Safety and Immunogenicity of a Live-attenuated Universal Flu Vaccine Followed by an Inactivated Universal Flu Vaccine

An MAE is an event for which the participant received medical attention such as hospitalization, an emergency room visit, or a visit to or from medical personnel. pIMDs are a subset of AEs that include autoimmune diseases and other inflammatory and/or neurologic disorders of interest which may or may not have an autoimmune etiology. LC-ILI is defined as at least 1 systemic symptom (fever or myalgia) AND at least 1 respiratory symptom (cough or sore throat), confirmed by polymerase chain reaction (PCR) assay. An SAE is an AE that met any of the following: * Death * Life threatening * Required inpatient hospitalization or prolongation of existing hospitalization * Results in persistent or significant incapacity or substantial disruption of the ability to conduct normal life functions * Results in congenital anomaly/birth defect * An important medical event that may jeopardize the well-being of the subject or require medical or surgical intervention to prevent an above outcome.

Time frame: Through Day 113 (28 days post-dose 2)

Population: Participants who received at least one study vaccination

Hematological and biochemical parameters assessed included hemoglobin, platelets, red blood cells, white blood cells (WBC), absolute neutrophil count (ANC), lymphocytes, monocytes, eosinophils, basophils, alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine, blood urea nitrogen (BUN) and BUN-to-creatinine ratio. Grading of laboratory parameters was based on the FDA Guidance for Industry: Toxicity Grading Scale for Healthy Adult and Adolescent Volunteers Enrolled in Preventive Vaccine Clinical Trials as Grade 1 (Mild), Grade 2 (Moderate), Grade 3 (severe), or Grade 4 (Potentially life-threatening). Grade 2 or higher: * BUN: \> 26 mg/dL * Creatinine: \> 1.7 mg/dL * ALT, AST: \> 2.5 × upper limit of normal (ULN) * Hemoglobin: \< 11.0 g/dL (females) or \< 12.5 g/dL (males) or change from baseline \> 1.5 g/dL * WBC: \> 15,000 cell/mm³ or \< 2,500 cell/mm³ * Lymphocytes: \< 750 cell/mm³ * ANC: \< 1,500 cell/mm³ * Eosinophils: \> 1,500 cell/mm³ * Platelets: \< 125,000 cell/mm³

Time frame: Days 8, 29, 85, 92, and 113

Population: Participants who received at least one study vaccination

Solicited adverse events were assessed by study staff for 60 minutes after each vaccination and and then by study participants daily for 7 days on a a diary card. Solicited general reactions included: * abdominal pain * arthralgia * cough * diarrhea * fatigue * fever * headache * myalgia * nausea * shivering * sore throat * vomiting * wheezing

Time frame: 7 days after each vaccination (Days 1-8 and Days 85-92)

Population: Participants who received the priming dose and booster dose

Solicited adverse events were assessed by study staff for 60 minutes after each vaccination and and then by study participants daily for 7 days on a a diary card. Solicited local reactions included: * Post LAIV dose: nasal congestion, rhinorrhea; * Post IIV dose: pain, redness, swelling.

Time frame: 7 days after each vaccination (Days 1-8 and Days 85-92)

Population: Participants who received the priming dose and booster dose

Unsolicited adverse events (AEs) are any AEs reported spontaneously by the participant, observed by the study personnel during study visits or those identified during review of medical records or source documents, such as diary cards. Participants were asked to record any unsolicited symptoms or other illness description in their diary card during the 28 days after each vaccination. All AEs, including clinical laboratory test results, were assessed by a study clinician and the study subject (as applicable) to quantify severity using a protocol-defined grading system as mild (mild symptoms, easily tolerated, not interfering with daily activities), moderate (causing some interference with daily activity), or severe (severe symptoms that prevent normal every day activities). The investigator assessed the relationship between study vaccines and the occurrence of each AE using clinical judgment.

Time frame: 28 days after each vaccination (Days 1 to 28 and Days 85 to 113)

Population: Participants who received at least one study vaccination

Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune response leading to lysis of antibody-coated target cells by immune effector cells and is triggered by the interaction between the Fc portion of an antibody and Fc-gamma receptors expressed on immune effector cells. This bioluminescent assay measured antibodies to a chimeric hemagglutinin (H6 head domain and H1 stalk domain) virus that mediate ADCC activity via the Fc-receptor. ADCC activity was measured by the area under the curve (AUC) of luminescence per serial dilution.

Time frame: Baseline (pre-dose 1), Month 1 (28 days post-dose 1), Month 4 (28 days post-dose 2), and Month 15 (12 months post-dose 2)

Population: Participants in the per-protocol population with available data at Baseline and each time point. The-per protocol population included all participants who received at least 1 vaccination and with no major deviations, including those considered likely to affect the immune response; data were included up to the time of the observed deviation event.

Anti-H1 hemagglutinin stalk immunoglobulin G (IgG) in saliva was quantified using an enzyme-linked immunosorbent assay (ELISA). The ELISA measured IgG antibodies against the H1 stalk domain by using a chimeric protein containing an exotic H6 hemagglutinin head domain that the vaccinees have not previously been exposed to (strain A/mallard/Sweden/81/02 \[H6N1\]) and the same H1 stalk domain as expressed by the vaccine (strain A/California/04/09 \[H1N1\]). The LLOQ for the assay was 1:10.

Time frame: Baseline (pre-dose 1), Month 1 (28 days post-dose 1), Month 4 (28 days post-dose 2), and Month 15 (12 months post-dose 2)

Population: Participants in the per-protocol population with available data at each time point. The per-protocol population included all participants who received at least 1 vaccination and with no major deviations, including those considered likely to affect the immune response; data were included up to the time of the observed deviation event.

Anti-H1 hemagglutinin stalk secretory immunoglobulin A (IgA) in saliva was quantified using an enzyme-linked immunosorbent assay (ELISA). The ELISA measured secretory IgA antibodies antibodies (actively secreted in the mucosa) against the H1 stalk domain in saliva using a chimeric protein containing an exotic H6 hemagglutinin head domain that the vaccinees have not previously been exposed to (strain A/mallard/Sweden/81/02 \[H6N1\]) and the same H1 stalk domain as expressed by the vaccine (strain A/California/04/09 \[H1N1\]). The LLOQ for the assay was 1:4.

Time frame: Baseline (pre-dose 1), Month 1 (28 days post-dose 1), Month 4 (28 days post-dose 2), and Month 15 (12 months post-dose 2)

Population: Participants in the per-protocol population with available data at each time point. The per-protocol population included all participants who received at least 1 vaccination and with no major deviations, including those considered likely to affect the immune response; data were included up to the time of the observed deviation event.

Anti-H1 hemagglutinin stalk total immunoglobulin A (IgA) in saliva was quantified using an enzyme-linked immunosorbent assay (ELISA). The ELISA measured total IgA antibodies antibodies (secreted through active and passive transfer processes in the mucosa) against the H1 stalk domain in saliva using a chimeric protein containing an exotic H6 hemagglutinin head domain that the vaccinees have not previously been exposed to (strain A/mallard/Sweden/81/02 \[H6N1\]) and the same H1 stalk domain as expressed by the vaccine (strain A/California/04/09 \[H1N1\]). The LLOQ for the assay was 1:10.

Time frame: Baseline (pre-dose 1), Month 1 (28 days post-dose 1), Month 4 (28 days post-dose 2), and Month 15 (12 months post-dose 2)

Population: Participants in the per-protocol population with available data at each time point. The per-protocol population included all participants who received at least 1 vaccination and with no major deviations, including those considered likely to affect the immune response; data were included up to the time of the observed deviation event.

This assay was performed to measure the neutralizing potential of cross-reactive antibodies to a Group 1 influenza against an H1N1 virus isolate that currently circulates in animals and is antigenically different from current human isolates. Anti-avian-swine H1N1 virus neutralizing antibodies were quantified using a microneutralization (MN) assay using Strain A/Swine/Jiangsu/40/2011 (asH1N1). The LLOQ for the assay was 1:10.

Time frame: Baseline (pre-dose 1), Month 1 (28 days post-dose 1), Month 4 (28 days post-dose 2), and Month 15 (12 months post-dose 2)

Population: Participants in the per-protocol population with available data at each time point. The per-protocol population included all participants who received at least 1 vaccination and with no major deviations, including those considered likely to affect the immune response; data were included up to the time of the observed deviation event.

To determine antibody breadth, assays were performed to measure the induction of cross-reactive IgG antibodies to a Group 1 influenza A virus with a heterosubtypic hemagglutinin distantly related to the currently circulating influenza A/H1 viruses, H18, the stalk domain of which shares about 65% amino acid identity with the H1 vaccine stalk domain. Anti-H18 full-length (ecto-domain) hemagglutinin IgG was quantified by ELISA using a recombinant antigen based on Strain A/flat-faced bat/Peru/033/10 (H18N11) HA. Titers are expressed as ELISA units (EU) per mL and were calculated on the basis of an internal standard to which units were arbitrarily assigned.

Time frame: Baseline (pre-dose 1), Month 1 (28 days post-dose 1), Month 4 (28 days post-dose 2), and Month 15 (12 months post-dose 2)

Population: Participants in the per-protocol population with available data at each time point. The per-protocol population included all participants who received at least 1 vaccination and with no major deviations, including those considered likely to affect the immune response; data were included up to the time of the observed deviation event.

Anti-H1 hemagglutinin stalk immunoglobulin A (IgA) was quantified using an enzyme-linked immunosorbent assay (ELISA). The ELISA measured IgA antibodies against the H1 stalk domain by using a chimeric protein containing an exotic H6 hemagglutinin head domain that the vaccinees have not previously been exposed to (strain A/mallard/Sweden/81/02 \[H6N1\]) and the same H1 stalk domain as expressed by the vaccine (strain A/California/04/09 \[H1N1\]) .

Time frame: Baseline (pre-dose 1), Month 1 (28 days post-dose 1), Month 4 (28 days post-dose 2), and Month 15 (12 months post-dose 2)

Population: Participants in the per-protocol population with available data at each time point. The per protocol population included all participants who received at least 1 vaccination and with no major deviations, including those considered likely to affect the immune response; data were included up to the time of the observed deviation event.

Anti-H1 hemagglutinin stalk immunoglobulin G (IgG) was quantified using an enzyme-linked immunosorbent assay (ELISA). The ELISA measured IgG antibodies against the H1 stalk domain by using a chimeric protein containing an exotic H6 hemagglutinin head domain that the vaccinees have not previously been exposed to (strain A/mallard/Sweden/81/02 \[H6N1\]) and the same H1 stalk domain as expressed by the vaccine (strain A/California/04/09 \[H1N1\]) . Titers are expressed as ELISA units (EU) per mL and were calculated on the basis of an internal standard to which units were arbitrarily assigned. The lower limit of quantitation (LLOQ) for the assay was 65.3 EU/mL.

Time frame: Baseline (pre-dose 1), Month 1 (28 days post-dose 1), Month 4 (28 days post-dose 2), Month 9 (6 months post-dose 2), and Month 15 (12 months post-dose 2)

Population: Participants in the per-protocol population with available data at each time point. The per protocol population included all participants who received at least 1 vaccination and with no major deviations, including those considered likely to affect the immune response; data were included up to the time of the observed deviation event.

Anti H1 hemagglutinin stalk neutralizing antibodies were quantified using a microneutralization (MN) assay using a virus that expresses a chimeric hemagglutinin that contains an exotic HA head domain (strain A/mallard/Sweden/81/2002 (\[H6N1\]) and the H1 stalk domain (strain A/California/04/2009 (\[H1N1pandemic\]) and an exotic neuraminidase, N5, for which humans are generally naïve (strain: A/mallard/Sweden/86/2003 \[H12N5\]). The LLOQ for the assay was 1:10.

Time frame: Baseline (pre-dose 1), Month 1 (28 days post-dose 1), Month 4 (28 days post-dose 2), and Month 15 (12 months post-dose 2)

Population: Participants in the per-protocol population with available data at each time point. The per protocol population included all participants who received at least 1 vaccination and with no major deviations, including those considered likely to affect the immune response; data were included up to the time of the observed deviation event.

To determine antibody breadth, assays were performed to measure the induction of cross-reactive IgG antibodies to a Group 1 influenza A virus with a heterosubtypic hemagglutinin, subtype H2, of which the stalk domain shares about 78% amino acid identity with the H1 vaccine stalk domain. Anti-H2 full-length (ecto-domain) hemagglutinin IgG was quantified by ELISA using a recombinant antigen based on A/mallard/Netherlands/5/1999 (H2N9) HA. Titers are expressed as ELISA units (EU) per mL and were calculated on the basis of an internal standard to which units were arbitrarily assigned.

Time frame: Baseline (pre-dose 1), Month 1 (28 days post-dose 1), Month 4 (28 days post-dose 2), and Month 15 (12 months post-dose 2)

Population: Participants in the per-protocol population with available data at each time point. The per-protocol population included all participants who received at least 1 vaccination and with no major deviations, including those considered likely to affect the immune response; data were included up to the time of the observed deviation event.

This assay was performed to measure the induction of antibodies reactive against the head domain of a group 1 influenza virus with a heterosubtypic hemagglutinin from a recent avian influenza virus. Anti-H5N8 virus neutralizing antibodies were quantified using a microneutralization (MN) assay using a reverse genetics (RG) reassortant virus based on PR8 for 6 genes with 2 surface proteins: HA and NA from A/Gyrfalcon/Washington/41088-6/2014 (H5N8). The LLOQ for the assay was 1:10.

Time frame: Baseline (pre-dose 1), Month 1 (28 days post-dose 1), Month 4 (28 days post-dose 2), and Month 15 (12 months post-dose 2)

Population: Participants in the per-protocol population with available data at each time point. The per-protocol population included all participants who received at least 1 vaccination and with no major deviations, including those considered likely to affect the immune response; data were included up to the time of the observed deviation event.

To determine antibody breadth, assays were performed to measure the induction of cross-reactive IgG antibodies to a Group 1 influenza A virus with a heterosubtypic hemagglutinin, subtype H9, the stalk domain of which shares about 59% amino acid identity with the H1 vaccine stalk domain. Anti-H9 full-length (ecto-domain) hemagglutinin IgG was quantified by ELISA using a recombinant antigen based on Strain A/chicken/Hong Kong/G9/1997 (H9N2) HA. Titers are expressed as ELISA units (EU) per mL and were calculated on the basis of an internal standard to which units were arbitrarily assigned. The LLOQ for the assay was 31 EU/mL.

Time frame: Baseline (pre-dose 1), Month 1 (28 days post-dose 1), Month 4 (28 days post-dose 2), and Month 15 (12 months post-dose 2)

Population: Participants in the per-protocol population with available data at each time point. The per-protocol population included all participants who received at least 1 vaccination and with no major deviations, including those considered likely to affect the immune response; data were included up to the time of the observed deviation event.

This assay was performed to measure the neutralizing potential of antibodies against the currently circulating human H1N1 isolate which is similar to the stalk used in study vaccines. Anti-human H1N1 virus neutralizing antibodies were quantified using a microneutralization (MN) assay using Strain A/Singapore/GP1908/2015 (IVR-180). The LLOQ for the assay was 1:10.

Time frame: Baseline (pre-dose 1), Month 1 (28 days post-dose 1), Month 4 (28 days post-dose 2), and Month 15 (12 months post-dose 2)

Population: Participants in the per-protocol population with available data at each time point. The per-protocol population included all participants who received at least 1 vaccination and with no major deviations, including those considered likely to affect the immune response; data were included up to the time of the observed deviation event.

Anti-H1 hemagglutinin stalk secretory immunoglobulin A (IgA) in saliva was quantified using an enzyme-linked immunosorbent assay (ELISA). The ELISA measured secretory IgA antibodies antibodies (actively secreted in the mucosa) against the H1 stalk domain in saliva using a chimeric protein containing an exotic H6 hemagglutinin head domain that the vaccinees have not previously been exposed to (strain A/mallard/Sweden/81/02 \[H6N1\]) and the same H1 stalk domain as expressed by the vaccine (strain A/California/04/09 \[H1N1\]). Mean geometric increase represents the fold-rise in antibody titer from Baseline to each post-baseline time point (ratio of post-baseline titer to Baseline titer).

Time frame: Baseline (pre-dose 1), Month 1 (28 days post-dose 1), Month 4 (28 days post-dose 2), and Month 15 (12 months post-dose 2)

Population: Participants in the per-protocol population with available data at each time point. The per-protocol population included all participants who received at least 1 vaccination and with no major deviations, including those considered likely to affect the immune response; data were included up to the time of the observed deviation event.

Anti-H1 hemagglutinin stalk total immunoglobulin A (IgA) in saliva was quantified using an enzyme-linked immunosorbent assay (ELISA). The ELISA measured total IgA antibodies (secreted through active and passive transfer processes in the mucosa) against the H1 stalk domain in saliva using a chimeric protein containing an exotic H6 hemagglutinin head domain that the vaccinees have not previously been exposed to (strain A/mallard/Sweden/81/02 \[H6N1\]) and the same H1 stalk domain as expressed by the vaccine (strain A/California/04/09 \[H1N1\]). Mean geometric increase represents the fold-rise in antibody titer from baseline to each post-baseline time point (ratio of post-baseline titer to Baseline titer).

Time frame: Baseline (pre-dose 1), Month 1 (28 days post-dose 1), Month 4 (28 days post-dose 2), and Month 15 (12 months post-dose 2)

Population: Participants in the per-protocol population with available data at each time point. The per-protocol population included all participants who received at least 1 vaccination and with no major deviations, including those considered likely to affect the immune response; data were included up to the time of the observed deviation event.

This assay was performed to measure the neutralizing potential of cross-reactive antibodies to a Group 1 influenza against an H1N1 virus isolate that currently circulates in animals and is antigenically different from current human isolates. Anti-avian-swine H1N1 virus neutralizing antibodies were quantified using a microneutralization (MN) assay using Strain A/Swine/Jiangsu/40/2011 (asH1N1). Mean geometric increase represents the fold-rise in antibody titer from Baseline to each post-baseline time point (ratio of post-baseline titer to Baseline titer).

Time frame: Baseline (pre-dose 1), Month 1 (28 days post-dose 1), Month 4 (28 days post-dose 2), and Month 15 (12 months post-dose 2)

Population: Participants in the per-protocol population with available data at each time point. The per-protocol population included all participants who received at least 1 vaccination and with no major deviations, including those considered likely to affect the immune response; data were included up to the time of the observed deviation event.

Anti-H1 hemagglutinin stalk immunoglobulin A (IgA) was quantified using an enzyme-linked immunosorbent assay (ELISA). The ELISA measured IgA antibodies against the H1 stalk domain by using a chimeric protein containing an exotic H6 hemagglutinin head domain that the vaccinees have not previously been exposed to (strain A/mallard/Sweden/81/02 \[H6N1\]) and the same H1 stalk domain as expressed by the vaccine (strain A/California/04/09 \[H1N1\]) . Mean geometric increase represents the fold-rise in antibody titer from baseline to each post-baseline time point (ratio of post-baseline titer to Baseline titer).

Time frame: Baseline (pre-dose 1), Month 1 (28 days post-dose 1), Month 4 (28 days post-dose 2), and Month 15 (12 months post-dose 2)

Population: Participants in the per-protocol population with available data at each time point. The per protocol population included all participants who received at least 1 vaccination and with no major deviations, including those considered likely to affect the immune response; data were included up to the time of the observed deviation event.

Anti-H1 hemagglutinin stalk immunoglobulin G (IgG) was quantified using an enzyme-linked immunosorbent assay (ELISA). The ELISA measured IgG antibodies against the H1 stalk domain by using a chimeric protein containing an exotic H6 hemagglutinin head domain that the vaccinees have not previously been exposed to (strain A/mallard/Sweden/81/02 \[H6N1\]) and the same H1 stalk domain as expressed by the vaccine (strain A/California/04/09 \[H1N1\]). Mean geometric increase represents the fold-rise in antibody titer from baseline to each post-baseline time point (ratio of post-baseline titer to Baseline titer).

Time frame: Baseline (pre-dose 1), Month 1 (28 days post-dose 1), Month 4 (28 days post-dose 2), Month 9 (6 months post-dose 2), and Month 15 (12 months post-dose 2)

Population: Participants in the per-protocol population with available data at each time point. The per protocol population included all participants who received at least 1 vaccination and with no major deviations, including those considered likely to affect the immune response; data were included up to the time of the observed deviation event.

Anti H1 hemagglutinin stalk neutralizing antibodies were quantified using a microneutralization (MN) assay using a virus that expresses a chimeric hemagglutinin that contains an exotic HA head domain (strain A/mallard/Sweden/81/2002 \[H6N1\]) and the H1 stalk domain (strain A/California/04/2009 \[H1N1pandemic\]) and an exotic neuraminidase, N5, for which humans are generally naïve (strain: A/mallard/Sweden/86/2003 \[H12N5\]). Mean geometric increase represents the fold-rise in antibody titer from baseline to each post-baseline time point (ratio of post-baseline titer to Baseline titer).

Time frame: Baseline (pre-dose 1), Month 1 (28 days post-dose 1), Month 4 (28 days post-dose 2), and Month 15 (12 months post-dose 2)

Population: Participants in the per-protocol population with available data at each time point. The per protocol population included all participants who received at least 1 vaccination and with no major deviations, including those considered likely to affect the immune response; data were included up to the time of the observed deviation event.

This assay was performed to measure the induction of antibodies reactive against the head domain of a group 1 influenza virus with a heterosubtypic hemagglutinin from a recent avian influenza virus. Anti-H5N8 virus neutralizing antibodies were quantified using a microneutralization (MN) assay using a reverse genetics (RG) reassortant virus based on PR8 for 6 genes with 2 surface proteins: HA and NA from A/Gyrfalcon/Washington/41088-6/2014 (H5N8). Mean geometric increase represents the fold-rise in antibody titer from Baseline to each post-baseline time point (ratio of post-baseline titer to Baseline titer).

Time frame: Baseline (pre-dose 1), Month 1 (28 days post-dose 1), Month 4 (28 days post-dose 2), and Month 15 (12 months post-dose 2)

Population: Participants in the per-protocol population with available data at each time point. The per-protocol population included all participants who received at least 1 vaccination and with no major deviations, including those considered likely to affect the immune response; data were included up to the time of the observed deviation event.

Anti-H1 hemagglutinin stalk immunoglobulin G (IgG) in saliva was quantified using an enzyme-linked immunosorbent assay (ELISA). The ELISA measured IgG antibodies in saliva against the H1 stalk domain by using a chimeric protein containing an exotic H6 hemagglutinin head domain that the vaccinees have not previously been exposed to (strain A/mallard/Sweden/81/02 \[H6N1\]) and the same H1 stalk domain as expressed by the vaccine (strain A/California/04/09 \[H1N1\]). Mean geometric increase represents the fold-rise in antibody titer from baseline to each post-baseline time point (ratio of post-baseline titer to Baseline titer).

Time frame: Baseline (pre-dose 1), Month 1 (28 days post-dose 1), Month 4 (28 days post-dose 2), and Month 15 (12 months post-dose 2)

Population: Participants in the per-protocol population with available data at each time point. The per-protocol population included all participants who received at least 1 vaccination and with no major deviations, including those considered likely to affect the immune response; data were included up to the time of the observed deviation event.

To determine antibody breadth, assays were performed to measure the induction of cross-reactive IgG antibodies to a Group 1 influenza A virus with a heterosubtypic hemagglutinin distantly related to the currently circulating influenza A/H1 viruses, subtype H18, the stalk domain of which shares about 65% amino acid identity with the H1 vaccine stalk domain. Anti-H18 full-length (ecto-domain) hemagglutinin IgG was quantified by ELISA using a recombinant antigen based on Strain A/flat-faced bat/Peru/033/10 (H18N11) HA. Mean geometric increase represents the fold-rise in antibody titer from Baseline to each post-baseline time point (ratio of post-baseline titer to Baseline titer).

Time frame: Baseline (pre-dose 1), Month 1 (28 days post-dose 1), Month 4 (28 days post-dose 2), and Month 15 (12 months post-dose 2)

Population: Participants in the per-protocol population with available data at each time point. The per-protocol population included all participants who received at least 1 vaccination and with no major deviations, including those considered likely to affect the immune response; data were included up to the time of the observed deviation event.

To determine antibody breadth, assays were performed to measure the induction of cross-reactive IgG antibodies to a Group 1 influenza A virus with a heterosubtypic hemagglutinin, subtype H2, of which the stalk domain shares about 78% amino acid identity with the H1 vaccine stalk domain. Anti-H2 full-length (ecto-domain) hemagglutinin IgG was quantified by ELISA using a recombinant antigen based on A/mallard/Netherlands/5/1999 (H2N9) HA. Mean geometric increase represents the fold-rise in antibody titer from Baseline to each post-baseline time point (ratio of post-baseline titer to Baseline titer).

Time frame: Baseline (pre-dose 1), Month 1 (28 days post-dose 1), Month 4 (28 days post-dose 2), and Month 15 (12 months post-dose 2)

Population: Participants in the per-protocol population with available data at each time point. The per-protocol population included all participants who received at least 1 vaccination and with no major deviations, including those considered likely to affect the immune response; data were included up to the time of the observed deviation event.

To determine antibody breadth, assays were performed to measure the induction of cross-reactive IgG antibodies to a Group 1 influenza A virus with a heterosubtypic hemagglutinin, subtype H9, the stalk domain of which shares about 59% amino acid identity with the H1 vaccine stalk domain. Anti-H9 full-length (ecto-domain) hemagglutinin IgG was quantified by ELISA using a recombinant antigen based on Strain A/chicken/Hong Kong/G9/1997 (H9N2) HA. Mean geometric increase represents the fold-rise in antibody titer from Baseline to each post-baseline time point (ratio of post-baseline titer to Baseline titer).

Time frame: Baseline (pre-dose 1), Month 1 (28 days post-dose 1), Month 4 (28 days post-dose 2), and Month 15 (12 months post-dose 2)

Population: Participants in the per-protocol population with available data at each time point. The per-protocol population included all participants who received at least 1 vaccination and with no major deviations, including those considered likely to affect the immune response; data were included up to the time of the observed deviation event.

This assay was performed to measure the neutralizing potential of antibodies against the currently circulating human H1N1 isolate which is similar to the stalk used in study vaccines. Anti-human H1N1 virus neutralizing antibodies were quantified using a microneutralization (MN) assay using Strain A/Singapore/GP1908/2015 (IVR-180). Mean geometric increase represents the fold-rise in antibody titer from Baseline to each post-baseline time point (ratio of post-baseline titer to Baseline titer).

Time frame: Baseline (pre-dose 1), Month 1 (28 days post-dose 1), Month 4 (28 days post-dose 2), and Month 15 (12 months post-dose 2)

Population: Participants in the per-protocol population with available data at each time point. The per-protocol population included all participants who received at least 1 vaccination and with no major deviations, including those considered likely to affect the immune response; data were included up to the time of the observed deviation event.

To detect viral shedding participants who received LAIV vaccine or intranasal sterile saline as the prime dose had nasal and oropharyngeal swabs collected on Days 1 to 5. Influenza type A virus ribonucleic acid (RNA) was detected using reverse transcription polymerase chain reaction (RT-PCR).

Time frame: Days 1 to 5

Population: Participants who received LAIV (Groups 1, 2, and 3) with evaluable samples

To study virus infectivity, nasal and oropharyngeal swab specimens that tested influenza A positive by RT-PCR were further tested for viability of virus in Madin Darby canine kidney (MDCK) cell culture and stained with monoclonal antibody specific to the cH8/1N1 LAIV virus to confirm detected virus is of vaccine origin.

Time frame: Days 1 to 5

Population: Participants who received LAIV (Groups 1, 2, and 3) and tested positive for influenza A virus by RT-PCR at any time during the 5 days post LAIV dose and with evaluable samples

An MAE is an event for which the participant received medical attention such as hospitalization, an emergency room visit, or a visit to or from medical personnel. pIMDs are a subset of AEs that include autoimmune diseases and other inflammatory and/or neurologic disorders of interest which may or may not have an autoimmune etiology. LC-ILI is defined as at least 1 systemic symptom (fever or myalgia) AND at least 1 respiratory symptom (cough or sore throat), confirmed by PCR assay. An SAE is an AE that met any of the following: * Death * Life threatening * Required inpatient hospitalization or prolongation of existing hospitalization * Resulted in persistent or significant incapacity or substantial disruption of the ability to conduct normal life functions * Resulted in congenital anomaly/birth defect * An important medical event that may jeopardize the well-being of the subject or require medical or surgical intervention to prevent an above outcome.

Time frame: From first dose to end of study, 588 days (21 months; 18 months post-dose 2)

Population: Participants who received at least one study vaccination

Hematological and biochemical parameters assessed included hemoglobin, platelets, red blood cells, white blood cells (WBC), absolute neutrophil count (ANC), lymphocytes, monocytes, eosinophils, basophils, alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine, blood urea nitrogen (BUN) and BUN-to-creatinine ratio. Grading of laboratory parameters was based on the FDA Guidance for Industry: Toxicity Grading Scale for Healthy Adult and Adolescent Volunteers Enrolled in Preventive Vaccine Clinical Trials as Grade 1 (Mild), Grade 2 (Moderate), Grade 3 (severe), or Grade 4 (Potentially life-threatening). Grade 2 or higher: * BUN: \> 26 mg/dL * Creatinine: \> 1.7 mg/dL * ALT, AST: \> 2.5 × upper limit of normal (ULN) * Hemoglobin: \< 11.0 g/dL (females) or \< 12.5 g/dL (males) or change from baseline \> 1.5 g/dL * WBC: \> 15,000 cell/mm³ or \< 2,500 cell/mm³ * Lymphocytes: \< 750 cell/mm³ * ANC: \< 1,500 cell/mm³ * Eosinophils: \> 1,500 cell/mm³ * Platelets: \< 125,000 cell/mm³

Time frame: Month 9 (6 months post-dose 2) and Month 15 (12 months post-dose 2)

Population: Participants who received at least one study vaccination

Anti-H1 hemagglutinin stalk immunoglobulin G (IgG) in saliva was quantified using an enzyme-linked immunosorbent assay (ELISA). The ELISA measured IgG antibodies in saliva against the H1 stalk domain by using a chimeric protein containing an exotic H6 hemagglutinin head domain that the vaccinees have not previously been exposed to (strain A/mallard/Sweden/81/02 \[H6N1\]) and the same H1 stalk domain as expressed by the vaccine (strain A/California/04/09 \[H1N1\]).

Time frame: Baseline (pre-dose 1), Month 1 (28 days post-dose 1), Month 4 (28 days post-dose 2), and Month 15 (12 months post-dose 2)

Population: Participants in the per-protocol population with available data at each time point. The per-protocol population included all participants who received at least 1 vaccination and with no major deviations, including those considered likely to affect the immune response; data were included up to the time of the observed deviation event.

Anti-H1 hemagglutinin stalk secretory immunoglobulin A (IgA) in saliva was quantified using an enzyme-linked immunosorbent assay (ELISA). The ELISA measured secretory IgA antibodies (actively secreted in the mucosa) against the H1 stalk domain in saliva by using a chimeric protein containing an exotic H6 hemagglutinin head domain that the vaccinees have not previously been exposed to (strain A/mallard/Sweden/81/02 \[H6N1\]) and the same H1 stalk domain as expressed by the vaccine (strain A/California/04/09 \[H1N1\]).

Time frame: Baseline (pre-dose 1), Month 1 (28 days post-dose 1), Month 4 (28 days post-dose 2), and Month 15 (12 months post-dose 2)

Population: Participants in the per-protocol population with available data at each time point. The per-protocol population included all participants who received at least 1 vaccination and with no major deviations, including those considered likely to affect the immune response; data were included up to the time of the observed deviation event.

Anti-H1 hemagglutinin stalk total immunoglobulin A (IgA) in saliva was quantified using an enzyme-linked immunosorbent assay (ELISA). The ELISA measured total IgA antibodies antibodies (secreted through active and passive transfer processes in the mucosa) against the H1 stalk domain in saliva using a chimeric protein containing an exotic H6 hemagglutinin head domain that the vaccinees have not previously been exposed to (strain A/mallard/Sweden/81/02 \[H6N1\]) and the same H1 stalk domain as expressed by the vaccine (strain A/California/04/09 \[H1N1\]).

Time frame: Baseline (pre-dose 1), Month 1 (28 days post-dose 1), Month 4 (28 days post-dose 2), and Month 15 (12 months post-dose 2)

Population: Participants in the per-protocol population with available data at each time point. The per-protocol population included all participants who received at least 1 vaccination and with no major deviations, including those considered likely to affect the immune response; data were included up to the time of the observed deviation event.

Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune response leading to lysis of antibody-coated target cells by immune effector cells and is triggered by the interaction between the Fc portion of an antibody and Fc-gamma receptors expressed on immune effector cells. This bioluminescent assay measured antibodies to a chimeric hemagglutinin (H6 head domain and H1 stalk domain) virus that mediate ADCC activity via the Fc-receptor. ADCC activity was measured by the area under the curve (AUC) of luminescence per serial dilution.

Time frame: Baseline (pre-dose 1), Month 1 (28 days post-dose 1), Month 4 (28 days post-dose 2), and Month 15 (12 months post-dose 2)

Population: Participants in the per-protocol population with available data at Baseline and each time point. The per-protocol population included all participants who received at least 1 vaccination and with no major deviations, including those considered likely to affect the immune response; data were included up to the time of the observed deviation event.

To determine antibody breadth, assays were performed to measure the induction of cross-reactive IgG antibodies to a Group 1 influenza A virus with a heterosubtypic hemagglutinin distantly related to the currently circulating influenza A/H1 viruses, subtype H18, the stalk domain of which shares about 65% amino acid identity with the H1 vaccine stalk domain. Anti-H18 full-length (ecto-domain) hemagglutinin IgG was quantified by ELISA using a recombinant antigen based on Strain A/flat-faced bat/Peru/033/10 (H18N11) HA.

Time frame: Baseline (pre-dose 1), Month 1 (28 days post-dose 1), Month 4 (28 days post-dose 2), and Month 15 (12 months post-dose 2)

Population: Participants in the per-protocol population with available data at each time point. The per-protocol population included all participants who received at least 1 vaccination and with no major deviations, including those considered likely to affect the immune response; data were included up to the time of the observed deviation event.

Anti-H1 hemagglutinin stalk immunoglobulin A (IgA) was quantified using an enzyme-linked immunosorbent assay (ELISA). The ELISA measured IgA antibodies against the H1 stalk domain by using a chimeric protein containing an exotic H6 hemagglutinin head domain that the vaccinees have not previously been exposed to (strain A/mallard/Sweden/81/02 \[H6N1\]) and the same H1 stalk domain as expressed by the vaccine (strain A/California/04/09 \[H1N1\]) .

Time frame: Baseline (pre-dose 1), Month 1 (28 days post-dose 1), Month 4 (28 days post-dose 2), and Month 15 (12 months post-dose 2)

Population: Participants in the per-protocol population with available data at each time point. The per protocol population included all participants who received at least 1 vaccination and with no major deviations, including those considered likely to affect the immune response; data were included up to the time of the observed deviation event.

Anti-H1 hemagglutinin stalk immunoglobulin G (IgG) was quantified using an enzyme-linked immunosorbent assay (ELISA). The ELISA measured IgG antibodies against the H1 stalk domain by using a chimeric protein containing an exotic H6 hemagglutinin head domain that the vaccinees have not previously been exposed to (strain A/mallard/Sweden/81/02 \[H6N1\]) and the same H1 stalk domain as expressed by the vaccine (strain A/California/04/09 \[H1N1\]) .

Time frame: Baseline (pre-dose 1), Month 1 (28 days post-dose 1), Month 4 (28 days post-dose 2), Month 9 (6 months post-dose 2), and Month 15 (12 months post-dose 2)

Population: Participants in the per-protocol population with available data at each time point. The per protocol population included all participants who received at least 1 vaccination and with no major deviations, including those considered likely to affect the immune response; data were included up to the time of the observed deviation event.

Anti H1 hemagglutinin stalk neutralizing antibodies were quantified using a microneutralization (MN) assay using a virus that expresses a chimeric hemagglutinin that contains an exotic HA head domain (strain A/mallard/Sweden/81/2002 (\[H6N1\]) and the H1 stalk domain (strain A/California/04/2009 (\[H1N1pandemic\]) and an exotic neuraminidase, N5, for which humans are generally naïve (strain: A/mallard/Sweden/86/2003 \[H12N5\]).

Time frame: Baseline (pre-dose 1), Month 1 (28 days post-dose 1), Month 4 (28 days post-dose 2), and Month 15 (12 months post-dose 2)

Population: Participants in the per-protocol population with available data at each time point. The per protocol population included all participants who received at least 1 vaccination and with no major deviations, including those considered likely to affect the immune response; data were included up to the time of the observed deviation event.

To determine antibody breadth, assays were performed to measure the induction of cross-reactive IgG antibodies to a Group 1 influenza A virus with a heterosubtypic hemagglutinin, subtype H2, of which the stalk domain shares about 78% amino acid identity with the H1 vaccine stalk domain. Anti-H2 full-length (ecto-domain) hemagglutinin IgG was quantified by ELISA using a recombinant antigen based on A/mallard/Netherlands/5/1999 (H2N9) HA.

Time frame: Baseline (pre-dose 1), Month 1 (28 days post-dose 1), Month 4 (28 days post-dose 2), and Month 15 (12 months post-dose 2)

Population: Participants in the per-protocol population with available data at each time point. The per-protocol population included all participants who received at least 1 vaccination and with no major deviations, including those considered likely to affect the immune response; data were included up to the time of the observed deviation event.

To determine antibody breadth, assays were performed to measure the induction of cross-reactive IgG antibodies to a Group 1 influenza A virus with a heterosubtypic hemagglutinin, subtype H9, the stalk domain of which shares about 59% amino acid identity with the H1 vaccine stalk domain. Anti-H9 full-length(ecto-domain) hemagglutinin IgG was quantified by ELISA using a recombinant antigen based on Strain A/chicken/Hong Kong/G9/1997 (H9N2) HA.

Time frame: Baseline (pre-dose 1), Month 1 (28 days post-dose 1), Month 4 (28 days post-dose 2), and Month 15 (12 months post-dose 2)

Population: Participants in the per-protocol population with available data at each time point. The per-protocol population included all participants who received at least 1 vaccination and with no major deviations, including those considered likely to affect the immune response; data were included up to the time of the observed deviation event.

This assay was performed to measure the neutralizing potential of antibodies against the currently circulating human H1N1 isolate which is similar to the stalk used in study vaccines. Anti-human H1N1 virus neutralizing antibodies were quantified using a microneutralization (MN) assay using Strain A/Singapore/GP1908/2015 (IVR-180).

Time frame: Baseline (pre-dose 1), Month 1 (28 days post-dose 1), Month 4 (28 days post-dose 2), and Month 15 (12 months post-dose 2)

Population: Participants in the per-protocol population with available data at each time point. The per-protocol population included all participants who received at least 1 vaccination and with no major deviations, including those considered likely to affect the immune response; data were included up to the time of the observed deviation event.

This assay was performed to measure the neutralizing potential of cross-reactive antibodies to a Group 1 influenza against an H1N1 virus isolate that currently circulates in animals and is antigenically different from current human isolates. Anti-avian-swine H1N1 virus neutralizing antibodies were quantified using a microneutralization (MN) assay using Strain A/Swine/Jiangsu/40/2011 (asH1N1).

Time frame: Baseline (pre-dose 1), Month 1 (28 days post-dose 1), Month 4 (28 days post-dose 2), and Month 15 (12 months post-dose 2)

Population: Participants in the per-protocol population with available data at each time point. The per-protocol population included all participants who received at least 1 vaccination and with no major deviations, including those considered likely to affect the immune response; data were included up to the time of the observed deviation event.

This assay was performed to measure the induction of antibodies reactive against the head domain of a group 1 influenza virus with a heterosubtypic hemagglutinin from a recent avian influenza virus. Anti-H5N8 virus neutralizing antibodies were quantified using a microneutralization (MN) assay using a reverse genetics (RG) reassortant virus based on PR8 for 6 genes with 2 surface proteins: HA and NA from A/Gyrfalcon/Washington/41088-6/2014 (H5N8).

Time frame: Baseline (pre-dose 1), Month 1 (28 days post-dose 1), Month 4 (28 days post-dose 2), and Month 15 (12 months post-dose 2)

Population: Participants in the per-protocol population with available data at each time point. The per-protocol population included all participants who received at least 1 vaccination and with no major deviations, including those considered likely to affect the immune response; data were included up to the time of the observed deviation event.

Anti-H1 hemagglutinin stalk immunoglobulin G (IgG) in saliva was quantified using an enzyme-linked immunosorbent assay (ELISA). The ELISA measured IgG antibodies in saliva against the H1 stalk domain by using a chimeric protein containing an exotic H6 hemagglutinin head domain that the vaccinees have not previously been exposed to (strain A/mallard/Sweden/81/02 \[H6N1\]) and the same H1 stalk domain as expressed by the vaccine (strain A/California/04/09 \[H1N1\]).

Time frame: Baseline (pre-dose 1), Month 1 (28 days post-dose 1), Month 4 (28 days post-dose 2), and Month 15 (12 months post-dose 2)

Population: Participants in the per-protocol population with available data at each time point. The per-protocol population included all participants who received at least 1 vaccination and with no major deviations, including those considered likely to affect the immune response; data were included up to the time of the observed deviation event.

Anti-H1 hemagglutinin stalk secretory immunoglobulin A (IgA) in saliva was quantified using an enzyme-linked immunosorbent assay (ELISA). The ELISA measured secretory IgA antibodies (actively secreted in the mucosa) against the H1 stalk domain in saliva by using a chimeric protein containing an exotic H6 hemagglutinin head domain that the vaccinees have not previously been exposed to (strain A/mallard/Sweden/81/02 \[H6N1\]) and the same H1 stalk domain as expressed by the vaccine (strain A/California/04/09 \[H1N1\]).

Time frame: Baseline (pre-dose 1), Month 1 (28 days post-dose 1), Month 4 (28 days post-dose 2), and Month 15 (12 months post-dose 2)

Population: Participants in the per-protocol population with available data at each time point. The per-protocol population included all participants who received at least 1 vaccination and with no major deviations, including those considered likely to affect the immune response; data were included up to the time of the observed deviation event.

Anti-H1 hemagglutinin stalk total immunoglobulin A (IgA) in saliva was quantified using an enzyme-linked immunosorbent assay (ELISA). The ELISA measured total IgA antibodies antibodies (secreted through active and passive transfer processes in the mucosa) against the H1 stalk domain in saliva using a chimeric protein containing an exotic H6 hemagglutinin head domain that the vaccinees have not previously been exposed to (strain A/mallard/Sweden/81/02 \[H6N1\]) and the same H1 stalk domain as expressed by the vaccine (strain A/California/04/09 \[H1N1\]).

Time frame: Baseline (pre-dose 1), Month 1 (28 days post-dose 1), Month 4 (28 days post-dose 2), and Month 15 (12 months post-dose 2)

Population: Participants in the per-protocol population with available data at each time point. The per-protocol population included all participants who received at least 1 vaccination and with no major deviations, including those considered likely to affect the immune response; data were included up to the time of the observed deviation event.

Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune response leading to lysis of antibody-coated target cells by immune effector cells and is triggered by the interaction between the Fc portion of an antibody and Fc-gamma receptors expressed on immune effector cells. This bioluminescent assay measured antibodies to a chimeric hemagglutinin (H6 head domain and H1 stalk domain) virus that mediate ADCC activity via the Fc-receptor. ADCC activity was measured by the area under the curve (AUC) of luminescence per serial dilution.

Time frame: Baseline (pre-dose 1), Month 1 (28 days post-dose 1), Month 4 (28 days post-dose 2), and Month 15 (12 months post-dose 2)

Population: Participants in the per-protocol population with available data at Baseline and each time point. The per-protocol population included all participants who received at least 1 vaccination and with no major deviations, including those considered likely to affect the immune response; data were included up to the time of the observed deviation event.

To determine antibody breadth, assays were performed to measure the induction of cross-reactive IgG antibodies to a Group 1 influenza A virus with a heterosubtypic hemagglutinin distantly related to the currently circulating influenza A/H1 viruses, subtype H18, the stalk domain of which shares about 65% amino acid identity with the H1 vaccine stalk domain. Anti-H18 full-length (ecto-domain) hemagglutinin IgG was quantified by ELISA using a recombinant antigen based on Strain A/flat-faced bat/Peru/033/10 (H18N11) HA.

Time frame: Baseline (pre-dose 1), Month 1 (28 days post-dose 1), Month 4 (28 days post-dose 2), and Month 15 (12 months post-dose 2)

Population: Participants in the per-protocol population with available data at each time point. The per protocol population included all participants who received at least 1 vaccination and with no major deviations, including those considered likely to affect the immune response; data were included up to the time of the observed deviation event.

Anti-H1 hemagglutinin stalk immunoglobulin A (IgA) was quantified using an enzyme-linked immunosorbent assay (ELISA). The ELISA measured IgA antibodies against the H1 stalk domain by using a chimeric protein containing an exotic H6 hemagglutinin head domain that the vaccinees have not previously been exposed to (strain A/mallard/Sweden/81/02 \[H6N1\]) and the same H1 stalk domain as expressed by the vaccine (strain A/California/04/09 \[H1N1\]) .

Time frame: Baseline (pre-dose 1), Month 1 (28 days post-dose 1), Month 4 (28 days post-dose 2), and Month 15 (12 months post-dose 2)

Population: Participants in the per-protocol population with available data at each time point. The per protocol population included all participants who received at least 1 vaccination and with no major deviations, including those considered likely to affect the immune response; data were included up to the time of the observed deviation event.

Anti-H1 hemagglutinin stalk immunoglobulin G (IgG) was quantified using an enzyme-linked immunosorbent assay (ELISA). The ELISA measured IgG antibodies against the H1 stalk domain by using a chimeric protein containing an exotic H6 hemagglutinin head domain that the vaccinees have not previously been exposed to (strain A/mallard/Sweden/81/02 \[H6N1\]) and the same H1 stalk domain as expressed by the vaccine (strain A/California/04/09 \[H1N1\]) .

Time frame: Baseline (pre-dose 1), Month 1 (28 days post-dose 1), Month 4 (28 days post-dose 2), Month 9 (6 months post-dose 2), and Month 15 (12 months post-dose 2)

Population: Participants in the per-protocol population with available data at each time point. The per protocol population included all participants who received at least 1 vaccination and with no major deviations, including those considered likely to affect the immune response; data were included up to the time of the observed deviation event.