles) and adjacent
`vasculature (arrowheads) wereseen, primarily composed ofneutrophils. In the
`single-dose 0.1 1g group, there were mild patchy expansionsofalveolar septae
`by mononuclear and polymorphonuclear cells. At day 4, lungs from PBScontrol
`mice exhibited moderate-to-marked expansionofalveolar septae(interstitial
`pattern) with decreased prominenceofadjacentalveolarspaces.Inthe 0.01pg
`two-dose group,inflammation was minimalto absent. Lungsin the 0.1p1g
`two-dose group showedmild, predominantly lymphocytic inflammation,
`associated with bronchioles and adjacentvasculature (arrowheads).In the
`single-dose 0.1 1g group there wasmild, predominantly lymphocytic
`inflammation around bronchovascular bundles (arrowheads).
`
`SARS-CoV-2 S(2P) protein formulated in alum (Extended Data Fig.8a, b).
`Following restimulation with peptide pools (one poolof overlapping
`peptides for each S subunit, S1 and S2) covering the entire S protein,
`splenocytes from mice immunized with mRNA-1273 secreted more
`IFN-y (a prototypic T,,1 cytokine) than IL-4,IL-5 or IL-13 (classical T,,2
`cytokines), whereas restimulation with SARS-CoV-2S(2P) protein with
`alum adjuvant induceda T,,2-biased response (Extended DataFig.8c, d).
`
`570 | Nature | Vol 586 | 22 October 2020
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`@ TwodosesmRNA-1273
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`pathogen sequencesalone". This study also providesa proofofconcept
`forthe prototype-pathogen approachto pandemic preparedness and
`responsethatis predicated on identifying generalizable solutionsfor
`medical counter measures within virus families or genera”, Although
`the response to the COVID-19 pandemic has been unprecedented in
`its speed and breadth,we envision further improvementsin rapid
`responsesto suchthreats. There are 24 othervirus families that are
`knownto infect humans, and sustained investigation ofthose potential
`threatswill improve our readiness for future pandemics“.
`
`Onlinecontent
`
`Any methods,additional references, Nature Research reporting sum-
`maries, source data, extended data, supplementary information,
`acknowledgements, peer review information;details of author con-
`tributions and competing interests; and statements of data and code
`availability are available at https://doi.org/10.1038/s41586-020-2622-0.
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`© This is a U.S. government work and not under copyright protectionin the U.S.; foreign
`copyright protection may apply 2020
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`Article
`
`with 10% FBS, 2mM glutamine and 1% penicillin-streptomycin at 37 °C
`and 5% CO,. Vero E6cells used in plaque assays to determine lung and
`nasal turbinate viraltitres were cultured in DMEM supplemented with
`10% Fetal Clone Il and 1% antibiotic-antimycotic at 37 °C and 5% CO2.
`Vero E6 cells used in plaque-reduction neutralization test (PRNT)
`assays were cultured in DMEM supplemented with 10% Fetal CloneII
`and amphotericin B (0.25 pg mI“) at 37 °C and 5% CO,. Lentivirus encod-
`ing hACE2-P2A-TMPRSS2 was madeto generate A549-hACE2-TMPRSS2
`cells, which were maintained in DMEM supplemented with 10% FBS
`and 1 pg mI“ puromycin. Expi293 cells were maintained in the manu-
`facturer’s suggested medium. BHK-21/WI-2 cells were obtained from
`Kerafast and cultured in DMEM with 5% FBS at 37 °C and 6-8% CO.,Cell
`lines were not authenticated.All cells lines were tested for mycoplasma
`and remained negative.
`
`In vitro mRNAexpression
`HEK293Tcells were transiently transfected with mRNA encoding
`SARS-CoV-2 wild-type S or S(2P) protein using a TranIT mRNAtrans-
`fection kit (Mirus). After 24 h, the cells were collected and resuspended
`in fluorescence-activated cell sorting (FACS) buffer (1x PBS, 3% FBS,
`0.05% sodium azide). To detect surface-protein expression,the cells
`were stained with 10 pg ml" ACE2-Flag (Sigma) or 10 pg ml’ CR3022
`in FACS bufferfor 30 min onice. Thereaft