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SUPPLEMENTARY APPENDIX Comparison of the Safety and Immunogenicity of a Novel Matrix-M-adjuvanted Nanoparticle Influenza Vaccine with a Quadrivalent Seasonal Influenza Vaccine in Older Adults: A Randomized Controlled Trial Corresponding Author: Vivek Shinde, MD Contents TOC \o "1-3" \h \z \u Contents PAGEREF _Toc47648487 \h 11Methods PAGEREF _Toc47648488 \h 31.1Eligibility Criteria PAGEREF _Toc47648489 \h 31.2Randomization and Stratification PAGEREF _Toc47648490 \h 31.3Blinding PAGEREF _Toc47648491 \h 31.4Ethics, Trial Registration, and Funding PAGEREF _Toc47648492 \h 31.5Trial Investigational Products PAGEREF _Toc47648493 \h 41.5.1qNIV PAGEREF _Toc47648494 \h 41.5.2Matrix-M PAGEREF _Toc47648495 \h 41.5.3Fluzone? Quadrivalent – Active Comparator PAGEREF _Toc47648496 \h 41.6Trial Objectives and Endpoints PAGEREF _Toc47648497 \h 41.7Assays for Assessment of Antibody and Cellular Immune Responses PAGEREF _Toc47648498 \h 51.7.1Antibody Responses Assessed by Hemagglutination Inhibition PAGEREF _Toc47648499 \h 51.7.2Antibody Responses Assessed by Hemagglutination Inhibition PAGEREF _Toc47648500 \h 61.7.3Cellular Immune Responses Assessed by Polyfunctional CD4+ T-Cell Responses With Intracellular Cytokine Staining (ICCS) Analysis PAGEREF _Toc47648501 \h 61.8Statistics PAGEREF _Toc47648502 \h 61.8.1Analysis Populations, Definitions PAGEREF _Toc47648503 \h 61.8.2Sample Size Justification PAGEREF _Toc47648504 \h 72Results PAGEREF _Toc47648505 \h 83References PAGEREF _Toc47648506 \h 32List of Figures TOC \h \z \c "Figure S" Figure S1. H3N2 phylogenetic tree and HAI testing plan for phase 3 PAGEREF _Toc47648507 \h 8Figure S2. Box plots for log10 scale counts of specific cytokine-expressing CD4+ effector T-cells against A/Kansas PAGEREF _Toc47648508 \h 11Figure S3. Box plots for log10 scale counts of specific cytokine-expressing CD4+ effector T-cells against B/Maryland PAGEREF _Toc47648509 \h 12Figure S4. RCD plots for proportion of specific cytokine-expressing CD4+ effector T-cells against A/Kansas PAGEREF _Toc47648510 \h 15Figure S5. RCD plots for proportion of specific cytokine-expressing CD4 + effector T-cells against B/Maryland PAGEREF _Toc47648511 \h 16Figure S6. Box plots for log10 scale counts of specific cytokine-expressing CD4+ total T-cells against A/Kansas PAGEREF _Toc47648512 \h 17Figure S7. Box plots for log10 scale counts of specific cytokine-expressing CD4+ total T-cells against B/Maryland PAGEREF _Toc47648513 \h 19Figure S8. RCD plots for proportion of specific cytokine-expressing CD4+ total T-cells against A/Kansas PAGEREF _Toc47648514 \h 21Figure S9. RCD plots for proportion of specific cytokine-expressing CD4+ total T-cells against B/Maryland PAGEREF _Toc47648515 \h 23Figure S10. Day 28 microneutralization baseline-adjusted ratio of GMTs using egg-adapted versus wild-type A/Singapore H3N2 from phase 2 trial16 PAGEREF _Toc47648516 \h 29Figure S11. Spaghetti plots representing CMI polyfunctional effector and total CD4+ T-cell responses by treatment group against A/Kansas and B/Maryland PAGEREF _Toc47648517 \h 30List of Tables TOC \h \z \c "Table S" Table S1. Mean concentrations of CD4+ effector and total T-cells, Day 7 fold-rise, and ratio of mean concentrations by treatment groups PAGEREF _Toc47648518 \h 9Table S2. CMI responses compared to other “enhanced” flu vaccines for older adults in terms of total CD4+: Day 7 GMFRs(post/pre) [E301 and Cowling et al. 2019] PAGEREF _Toc47648519 \h 25Table S3. Any and severe solicited local and systemic AEs through Day 28 PAGEREF _Toc47648520 \h 26Table S4. Comparison of safety data from sponsor’s phase 2 and 3 trial with IIV3-HD (Fluzone? High Dose [HD]) and IIV4 (Fluzone? Quadrivalent) PAGEREF _Toc47648521 \h 27MethodsEligibility CriteriaParticipants were adult male or females ≥65 years of age and clinically stable as assessed by 1) ambulatory status, defined as living independently in the community or in a residential facility providing minimal assistance (eg, meal preparation and transport); 2) absence of changes in medical therapy within 1 month due to treatment failure or toxicity; 3) absence of medical events qualifying as serious adverse events (SAEs) within the prior 2 months; and 4) absence of known, current, and life-limiting diagnoses that rendered survival to completion of the protocol unlikely in the opinion of the investigator. Participants were willing and able to give informed consent prior to trial enrollment, and were able to attend trial visits, comply with trial requirements, and provide timely, reliable, and complete reports of adverse events (AEs).Exclusionary criteria included participation in research involving investigational product (drug/biologic/device) within 45 days before planned date of study vaccination, or in any previous Novavax influenza vaccine clinical trial(s); history of a serious reaction to prior influenza vaccination, known allergy to constituents of Fluzone? Quadrivalent or polysorbate?80; history of Guillain-Barré syndrome (GBS) within 6 weeks following a previous influenza vaccine; receipt of any vaccine in the 4 weeks preceding the trial vaccination and any influenza vaccine within 6?months preceding the trial vaccination; presence of any known or suspected immunosuppressive illness, congenital or acquired, based on medical history and/or physical examination; chronic administration (defined as more than 14 continuous days) of immunosuppressants or other immune-modifying drugs within 6 months prior to the administration of the trial vaccine (an immunosuppressant dose of glucocorticoid was defined as a systemic dose ≥10 mg of prednisone per day or equivalent. The use of topical, inhaled, and nasal glucocorticoids was permitted); administration of immunoglobulins and/or any blood products within the 3 months preceding the administration of the trial vaccine; acute disease at the time of enrollment (defined as the presence of a moderate or severe illness with or without fever, or an oral temperature ≥38.0°C, on the planned day of vaccine administration); any condition that in the opinion of the investigator would pose a health risk to the participant if enrolled or could interfere with evaluation of the vaccine or interpretation of trial results (including neurologic or psychiatric conditions deemed likely to impair the quality of safety reporting); known disturbance of coagulation; and suspicion or recent history (within 1 year of planned vaccination) of alcohol or other substance abuse.Randomization and StratificationParticipant randomization was conducted using an interactive web randomization system (IWRS). Stratification was by age, and history of receipt of the 2018-2019 influenza vaccine.Blinding All participants were assigned an identification number using an electronic data capture (EDC) system. Treatment assignments were known only to the responsible unblinded vaccine administrators at the trial center, who referred to the randomization schedule for the correct treatment assignment to each participant. These persons, identified prior to trial dosing, did not perform any trial assessments post-dosing. Participants and clinical site staff other than vaccine administrators remained blinded for the duration of the trial. Safety follow-up continues at the time of this summary; therefore, sponsor clinical personnel have been provided unblinded data at the treatment group level only and subjects, clinical site staff, and the clinical trial monitoring staff remain fully blinded as to participant treatment assignments. Ethics, Trial Registration, and FundingThe trial was reviewed by the Advarra Institutional Review Board (Columbia, Maryland, USA) and conducted in accordance with Good Clinical Practices based on the Declaration of Helsinki and International Conference on Harmonisation guidelines. All participants provided informed consent. The trial was funded by the sponsor, Novavax, Inc. (Gaithersburg, MD, USA). The trial is registered at (NCT04120194).Trial Investigational ProductsqNIVThe quadrivalent nanoparticle influenza vaccine (qNIV) based on purified, recombinant full-length hemagglutinin (HA) protein that self-assembles into distinct HA nanoparticle structures of approximately 20 to 40 nm.PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5TbWl0aDwvQXV0aG9yPjxZZWFyPjIwMTc8L1llYXI+PFJl

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ADDIN EN.CITE.DATA 1,2 The baculovirus/Spodoptera frugiperda (Sf9) insect cell system was used to clone and express recombinant influenza HAs from the influenza strains recommended for the 2019-2020 Northern Hemisphere influenza season: A/Brisbane/02/2018 (H1N1) pdm09; A/Kansas/14/2017?(H3N2); B/Maryland/15/2016 (Victoria lineage); B/Phuket/3073/2013 (Yamagata lineage), ADDIN EN.CITE <EndNote><Cite><Author>WHO</Author><Year>2016</Year><RecNum>9</RecNum><DisplayText><style face="superscript">3</style></DisplayText><record><rec-number>9</rec-number><foreign-keys><key app="EN" db-id="fzfwwa9vsspeexevpxnxaasddzrzewww5stz" timestamp="1499352193">9</key></foreign-keys><ref-type name="Web Page">12</ref-type><contributors><authors><author>WHO </author></authors></contributors><titles><title>Recommended composition of influenza virus vaccines for use in the 2016-17 northern hemisphere influenza season </title></titles><volume>2016</volume><number>July 6 </number><dates><year>2016</year></dates><publisher>World Health Organization </publisher><urls></urls></record></Cite></EndNote>3,4 using wild-type virus HA gene sequences. Accession numbers are provided below: A/Brisbane/02/2018 (H1N1) pdm09; EPI1312884 A/Kansas/14/2017?(H3N2) EPI1146345B/Maryland/15/2016 EPI892419 (Victoria lineage)B/Phuket/3073/2013 EPI608074 (Yamagata lineage)??Matrix-MMatrix-M is a saponin-based adjuvant, which is co-administered with an antigen to induce an enhanced immune response. The mechanism of Matrix-M is not fully defined, but it has been associated with a potent induction of leukocyte activation and migration into the draining lymph nodes. Even though there is no clear evidence of a specific pattern-recognition receptor being stimulated by Matrix-M, it has been shown to induce a strong T-cell response in multiple virus vaccine models.Matrix-M is manufactured by mixing defined, partially purified extracts of the bark of the Quillaja saponaria Molina tree, termed Fraction-A and Fraction-C, with cholesterol and phosphatidylcholine in the presence of a detergent.5 qNIV drug product formulated with Matrix-M adjuvant was filled directly in 0.75 mL volumes into 2.0 mL glass vials at appropriate concentrations. The buffer composition was 25?mM sodium phosphate, 150 mM sodium chloride, 100 mM arginine hydrochloride, 5% w/v trehalose, and 0.03% PS80, pH 7.5. The final antigen and adjuvant co-formulated drug product was filled into 2R single-use glass vials. Fluzone? Quadrivalent – Active ComparatorThe active comparator in the trial was the 2019-2020 Fluzone Quadrivalent (Sanofi Pasteur). According to the manufacturer’s label, Fluzone contained 15 ?g HA per 4 strains. Fluzone was administered based on manufacturer’s instructions.6Trial Objectives and Endpoints The primary objectives of the trial were: a) to demonstrate the non-inferior immunogenicity of 240 ?g qNIV co-formulated with 75??g Matrix-M1 adjuvant, relative to a US-licensed comparator, IIV4, in clinically stable adults ≥65 years of age, in terms of hemagglutination inhibition (HAI) (assayed with egg-propagated virus) antibody responses to all qNIV homologous influenza strains (ie, 2 influenza A and 2 influenza B strains) at Day 28 post-vaccination and b) to describe the safety profile of 240 ?g qNIV co-formulated with 75 ?g Matrix-M1 adjuvant, and the comparator in clinically stable adults ≥65 years of age. The safety profile included solicited shortterm reactogenicity; 28-day all AE profile; 1-year post-injection medically attended AE (MAE), SAE, and significant new medical condition (SNMC), including immunologically mediated AEsof special interest (AESIs).The secondary objective of the trial was to describe the immunogenicity of 240 ?g qNIV, co-formulated with 75 ?g Matrix-M1 adjuvant, and of a US-licensed comparator, IIV4, in clinically stable adults?≥65 years of age, in terms of HAI (assayed with both egg-propagated virus and wildtype virus-like particle [VLP] reagents) antibody responses to all qNIV homologous influenza strains (2 influenza A and 2 influenza B strains) and at least 1 antigenically drifted A or B strain in terms of geometric mean titers (GMTs), geometric mean ratio (GMR), seroconversion rate (SCR), seroprotection rate (SPR), and the baseline adjusted ratio of GMTs (GMTR) between treatment arms at Day 28.The exploratory cell-mediated immune (CMI) objective of the trial was to describe the quality and amplitude of CMI responses in healthy adults ≥65 years of age to vaccination with 240 ?g qNIV co-formulated with 75 ?g Matrix-M1 adjuvant, as measured by functional T-cell responses based on intracellular cytokine analysis. Additional markers of CMI (eg, memory B-cells and/or other T-cell subsets) may be evaluated depending on availability/recovery of cell volume. Due to the laborious nature of the cellular assays, they will be performed on subjects from a limited number of participating sites, and results may be reported as an addendum to the main clinical study report. The primary endpoints of the trial were:Comparative HAI antibody responses (assayed with egg-propagated virus) on Day 28, summarized in terms of the ratio of GMTs AND SCR difference between subjects receiving qNIV or IIV4 for all 4 vaccine-homologous influenza strains (ie, 2 influenza A and 2 influenza B strains). Non-inferiority for each homologous strain was demonstrated if:The lower bound of the 2-sided 95% confidence interval (CI) on the ratio of the GMTs (GMTqNIV/GMTIIV4) is ≥ 0.67 ANDThe lower bound of the 2-sided 95% CI on the difference between the SCRs (SCRqNIV-SCRIIV4) is ≥-10% Number and percentage (95% CI) of subjects with solicited local and systemic AEs over the 7?days post-injection (ie, Day 0 through Day 6, inclusive); all AEs through 28 days post-injection (ie, Day?0 through Day 27, inclusive); and MAEs, SAEs, and SNMCs – including AESIs – through 1?year post-injection. The secondary endpoints of the trial were HAI antibody titers (assayed with both egg-propagated virus and wild-type VLP reagents) at Day 0 and Day 28 specific for vaccine-homologous A and B strain(s), and antigenically drifted influenza strains. Derived/calculated endpoints based on these data included: GMT – defined as the antilog of the mean of the log-transformed HAI titers on Days 0 and?28 GMR – defined as the ratio of post-vaccination to prevaccination (Day 0) HAI GMTs (GMRPost/Pre) on Day 28 SCR – defined as proportion of subjects in a given treatment group with either a baseline reciprocal (Day 0) titer of <10 and a post-vaccination reciprocal titer?≥40, or a baseline reciprocal (Day 0) titer of ≥10 and a post-vaccination titer ≥4-fold higher on Day 28 SPR – defined as the proportion of subjects with a reciprocal HAI titer?≥40 on Day 28 Ratio of GMTs between treatment arms at Day 28 post-vaccination (adjusted for intergroup variation in baseline [pre-vaccination] titers)Exploratory CMI endpoints were counts and/or proportions of Days 0, 7, 28, and 182 peripheral blood effector memory T-cell populations that secrete 1 or more of IL-2, CD40L, IFN-γ, and TNF-α cytokines following in vitro re-stimulation with HA in subjects selected for cellular immune response monitoring. Assays for Assessment of Antibody and Cellular Immune ResponsesAntibody Responses Assessed by Hemagglutination InhibitionAssay Using Egg-propagated Reagents (Egg-based HAI)Briefly, Day 0 and Day 28 human sera treated with receptor destroying enzyme (RDE; Denka Seiken Co, Campbell, CA) overnight at 37?C to remove non-specific inhibitors of hemagglutination, heat inactivated at 56?C for 30 min, diluted to 1:10 with Dulbecco's phosphate-buffered saline (DPBS), plated into microtiter wells, followed by a series of 2-fold dilutions of serum for a total of 10 dilutions. Four HA units of egg-based virus in dilution buffer containing 80 nM of Oseltamivir and indicator 0.75% human red blood cell (RBC) suspension were added to designated wells in 2 sequential steps, with mixing and incubation at each step in round-bottom 96-well plates. HAI antibody titers were determined as the reciprocal of the highest dilution of serum that completely inhibited hemagglutination. The serum HAI titer was calculated from the GMT of duplicate test results. Positive and negative serum samples were included in each assay run to serve as assay quality controls. Plates were read on Cypher One Haemagglutination Analyzer (InDevR, Inc., Boulder, CO) using software to capture plate images.Antibody Responses Assessed by Hemagglutination InhibitionAssay Using Wild-type VLP Reagents (wt-HAI)Due to the documented inability of recent A(H3N2) strains to agglutinate avian or smallmammal RBC reagents in HAI assays as a consequence of evolutionary changes in contemporary viral HA preference for binding to specific sialic acid receptors present on human cells, and, in addition, the presence of immunologically significant mutations induced by egg passage in the HA of these strains, vaccine immunogenicity was assessed by the classical HAI qualified method, described in Section 1.7.1, but adapted with 140-190 nm recombinant wild-type hemagglutinin virus-like particles (wt-HA-VLPs), reflecting the amino acid sequence of circulating virus, as the agglutinating agent and human type-O RBCs(Biological Specialty Corporation, Colmar, PA) as the agglutination target in order to restore assessment of HAI antibody activity.2,7,15 Wt-HA-VLPs were constructed with wild-type HA, neuraminidase (NA), and M1 genes and produced inbaculovirus/Sf9 cell expression system. Cellular Immune Responses Assessed by Polyfunctional CD4+ T-Cell Responses With Intracellular Cytokine Staining (ICCS) Analysis IFN-r, TNF-a, and IL-2 were measured because they are classical type I cytokines, and CD40L is a marker for activated T-cells.8,9 These are well-established markers for measuring antigen-specific T-cell response. T-cells that simultaneously produce 2 or more of these cytokines are defined as polyfunctional T-cells.10 The number of polyfunctional T-cells has been shown to be associated with better protective effector activity and immunologic memory.11,12,13Briefly, human peripheral blood mononuclear cells (PBMCs) were cultured overnight after thawing. Cells with a viability >85% proceeded to the following assays. Human PBMCs were cultured in 96-well U-bottom plates at a density of 1-2 x 106 cells/well and treated with the HA protein (A/Kansas/14/2017 [H3N2] or B/Maryland/15/2016 [Victoria lineage]; 5 ?g/mL), PMA + ionomycin (a positive control for T-cell activation), or medium only (negative control). After incubation at 37°C for 6 hours in the presence of BD GolgiPlug? and BDGolgiStop? (BD Biosciences, San Jose, CA) for the last 4 hours of culturing, cells were labeled for surface markers (CD3, CD4, CD8, CD45RA, and CCR7 [BD Biosciences]) and the LIVE/DEAD indicator dye (Life Technologies/Thermo Fisher Scientific, Waltham, MA) was added. The intracellular cytokines/ligand were detected by antibodies specific for IFN-γ, TNF-α, IL-2, and CD40L [BD Biosciences]. The samples were processed using a LSR-Fortessa flow cytometer (Becton Dickinson, San Jose, CA). Data were analyzed using Flowjo software version Xv10 (Tree Star Inc., Ashland, OR). Data shown were gated on effector (including effector memory) CD4+ T-cell population (CD45RA-CCR7-), or total CD4+ T-cell population.Statistics Analysis Populations, DefinitionsThe safety population included all trial participants that provided consent, were randomized, and received the test article. The safety population was used for all safety analyses and was analyzed as actually treated. The per protocol (PP) population was the primary population for immunogenicity analysis and included randomized participants who received the assigned dose of the test article according to the protocol, had HAI serology results at Day 0 and Day 28, and had no major protocol deviations. The intent-to-treat (ITT) population included all participants in the safety population that provided any HAI serology data.HAI antibody responses were summarized as GMTs, defined as the antilog of the mean of the log-transformed HAI titers on Day 0 and Day 28 and as SCRs, defined as proportion of participants in a given treatment group with either a baseline reciprocal (Day 0) titer of <10 and a post-vaccination reciprocal titer ≥40, or a baseline reciprocal (Day 0) titer of ≥10 and a post-vaccination titer ≥4-fold higher on Day 28.The immunologic non-inferiority of qNIV to IIV4 was demonstrated if the lower bound of the 2-sided 95% CI of the Day 28 post-vaccination ratio of the GMTs between the 2 treatment groups (GMTRqNIV/ IIV4) was ≥0.67 and the SCR difference success criterion required that the lower bound of the 2-sided 95% CI of the difference in SCR between the 2 groups at Day 28 (SCRqNIV?SCRIIV4) was ≥-10%. Two-sided 95% CIs for Day 28 post-vaccination GMTRqNIV/ IIV4 were based on a log normal distribution. The log10 values were used to construct a CI using the analysis of covariance (ANCOVA) with treatment group and baseline at Day 0 (adjusted for intergroup variation in baseline [prevaccination] titers) as the covariates under 2-sided type I error rate of 0.05. No type I error rate adjustments were made. The mean difference and the corresponding CI limits were exponentiated to obtain the GMTR and the corresponding CI. Two-sided 95% CIs for the difference of the SCRs between qNIV and IIV4 were based on the Newcombe hybrid score (METHOD = SCORE riskdiffoption for PROC FREQ in SAS).CD4+ T-cells responding to in vitro stimulation with influenza HA antigens (separately with A/Kansas and B/Maryland) by expressing IFN-γ either as a single marker or as polyfunctional arrays of at least 2, 3, or 4 cytokines/activation markers consisting of: IFN-γ, tumor necrosis factor alpha (TNF-α), interleukin-2 (IL-2), or CD40L were reported as median counts and geometric mean counts (GMCs) per million cells. Geometric mean fold-rises (GMFRspost/pre) in counts on post-vaccination Day 7 relative to pre-vaccination Day 0 within treatment group and baseline-adjusted GMCs ratio (GMCRqNIV/IIV4) at Day 7 ratio between treatment groups were calculated. Sample Size Justification This trial had a single comparison between qNIV and IIV4 standard deviation (SD) at Day 28 post-vaccination for the primary immunogenicity objective. No adjustment of the type I error rate for multiple comparisons was warranted since the simultaneous successes of all 8 comparisons planned for the 4 strains contained in the vaccines and the 2 endpoints (GMT and SCR) were required for the demonstration of the primary non-inferiority objective of the study. Non-inferiority was defined as the lower bound of the 2-sided 95% CI on the ratio of GMTs (GMTRqNIV/ IIV4) ≥0.67 (ie, unadjusted 1-sided p-value <0.025 against the null hypothesis of H0: GMTRqNIV/ IIV4 <0.67) and the lower bound of the 2-sided 95% CI on the difference of the SCRs (qNIV?IIV4 SD) ≥-10% (ie, unadjusted 1-sided p-value <0.025 against the null hypothesis of H0: difference of SCRs <-10%). For the calculation of sample size estimation, we used assumptions of true differences, ie, the ratio of GMTs (GMTRqNIV/ IIV4) was 1.0 for all 4 homologous strains, and the difference of the SCRs was -2% for each of the 4 homologous strains, based on the average performance in the previous phase 2 trial (qNIV-E-201). To achieve an overall 90% power (ie, ~97.5% power for each of the 4 strains) to demonstrate a non-inferiority margin for SCR difference of -10%, the SCR of reference group (IIV4 SD) was assumed to be 0.5 and the SCR of treatment group (qNIV) was assumed to be 0.48 under the null hypothesis of inferiority (H0: difference of SCRs <-10%). The significance level of the testing was 0.025. Sample size was estimated to be 1195 in each group by assuming equal size in each group. The sample size accounted for a 10% attrition rate for the PP population such that the primary analysis population for all immunogenicity endpoints would be 1325 in each group.The calculated power for each strain, unadjusted for multiple comparisons, was close to 100% to detect a 1.5-fold difference in GMTs. For 4 strains, this study was designed to exclude a GMTRqNIV/ IIV4 of <0.67 with 100% power. Therefore, for demonstrating non-inferiority for 4 strains with the true GMTRqNIV/ IIV4 of 1.0 and the true SCR differences of -2%, this study provided ≥90% overall power.For safety endpoints, the probability of observing at least 1 AE among 1325 qNIV participants was >90% if the true rate of such events was 0.18%. Observing no AEs of interest (eg, vaccine-related SAE) would represent an upper bound of the 1-sided 95% CI on the percentage of such event of 0.2%.Results Figure S SEQ Figure_S \* ARABIC 1. H3N2 phylogenetic tree and HAI testing plan for phase 3Six heterologous, genetically and/or antigenically distinct A(H3N2) strains were selected from across the phylogenetic tree representing multiple distinct clades and subclades. Table S SEQ Table_S \* ARABIC 1. Mean concentrations of CD4+ effector and total T-cells, Day 7 fold-rise, and ratio of mean concentrations by treatment groupsInfluenza Virus Strain A/KansasB/MarylandCytokine ExpressionDouble+Triple+Quadruple +IFN-γ+Double+Triple+Quadruple +IFN-γ+TreatmentqNIV CD4+Effector Cells VariableN = 66 Day 0 GMC (95% CI)763.5(605.8-962.2)479.1 (365.9-627.2)188.0(146.5-241.2)342.7 (273.8- 428.9)353.6(284.4-439.7)222.4(178.9-276.6)93.4(75.9-115.0)212.9(172.5- 262.7)N = 68 Day 7 GMC (95% CI)2487.6(2000.0-3094.0)1706.8(1324.7-2199.2)751.7(580.7-973.1)1601.3 (1274.4-2012.0)1668.6(1343.0-2073.2)971.8(769.8-1226.8)357.7(275.6-464.4)1364.7 (1090.9- 1707.3)Day 7 GMFR(post/pre) (95% CI)p-value 3.1(2.5-3.8)<0.0013.4(2.6-4.4)<0.0013.9(3.0-5.1)<0.0014.6 (3.6-5.8)<0.0014.9(3.8-6.3)<0.0014.5(3.5-5.9)<0.0014.0(3.1-5.1)<0.0016.7(5.2-8.5)<0.001IIV4 N = 68 Day 0 GMC (95% CI)747.0 (611.7-912.2)493.3(400.0-608.3)189.4(148.6-241.3)354.8 (292.1- 430.9)371.3(299.8-460.0)237.3(187.4-300.6)108.4(85.2-137.9)219.6 (173.9- 277.3)N = 67 Day 7 GMC (95% CI)992.2(783.4-1256.6)656.1(510.6-843.1)257.5(195.9-338.3)550.6 (430.9- 703.5)740.8(598.3-917.2)459.4(365.9-576.9)183.5(141.8-237.4)578.4 (468.1- 714.6)Day 7 GMFR(post/pre) (95% CI)p-value 1.3(1.1-1.6)0.0071.3(1.1-1.6)0.0101.4(1.1-1.7)0.0051.6 (1.3-1.9)<0.0012.0(1.6-2.4)<0.0011.9(1.5-2.3)<0.0011.7 (1.4-2.0)<0.0012.6 (2.1-3.3)<0.001Day 7 Baseline adjusted ratio of GMCR(qNIV/IIV4) (95% CI)p-value2.41 (1.84-3.14)<0.001 2.60(1.92-3.51)<0.0012.89(2.12-3.94)<0.0012.93 (2.21-3.88)<0.0012.41(1.85-3.16)<0.0012.30(1.73-3.05)<0.0012.26(1.66-3.07)<0.0012.49 (1.88-3.29)<0.001Total CD4+ CellsqNIVN = 66 Day 0 GMC (95% CI)678.7(555.6-829.2)412.8(326.1-522.6)159.1(126.6-200.0)280.6 (226.4- 347.7)246.3(202.9-299.1)156.7(125.4-195.9)69.9(56.0-87.2)135.2 (106.8-171.3)N = 68 Day 7 GMC (95% CI)2189.5(1796.6-2668.4)1579.2(1280.8-1947.0)709.2(562.6-893.9)1425.8(1161.2- 1750.7)1214.6(982.9-1500.9)770.3(618.6-959.2)305.5(239.1-390.4)990.6(796.2-1232.5)Day 7 GMFR(post/pre) (95% CI)p-value 3.1(2.6-3.8)<0.0013.7(2.9-4.7)<0.0014.4(3.4-5.6)<0.0015.1 (3.9-6.5)<0.0015.2(4.2-6.5)<0.0015.2(4.1-6.7)<0.0014.7(3.7-6.0)<0.0017.9 (6.1-10.2)<0.001IIV4 N = 68 Day 0 GMC (95% CI)651.9(541.0-785.5)427.0(355.5-512.8)159.5(126.9-200.4)298.6 (247.9- 359.6)261.1(205.5-331.8)180.2(142.2-228.5)84.9(66.2-108.9)160.3(123.4-208.1)N = 67 Day 7 GMC (95% CI)872.9(698.6-1090.8)570.1(447.6-726.2)239.8(185.8-309.5)464.1 (364.2- 591.4)535.2(430.5-665.3)352.3(283.3-438.2)147.6(115.2-189.2)407.9 (321.9-516.9)Day 7 GMFR(post/pre) (95% CI)p-value 1.4(1.1-1.6)0.0031.3(1.1-1.6)0.0041.5(1.2-1.9)<0.0011.6 (1.3-1.9)<0.0012.1(1.7-2.5)<0.0011.9(1.6-2.4)<0.0011.7(1.4-2.1)<0.0012.5 (2.0-3.3)<0.001Day 7 Baseline adjusted ratio of GMCR(qNIV/IIV4) (95% CI)p-value 2.41(1.86-3.12)<0.0012.78(2.09-3.69)<0.0012.94(2.19-3.94)<0.0013.17(2.36-4.26)<0.0012.47(1.91-3.20)<0.0012.49(1.91-3.25)<0.0012.53(1.90-3.35)<0.0012.78(2.09-3.71)<0.001Abbreviations: CI, confidence interval; GMC, geometric mean count; GMCR(qNIV/IIV4), geometric mean count ratio between qNIV and IIV4, GMFR(post/pre), geometric mean fold-rise of GMCs post vs pre vaccination. Double+/Triple+/Quadruple+: at least 2/3/all of CD40L+, IFN-γ+, TNF-α+, and IL-2+, respectively. Cell-mediated immune (CMI) response endpoints were performed on a subset of approximately 140 participants from several pre-designated clinical sites. GMC was defined as the antilog of the mean of the log-transformed counts for a given treatment group and time-point. For CD4+ effector T-cells, the lower limit of quantitation (LLoQ) was set as 70. If cytokine was <70, log10 scale of cytokines counts was recorded as log10 scale half LLoQ, which is 35. For CD4 + total T-cells, LLoQ was set as 40. If cytokine was <40, log10 scale of cytokines counts was recorded as log10 scale half LLoQ, which is 20. For IFN-Y, LLoQ was set as 110 for effector CD4+ T-cells, and 30 for total CD4+ T-cells. Thus, if cytokines count was <LLoQ, log10 scale of cytokines count was recorded as log10 scale half LLoQ, which is 55 and 15, respectively.GMFR(post/pre) was defined as the ratio of 2 GMCs within treatment group at 2 different time-points (ie, between Day 7 and Day 0). 95% CI and p-value were obtained by paired t-test of GMR=1. Baseline-adjusted GMCR(qNIV/IIV4) was defined as the ratio of 2 GMCs for a comparison of 2 specified treatment groups at the specified time-points. A mixed-effects model with treatment group and baseline counts of covariates was performed. The ratios of geometric least square (LS) means and 95% CI for the ratios were calculated by back transforming the mean differences and 95% confidence limits for the differences of log (base 10) transformed total counts of triple cytokine or double cytokine between 2 specified treatment groups. Figure S SEQ Figure_S \* ARABIC 2. Box plots for log10 scale counts of specific cytokine-expressing CD4+ effector T-cells against A/KansasCell-mediated immune (CMI) responses were measured by intracellular cytokine staining (ICCS). Counts of peripheral blood CD4+ T-cells expressing IL-2, IFN-γ, TNF-α, and/or CD40L+ cytokines were measured following in vitro re-stimulation with A/Kansas-specific recombinant wild-type sequence hemagglutinins (HAs).Box plots are shown for counts of cells per 106 producing IFN-γ, TNF-α, IL-2, and/or CD40L+, or combinations thereof. Responses were evaluated using peripheral blood mononuclear cells (PBMCs) obtained from a subgroup of participants on Day 0 (pre-vaccination) and Day 7. The box plots represent the interquartile range (±3 standard deviations); the solid horizontal black line represents the median and the number indicates the median count of CD4+ effector T-cells expressing a particular combination of markers, and the open diamond represents the mean. Figure S SEQ Figure_S \* ARABIC 3. Box plots for log10 scale counts of specific cytokine-expressing CD4+ effector T-cells against B/MarylandCell-mediated immune (CMI) responses were measured by intracellular cytokine staining (ICCS). Counts of peripheral blood CD4+ T-cells expressing IL-2, IFN-γ, TNF-α, and/or CD40L+ cytokines were measured following in vitro re-stimulation with B/Maryland-specific recombinant wild-type sequence hemagglutinins (HAs).Box plots are shown for counts of cells per 106 producing IFN-γ, TNF-α, IL-2, and/or CD40L+, or combinations thereof. Responses were evaluated using peripheral blood mononuclear cells (PBMCs) obtained from a subgroup of participants on Day 0 (pre-vaccination) and Day 7. The box plots represent the interquartile range (±3 standard deviations); the solid horizontal black line represents the median and the number indicates the median count of CD4+ effector T-cells expressing a particular combination of markers, and the open diamond represents the mean.Figure S SEQ Figure_S \* ARABIC 4. RCD plots for proportion of specific cytokine-expressing CD4+ effector T-cells against A/Kansas201041038100IFN-γ+0IFN-γ+161290010795Triple Cytokine+0Triple Cytokine+178181039370Double Cytokine+00Double Cytokine+163195048895Quadruple Cytokine+Quadruple Cytokine+Figure S SEQ Figure_S \* ARABIC 5. RCD plots for proportion of specific cytokine-expressing CD4 + effector T-cells against B/Maryland213169520691IFN-γIFN-γ159575543180Triple Cytokine+Triple Cytokine+170587428575Double Cytokine+Double Cytokine+138620528575Quadruple Cytokine+Quadruple Cytokine+Figure S SEQ Figure_S \* ARABIC 6. Box plots for log10 scale counts of specific cytokine-expressing CD4+ total T-cells against A/KansasCell-mediated immune (CMI) responses were measured by intracellular cytokine staining (ICCS). Counts of peripheral blood CD4+ T-cells expressing IL-2, IFN-γ, TNF-α, and/or CD40L+ were measured following in vitro re-stimulation with A/Kansas-specific recombinant wild-type sequence hemagglutinins (Has). Box plots are shown for counts of cells per 106 producing IFN-γ, TNF-α, IL-2, and/or CD40L, or combinations thereof. Responses were evaluated using peripheral blood mononuclear cells (PBMCs) obtained from a subgroup of participants on Day 0 (pre-vaccination) and Day 7. The box plots represent the interquartile range (±3 standard deviations); the solid horizontal black line represents the median and the number indicates the median count of CD4+ total T-cells expressing a particular combination of markers, and the open diamond represents the mean.Figure S SEQ Figure_S \* ARABIC 7. Box plots for log10 scale counts of specific cytokine-expressing CD4+ total T-cells against B/MarylandCell-mediated immune (CMI) responses were measured by intracellular cytokine staining (ICCS). Counts of peripheral blood CD4+ T-cells expressing IL-2, IFN-γ, TNF-α, and/or CD40L+ were measured following in vitro re-stimulation with B/Maryland-specific recombinant wild-type sequence hemagglutinins (Has). Box plots are shown for counts of cells per 106 producing IFN-γ, TNF-α, IL-2, and/or CD40L, or combinations thereof. Responses were evaluated using peripheral blood mononuclear cells (PBMCs) obtained from a subgroup of participants on Day 0 (pre-vaccination) and Day 7. The box plots represent the interquartile range (±3 standard deviations); the solid horizontal black line represents the median and the number indicates the median count of CD4+ total T-cells expressing a particular combination of markers, and the open diamond represents the mean.Figure S SEQ Figure_S \* ARABIC 8. RCD plots for proportion of specific cytokine-expressing CD4+ total T-cells against A/Kansas196596036068IFN-γIFN-γ178015948260Triple Cytokine+Triple Cytokine+178990918415Double Cytokine+0Double Cytokine+151853919685Quadruple Cytokine+Quadruple Cytokine+Figure S SEQ Figure_S \* ARABIC 9. RCD plots for proportion of specific cytokine-expressing CD4+ total T-cells against B/Maryland185039030099IFN-γIFN-γ168973536195Triple Cytokine+Triple Cytokine+170942044821Double Cytokine+Double Cytokine+153162048387Quadruple Cytokine+0Quadruple Cytokine+Table S SEQ Table_S \* ARABIC 2. CMI responses compared to other “enhanced” flu vaccines for older adults in terms of total CD4+: Day 7 GMFRs(post/pre) [E301 and Cowling et al. 2019]qNIV(E301)IIV4 (Fluzone Quad)[Sanofi](E301) IIV “FluQuadri”[Sanofi](Cowling et al)14IIV3-HD Fluzone HD[Sanofi] (Cowling et al) 14RIV4Flublok[Sanofi] (Cowling et al) 14aIIV3FLUAD[Seqirus] (Cowling et al) 14StrainParameterDay 7 GMFRH3N2: A/Kansas (E301) or A/Switzerland (Cowling et al)IFN-γ+5.11.61.202.042.591.82B-Vic: B/Maryland (E301) or B/Brisbane (Cowling et al)IFN-γ+7.92.51.682.161.382.56Table S SEQ Table_S \* ARABIC 3. Any and severe solicited local and systemic AEs through Day 28TreatmentqNIVN=1333IIV4N=1319Count (% of participants)Subjects with at least one solicited TEAE551 (41.3)420 (31.8)Severe 21 (1.6)13 (1.0)Category Verbatim TermLocalBruising38 (2.9)29 (2.2)Severe1 (0.1)2 (0.2)Pain341 (25.6)212 (16.1)Severe3 (0.2)0 (0.0)Redness67 (5.0)34 (2.6)Severe3 (0.2)0 (0.0)Swelling84 (6.3)41 (3.1)Severe4 (0.3)0 (0.0)GeneralChills66 (5.0)44 (3.3)Severe5 (0.4)0 (0.0)Fatigue125 (9.4)93 (7.1)Severe5 (0.4)3 (0.2)Headache142 (10.7)104 (7.9)Severe3 (0.2)2 (0.2)Joint Pain77 (5.8)47 (3.6)Severe4 (0.3)0 (0.0)Muscle Pain167 (12.5)106 (8.0)Severe7 (0.5)1 (0.1)Oral Temperature5 (0.4)4 (0.3)Severe0 (0.0)0 (0.0)GastrointestinalDiarrhea51 (3.8)58 (4.4)Severe2 (0.2)2 (0.2)Nausea35 (2.6)23 (1.7)Severe1 (0.1)1 (0.1)Vomiting12 (0.9)9 (0.7)Severe0 (0.0)0 (0.0)Respiratory/FacialChest Tightness10 (0.8)13 (1.0)Severe1 (0.1)0 (0.0)Cough65 (4.9)56 (4.2)Severe3 (0.2)3 (0.2)Difficulty Breathing13 (1.0)13 (1.0)Severe0 (0.0)0 (0.0)Difficulty Swallowing7 (0.5)14 (1.1)Severe0 (0.0)0 (0.0)Eye Redness13 (1.0)18 (1.4)Severe0 (0.0)2 (0.2)Eyelid Swelling4 (0.3)8 (0.6)Severe0 (0.0)0 (0.0)Facial Swelling3 (0.2)3 (0.2)Severe0 (0.0)0 (0.0)Hoarseness29 (2.2)21 (1.6)Severe1 (0.1)1 (0.1)Sore Throat42 (3.2)42 (3.2)Severe2 (0.2)2 (0.2)Wheezing17 (1.3)17 (1.3)Severe0 (0.0)1 (0.1)Note: TEAE = treatment-emergent adverse event.N = number of participants who received test article at Day 0. n = number of participants in each specified category of adverse events.% = (n/N)*100.Treatment group in safety population was based on the actual dose(s) received. Participants with multiple occurrences of the same event were counted once for that event. Solicited adverse events reported by participants (via diary or spontaneously) with a recorded start date within the 7-day post-vaccination window are provided. For fever: severe = >38.9°C.Table S SEQ Table_S \* ARABIC 4. Comparison of safety data from sponsor’s phase 2 and 3 trial with IIV3-HD (Fluzone? High Dose [HD]) and IIV4 (Fluzone? Quadrivalent) qNIV (Phase 3)IIV4(Phase 3)IIV3-HD(Phase 2)qNIV(Phase 2) N=1333N=1319N=153N=156% of Participants with EventsAny treatment-emergent adverse event (TEAE)49.441.847.148.7Any solicited TEAE41.331.837.937.8Local solicited27.918.426.121.8Severe local solicited0.60.200Systemic solicited27.722.124.228.8Severe systemic solicited1.10.81.33.2Unsolicited TEAE18.618.320.317.9Severe unsolicited1.70.90.71.9Severe & related unsolicited0.80.200Medically attended unsolicited7.47.98.57.1Serious adverse events (SAEs)0.80.40.70.6Note: phase 2 data.16 Figure S SEQ Figure_S \* ARABIC 10. Day 28 microneutralization baseline-adjusted ratio of GMTs using egg-adapted versus wild-type A/Singapore H3N2 from phase 2 trial16Full strain name: A/Singapore/INFIMH-16-0019/2016 (H3N2), either egg-adapted or wild-type. Description: In the previous phase 2 study, we corroborated the discrepancy in findings between using egg-adapted versus wild-type HAI reagents by showing a similar pattern of differential antibody responses for egg-adapted versus wild-type virus reagents in a microneutralization (MN) assay, wherein we observed that qNIV induced substantially higher MN antibodies than IIV3-HD against a vaccine-homologous wild-type sequenced A/Singapore/INFIMH-16-0019/2016 (H3N2) virus, whereas when MN antibodies were assessed against an egg-derived variant of the same virus, IIV3-HD appeared to induce higher level of MN antibodies, thus orthogonally underscoring the problem of employing egg-derived reagents in serological assays.16Abbreviations: GMT, geometric mean titer; MN, microneutralization; qNIV, quadrivalent recombinant nanoparticle influenza vaccine; IIV3-HD, trivalent high-dose inactivated influenza vaccine; B, treatment group B of phase 2 trial, which contained qNIV with 60 ?g HA/4 strains plus 50 ?g Matrix-M; C, treatment group C, which contained qNIV with 60 ?g HA/4 strains plus 75 ?g Matrix-M; F, treatment group F, which contained IIV3-HD. Figure S SEQ Figure_S \* ARABIC 11. Spaghetti plots representing CMI polyfunctional effector and total CD4+ T-cell responses by treatment group against A/Kansas and B/Maryland Cell-mediated immune (CMI) responses were measured by intracellular cytokine staining (ICCS). Counts of peripheral blood CD4+ T-cells expressing either IFN- γ or ≥2 cytokine/activation markers (ie, IL-2, IFN-γ, TNF-α, and/or CD40L+) were measured following in vitro re-stimulation with A/Kansas or B/Maryland-specific recombinant wild-type sequence hemagglutinins (HAs). References ADDIN EN.REFLIST 1.Smith G, Liu Y, Flyer D, et al. Novel hemagglutinin nanoparticle influenza vaccine with Matrix-M adjuvant induces hemagglutination inhibition, neutralizing, and protective responses in ferrets against homologous and drifted A(H3N2) subtypes. Vaccine. 2017;35(40):5366-5372.2.Shinde V, Fries L, Wu Y, et al. Improved titers against influenza drift variants with a nanoparticle vaccine. N Engl J Med. 2018;378(24):2346-2348.3.World Health Organization. Recommended composition of influenza virus vaccines for use in the 2016-17 northern hemisphere influenza season. 2016. . Accessed July 6, 2016.4. Vaccines Related Biological Products Advisory Committee. Summary Minutes: 152nd Vaccines and Related Biological Products Advisory Committee. . Accessed March 27, 2019.5.Bengtsson LK, Morein B, Osterhaus AD. ISCOM technology-based Matrix M? adjuvant: success in future vaccines relies on formulation. Expert Rev Vaccines. 2011;10(4):401-403.6.Fluzone? Quadrivalent. [package insert]. Swiftwater, PA: Sanofi Pasteur Inc.; 2020. . Accessed April 15, 2020.7.Zost SJ, Parkhouse K, Gumina ME, et al. Contemporary H3N2 influenza viruses have a glycosylation site that alters binding of antibodies elicited by egg-adapted vaccine strains. ProcNatl Acad Sci U S A.2017;114(47):12578-12583.8.Foulds KE, Chang-you, W, Seder RA. Th1 memory: implications for vaccine development. Immunol Rev. 2006;211:58-66. 9.Spellberg B, Edwards JE. Type 1/type 2 Immunity in infectious diseases. Clin Infect Dis. 2001;32(1):76-102.10.Chattopadhyay PK, Yu J, Roederer M.?A live-cell assay to detect antigen-specific CD4+ T cells with diverse cytokine profiles.?Nat Med.?2005;11:1113-1117.?11.Darrah PA, Patel DT,? De Luca PM,?et al. Multifunctional TH1 cells define a correlate of vaccine-mediated protection against Leishmania major. Nat Med. 2007;13(7):843-850.12.Cunningham AL, Heineman TC, Lal H, et al. Immune responses to a recombinant glycoprotein E herpes zoster vaccine in adults aged 50 years or older.?J Infect Dis. 2018;217(11):1750-1760.13.Mordmüller B, Surat G, Lagler H et al. Sterile protection against human malaria by chemoattenuated PfSPZ vaccine.?Nature.?2017;542(7642):445-449.?14.Cowling BJ, Perera RAPM, Valkenburg SA, et al. Comparative immunogenicity of several enhanced influenza vaccine options for older adults: a randomized, controlled trial. Clin Infect Dis. 2019. . Bruce-Staskal PJ, Woods RM, Borisov OV, et al. Hemagglutinin from multiple divergent influenza A and B viruses bind to a distinct 2 branched, sialylated poly-LacNAc glycan by surface plasmon resonance. Vaccine. 2020. In review. 16.Shinde VS, Cai R, Plested J, et al. Induction of broadly cross-reactive hemagglutination inhibiting antibody and polyfunctional CD4+ T-cell responses against wild-type A(H3N2) hemagglutinins by a recombinant saponin adjuvanted nanoparticle vaccine. Clin Infect Dis. 2020. In review. ................
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