A Phase 4 interventional study of Influenza Virus Vaccine Inactivated in Influenza and Influenza Immunisation, sponsored by National Institute of Allergy and Infectious Diseases (NIAID). Terminated at 1 site in United States. Open to participants aged 18 Years to 49 Years, including healthy volunteers. Per ClinicalTrials.gov, last updated 2023-10-25.
Sponsored by National Institute of Allergy and Infectious Diseases (NIAID) · Phase 4, Interventional, and Prevention
This was an open label, single arm, Phase IV study of longitudinal immunologic responses to influenza vaccine in healthy adult participants, with the aim of enrolling up to 70 participants. This study enrolled males and non-pregnant females, 18-49 years old, inclusive. The participants were screened at enrollment with a history and physical exam and laboratory testing to ensure they were healthy enough to participate. Total enrollment was 60 participants.
Qualifying participants were vaccinated with an FDA approved seasonal inactivated influenza vaccine (IIV) according to the package insert. The study enrolled a total 60 participants. The primary objective of the study was to characterize HA-specific plasmablasts and memory B cells after influenza vaccination.
Note: Due to the Coronavirus Disease 2019 (COVID-19) pandemic, all non-essential research was halted in mid-March 2020. New enrollments were placed on hold for this study. Follow-up visits were also halted, which impacted the timing of participants' subsequent follow-up visits. Five participants had their Day 180 visits halted due to the COVID-19 pandemic.
This was an open label, single arm, Phase IV study of longitudinal immunologic responses to influenza vaccine in healthy adult participants. This study enrolled males and non-pregnant females, 18-49 years old. The primary objective of the study was to characterize hemagglutinin (HA)-specific plasmablasts and memory B cells after influenza vaccination. The secondary objective was to investigate the longevity of humoral immunity to influenza virus in humans. Total enrollment was 60 participants.
This was a multi-year study. Participant duration in this study was 180 days. Participants were screened at enrollment with a history and physical exam and laboratory testing to ensure they were healthy enough to participate. Qualifying participants were vaccinated with an FDA approved seasonal inactivated influenza vaccine (IIV) according to the package insert. Approximately 450 ml of blood was collected for the research assays during the course of the study. Specifically, 16 ml was be collected for screening; 48ml was collected at enrollment; 96ml was collected at visit Days 7 and 14; and 64 ml was collected at 28, 90, and 180 days post vaccination.
A total of 60 participants were enrolled over the course of the study. Participants who completed the study were given the option to re-enroll in subsequent years as long as they continued to meet all inclusion/exclusion criteria. Re-enrolling participants were re-consented, given new participants identifiers, and counted towards the enrollment number goal for each year of participation. Safety was assessed from the time of study enrollment through the last study visit, via monitoring of vital signs, and querying for change in health status.
Note: Due to the Coronavirus Disease 2019 (COVID-19) pandemic, all non-essential research was halted in mid-March 2020. New enrollments were placed on hold for this study. Follow-up visits were also halted, which impacted the timing of participants' subsequent follow-up visits. Five participants had their Day 180 visits halted due to the COVID-19 pandemic.
A request was submitted to the Emory University Institutional Review Board to extend the missed visit windows for the Day 180 visit for a maximum of up to 90 days, to ensure that ample time would be available to bring participants back for their missed visits. Enrollment for this study ended on September 30, 2020, before research activities could resume at Emory.
2,214 studies on the registry are indexed under Influenza, Human; 163 are open to participants now.
This study's enrollment of 60 is below the median of 238 across 1,853 interventional studies indexed under Influenza, Human.
Browse Influenza, Human studies →National Institute of Allergy and Infectious Diseases (NIAID) is the lead sponsor of 2,401 studies on the registry; 179 are open to participants now.
Of its 396 completed or terminated interventional studies of FDA-regulated products, 294 (74%) have results posted.
Counted across the registry records on this site, refreshed daily.
Screening labs within normal limits per the local laboratory normal ranges or considered to be not clinically significant by the investigator. Normal laboratory ranges are as listed below:
Hematology:
Chemistries:
Exclusion Criteria:
0.5 ml of seasonal inactivated influenza vaccine (IIV) was administered intramuscularly (IM) on Day 0 of each study season, for this multi-year study. A total of 60 participants were enrolled in this study.
Biological: Influenza Virus Vaccine Inactivated
A synthetic vaccine consisting of three inactivated influenza viruses: two different influenza type A strains and one influenza type B strain. Trivalent influenza vaccine is formulated annually, based on influenza strains projected to be prevalent in the upcoming flu season.
Percentage of Subjects Achieving Seroconversion (Pre-vaccination Hemagglutination Inhibition (HI) Titer <1:10 and a Post-vaccination HI Titer >1:40 or a Pre-vaccination HI Titer >1:10 and a Minimum Four-fold Rise in Post-vaccination HI Antibody Titer)
Hemagglutination inhibition titers were measured against reference influenza A/H1N1 and A/H3N2 strains, as well as influenza B Yamagata lineage influenza strains. Strains were matched to the composition of the vaccine received by each subject. For H1N1 and H3N2, hemagglutination was performed using receptor destroying enzyme (RDE)-treated plasma and turkey red blood cells following the World Health Organization (WHO) standard protocol. For influenza B, guinea pig red blood cells were used instead.
Time frame: Day 28
Percentage of Subjects With Pre-vaccination Titer <1:40
Hemagglutination inhibition titers were measured against reference influenza A/H1N1 and A/H3N2 strains, as well as influenza B Yamagata lineage influenza strains. Strains were matched to the composition of the vaccine received by each subject. For H1N1 and H3N2, hemagglutination was performed using RDE-treated plasma and turkey red blood cells following the WHO standard protocol. For influenza B, guinea pig red blood cells were used instead.
Time frame: Day 0
Endpoint Immunoglobulin G (IgG) Titers for Serum Antibody Responses Directed Against the Full Length A/H1N1 Hemagglutinin (HA) Protein
ELISAs were performed using serial dilutions of plasma incubated on Maxisorp ELISA plates coated with 50 nanograms per well of H1N1 hemagglutinin protein from strain A/Michigan/45/2015. Plasma samples were drawn from donors who received the 2017-18 or 2018-19 influenza vaccine. Bound serum IgG was detected using 1:1000 diluted goat anti-human IgG-horseradish peroxidase (HRP) from Jackson Immunoresearch and plates were developed using o-phenylenediamine (OPD) substrate and developed for 7 minutes. The ELISA endpoint titer was defined as the reciprocal dilution of plasma that produced a background-subtracted optical density at 490 (OD490) value of 0.3, corresponding to 10% of maximum binding. Endpoint titers were determined by interpolation from the serial dilution curve results using Graphpad Prism software.
Time frame: Day 0
Endpoint IgG Titers for Serum Antibody Responses Directed Against the Full Length A/H1N1 Hemagglutinin (HA) Protein
ELISAs were performed using serial dilutions of plasma incubated on Maxisorp ELISA plates coated with 50 nanograms per well of H1N1 hemagglutinin protein from strain A/Michigan/45/2015. Plasma samples were drawn from donors who received the 2017-18 or 2018-19 influenza vaccine. Bound serum IgG was detected using 1:1000 diluted goat anti-human IgG-HRP from Jackson Immunoresearch and plates were developed using OPD substrate and developed for 7 minutes. The ELISA endpoint titer was defined as the reciprocal dilution of plasma that produced a background-subtracted OD490 value of 0.3, corresponding to 10% of maximum binding. Endpoint titers were determined by interpolation from the serial dilution curve results using Graphpad Prism software.
Time frame: Day 180
Endpoint IgG Titers for Serum Antibody Responses Directed Against the Full Length A/H1N1 Hemagglutinin (HA) Protein
ELISAs were performed using serial dilutions of plasma incubated on Maxisorp ELISA plates coated with 50 nanograms per well of H1N1 hemagglutinin protein from strain A/Michigan/45/2015. Plasma samples were drawn from donors who received the 2017-18 or 2018-19 influenza vaccine. Bound serum IgG was detected using 1:1000 diluted goat anti-human IgG-HRP from Jackson Immunoresearch and plates were developed using OPD substrate and developed for 7 minutes. The ELISA endpoint titer was defined as the reciprocal dilution of plasma that produced a background-subtracted OD490 value of 0.3, corresponding to 10% of maximum binding. Endpoint titers were determined by interpolation from the serial dilution curve results using Graphpad Prism software.
Time frame: Day 28
Endpoint IgG Titers for Serum Antibody Responses Directed Against the H1N1 Hemagglutinin (HA) Stem Region
ELISAs were performed using serial dilutions of plasma incubated on Maxisorp ELISA plates coated with 50 nanograms per well of a chimeric HA protein containing the head domain from an H6 influenza virus and the stem domain from A/California/07/2009. Plasma samples were drawn from donors who received the 2017-18 or 2018-19 influenza vaccine. Bound serum IgG was detected using 1:1000 diluted goat anti-human IgG-HRP from Jackson Immunoresearch and plates were developed using OPD substrate and developed for 7 minutes. The ELISA endpoint titer was defined as the reciprocal dilution of plasma that produced a background-subtracted OD490 value of 0.3, corresponding to 10% of maximum binding. Endpoint titers were determined by interpolation from the serial dilution curve results using Graphpad Prism software.
Time frame: Day 0
Endpoint IgG Titers for Serum Antibody Responses Directed Against the H1N1 Hemagglutinin (HA) Stem Region
ELISAs were performed using serial dilutions of plasma incubated on Maxisorp ELISA plates coated with 50 nanograms per well of a chimeric HA protein containing the head domain from an H6 influenza virus and the stem domain from A/California/07/2009. Plasma samples were drawn from donors who received the 2017-18 or 2018-19 influenza vaccine. Bound serum IgG was detected using 1:1000 diluted goat anti-human IgG-HRP from Jackson Immunoresearch and plates were developed using OPD substrate and developed for 7 minutes. The ELISA endpoint titer was defined as the reciprocal dilution of plasma that produced a background-subtracted OD490 value of 0.3, corresponding to 10% of maximum binding. Endpoint titers were determined by interpolation from the serial dilution curve results using Graphpad Prism software.
Time frame: Day 180
Endpoint IgG Titers for Serum Antibody Responses Directed Against the H1N1 Hemagglutinin (HA) Stem Region.
ELISAs were performed using serial dilutions of plasma incubated on Maxisorp ELISA plates coated with 50 nanograms per well of a chimeric HA protein containing the head domain from an H6 influenza virus and the stem domain from A/California/07/2009. Plasma samples were drawn from donors who received the 2017-18 or 2018-19 influenza vaccine. Bound serum IgG was detected using 1:1000 diluted goat anti-human IgG-HRP from Jackson Immunoresearch and plates were developed using OPD substrate and developed for 7 minutes. The ELISA endpoint titer was defined as the reciprocal dilution of plasma that produced a background-subtracted OD490 value of 0.3, corresponding to 10% of maximum binding. Endpoint titers were determined by interpolation from the serial dilution curve results using Graphpad Prism software.
Time frame: Day 28
Expression of Human Influenza-specific Monoclonal Antibodies From Plasmablasts
Monoclonal antibodies were cloned from sorted H1 hemagglutinin-binding cells expressing high levels of the CD38 protein (CD38hi) plasmablasts isolated from a single donor on day 7 post vaccination.
Time frame: Day 7
Frequency of Hemagglutinin (HA) Head-specific IgG Secreting Memory B Cells Per Total IgG Secreting Cells
Peripheral blood mononuclear cells from volunteers who received the 2017-18 or 2018-19 influenza vaccine were stimulated with R-848/interleukin (IL)-2 to induce polyclonal plasmablast differentiation. Total H1-specific memory B cells were detected using hemagglutinin protein from A/Michigan/45/2015 as an ELISPOT capture antigen and IgG detection. Frequencies were expressed as a percentage of the spots observed using total IgG capture antibody. Head specific memory B cell frequencies were calculated by subtracting the percentage of stem-specific IgG memory B cells from the percentage of total H1-specific cells.
Time frame: Day 0
Frequency of Hemagglutinin (HA) Head-specific IgG Secreting Memory B Cells Per Total IgG Secreting Cells
Peripheral blood mononuclear cells from volunteers who received the 2017-18 or 2018-19 influenza vaccine were stimulated with R-848/IL-2 to induce polyclonal plasmablast differentiation. Total H1-specific memory B cells were detected using hemagglutinin protein from A/Michigan/45/2015 as an ELISPOT capture antigen and IgG detection. Frequencies were expressed as a percentage of the spots observed using total IgG capture antibody. Head specific memory B cell frequencies were calculated by subtracting the percentage of stem-specific IgG memory B cells from the percentage of total H1-specific cells.
Time frame: Day 180
Frequency of Hemagglutinin (HA) Head-specific IgG Secreting Memory B Cells Per Total IgG Secreting Cells
Peripheral blood mononuclear cells from volunteers who received the 2017-18 or 2018-19 influenza vaccine were stimulated with R-848/IL-2 to induce polyclonal plasmablast differentiation. Total H1-specific memory B cells were detected using hemagglutinin protein from A/Michigan/45/2015 as an ELISPOT capture antigen and IgG detection. Frequencies were expressed as a percentage of the spots observed using total IgG capture antibody. Head specific memory B cell frequencies were calculated by subtracting the percentage of stem-specific IgG memory B cells from the percentage of total H1-specific cells.
Time frame: Day 28
Frequency of Hemagglutinin (HA) Stem-specific IgG Secreting Memory B Cells Per Total IgG Secreting Cells
Peripheral blood mononuclear cells from volunteers who received the 2017-18 or 2018-19 influenza vaccine were stimulated with R-848/IL-2 to induce polyclonal plasmablast differentiation. Stem-specific memory B cells were detected using a chimeric hemagglutinin containing an H6 influenza head domain and the stem domain from A/California/07/2009 as an ELISPOT capture antigen. Frequencies were expressed as a percentage of the spots observed using total IgG capture antibody.
Time frame: Day 0
Frequency of Hemagglutinin (HA) Stem-specific IgG Secreting Memory B Cells Per Total IgG Secreting Cells
Peripheral blood mononuclear cells from volunteers who received the 2017-18 or 2018-19 influenza vaccine were stimulated with R-848/IL-2 to induce polyclonal plasmablast differentiation. Stem-specific memory B cells were detected using a chimeric hemagglutinin containing an H6 influenza head domain and the stem domain from A/California/07/2009 as an ELISPOT capture antigen. Frequencies were expressed as a percentage of the spots observed using total IgG capture antibody.
Time frame: Day 180
Frequency of Hemagglutinin (HA) Stem-specific IgG Secreting Memory B Cells Per Total IgG Secreting Cells
Peripheral blood mononuclear cells from volunteers who received the 2017-18 or 2018-19 influenza vaccine were stimulated with R-848/IL-2 to induce polyclonal plasmablast differentiation. Stem-specific memory B cells were detected using a chimeric hemagglutinin containing an H6 influenza head domain and the stem domain from A/California/07/2009 as an ELISPOT capture antigen. Frequencies were expressed as a percentage of the spots observed using total IgG capture antibody.
Time frame: Day 28
Function of Human Influenza-specific Monoclonal Antibodies Assessed by Virus Neutralization Assay
Madin-Darby canine kidney (MDCK) cells in 96-well plates were infected in duplicate for 5 hours with H1N1 influenza virus (strain A/Michigan/45/2015) that had been pre-incubated for 15 minutes at 37 degrees with influenza monoclonal antibodies. Pre-incubations were performed using serial 2-fold dilutions of each monoclonal antibody, beginning at a concentration of 30 micrograms per milliliter. Virus was used at a dose sufficient to yield 5% infection in the absence of antibody. Infection was assessed after 5 hours in methanol-permeabilized cells by staining with an influenza nucleoprotein-specific mouse antibody (HB65), then phycoerythrin (PE)-conjugated anti-mouse IgG, followed by identification of PE-positive cells by flow cytometry. The 50% neutralizing titer reported is equivalent to the lowest monoclonal antibody concentration that resulted in a greater than or equal to 2-fold reduction in viral infection relative to the no-antibody control.
Time frame: Day 7
Function of Human Influenza-specific Monoclonal Antibodies Assessed by Enzyme-Linked Immunosorbent Assay (ELISA)
Influenza-specific mAbs were assessed for HA binding by ELISA using recombinant HA protein from H1N1 strain A/California/07/2009. 50 ng of HA were coated per well in Maxisorp ELISA plates and serial dilutions of mAb were added. The concentration of each mAb that gave 50% maximal binding (EC50) was determined.
Time frame: Day 7
Function of Human Influenza-specific Monoclonal Antibodies Assessed by Hemagglutination Inhibition (HAI) Assay
The HAI titer for each antibody was determined using the World Health Organization (WHO) standard protocol using H1N1 strain A/Michigan/45/2015.
Time frame: Day 7
Quadrivalent Influenza Vaccine Immunoglobulin Gamma-1 (IgG1) ELISA Endpoint Titer
ELISA titers were measured against the matched seasonal quadrivalent inactivated influenza vaccine received by each subject using a secondary antibody specific for human IgG1. The endpoint ELISA titer was defined as the reciprocal dilution of plasma that gave an optical density (OD) reading of 0.2, which corresponded to \~3 standard deviations above the background OD for ELISA wells incubated without plasma.
Time frame: Day 0
Quadrivalent Influenza Vaccine IgG1 ELISA Endpoint Titer
ELISA titers were measured against the matched seasonal quadrivalent inactivated influenza vaccine received by each subject using a secondary antibody specific for human IgG1. The endpoint ELISA titer was defined as the reciprocal dilution of plasma that gave an optical density (OD) reading of 0.2, which corresponded to \~3 standard deviations above the background OD for ELISA wells incubated without plasma.
Time frame: Day 180
Quadrivalent Influenza Vaccine IgG1 ELISA Endpoint Titer
ELISA titers were measured against the matched seasonal quadrivalent inactivated influenza vaccine received by each subject using a secondary antibody specific for human IgG1. The endpoint ELISA titer was defined as the reciprocal dilution of plasma that gave an optical density (OD) reading of 0.2, which corresponded to \~3 standard deviations above the background OD for ELISA wells incubated without plasma.
Time frame: Day 28
Plasmablast Responses Against Hemagglutinin (HA) Head Antigens Assessed by Direct ex Vivo Enzyme-linked Immunospot (ELISPOT)
Plasmablasts (PB) were enumerated directly from frozen PBMC without stimulation. Total H1-specific cells were detected using hemagglutinin protein from A/Michigan/45/2015 as an ELISPOT capture antigen and IgG detection. Frequencies were expressed as the number of spots observed per million peripheral blood mononuclear cells. Head specific PB frequencies were calculated by subtracting the number of stem-specific PB per million peripheral blood mononuclear cells from the number of total H1-specific PB per million cells.
Time frame: Day 7
Plasmablast Responses Against Hemagglutinin (HA) Stem Antigens Assessed by Direct ex Vivo Enzyme-linked Immunospot (ELISPOT)
Plasmablasts (PB) were enumerated directly from frozen PBMC without stimulation. Stem-specific cells were detected using a chimeric hemagglutinin containing an H6 influenza head domain and the stem domain from A/California/07/2009 as an ELISPOT capture antigen and IgG detection. Frequencies were expressed as the number of spots observed per million peripheral blood mononuclear cells.
Time frame: Day 7
The following methods were used to recruit participants: traditional flyers posted throughout campus, word of mouth referrals, and social media posts. In addition, listserves, and campus newsletters were also leveraged to expand our participant reach.
| Milestone | Seasonal Flu Vaccine/Healthy Adult Volunteers |
|---|---|
| Started | 60 |
| Completed | 45 |
| Not completed | 15 |
| Withdrew: 15 participants were unable to complete study. research was halted due to coronavirus disease 2019. | 15 |
Hemagglutination inhibition titers were measured against reference influenza A/H1N1 and A/H3N2 strains, as well as influenza B Yamagata lineage influenza strains. Strains were matched to the composition of the vaccine received by each subject. For H1N1 and H3N2, hemagglutination was performed using receptor destroying enzyme (RDE)-treated plasma and turkey red blood cells following the World Health Organization (WHO) standard protocol. For influenza B, guinea pig red blood cells were used instead.
| Participants | Seasonal Flu Vaccine/Healthy Adult Volunteers |
|---|---|
| H1N1 seroconversion | 28 |
| H3N2 seroconversion | 30 |
| Influenza B Yamagata lineage seroconversion | 32 |
Hemagglutination inhibition titers were measured against reference influenza A/H1N1 and A/H3N2 strains, as well as influenza B Yamagata lineage influenza strains. Strains were matched to the composition of the vaccine received by each subject. For H1N1 and H3N2, hemagglutination was performed using RDE-treated plasma and turkey red blood cells following the WHO standard protocol. For influenza B, guinea pig red blood cells were used instead.
| Participants | Seasonal Flu Vaccine/Healthy Adult Volunteers |
|---|---|
| A/H1N1 HAI titer <1:40 on day 0 | 24 |
| A/H3N2 HAI titer <1:40 on day 0 | 25 |
| B Yamagata lineage HAI titer <1:40 on day 0 | 13 |
ELISAs were performed using serial dilutions of plasma incubated on Maxisorp ELISA plates coated with 50 nanograms per well of H1N1 hemagglutinin protein from strain A/Michigan/45/2015. Plasma samples were drawn from donors who received the 2017-18 or 2018-19 influenza vaccine. Bound serum IgG was detected using 1:1000 diluted goat anti-human IgG-horseradish peroxidase (HRP) from Jackson Immunoresearch and plates were developed using o-phenylenediamine (OPD) substrate and developed for 7 minutes. The ELISA endpoint titer was defined as the reciprocal dilution of plasma that produced a background-subtracted optical density at 490 (OD490) value of 0.3, corresponding to 10% of maximum binding. Endpoint titers were determined by interpolation from the serial dilution curve results using Graphpad Prism software.
| H1 HA ELISA endpoint reciprocal dilution | Seasonal Flu Vaccine/Healthy Adult Volunteers |
|---|---|
| Endpoint Immunoglobulin G (IgG) Titers for Serum Antibody Responses Directed Against the Full Length A/H1N1 Hemagglutinin (HA) Protein | 11851 (4170 to 39548) |
ELISAs were performed using serial dilutions of plasma incubated on Maxisorp ELISA plates coated with 50 nanograms per well of H1N1 hemagglutinin protein from strain A/Michigan/45/2015. Plasma samples were drawn from donors who received the 2017-18 or 2018-19 influenza vaccine. Bound serum IgG was detected using 1:1000 diluted goat anti-human IgG-HRP from Jackson Immunoresearch and plates were developed using OPD substrate and developed for 7 minutes. The ELISA endpoint titer was defined as the reciprocal dilution of plasma that produced a background-subtracted OD490 value of 0.3, corresponding to 10% of maximum binding. Endpoint titers were determined by interpolation from the serial dilution curve results using Graphpad Prism software.
| H1 HA ELISA endpoint reciprocal dilution | Seasonal Flu Vaccine/Healthy Adult Volunteers |
|---|---|
| Endpoint IgG Titers for Serum Antibody Responses Directed Against the Full Length A/H1N1 Hemagglutinin (HA) Protein | 48445 (30675 to 78013) |
ELISAs were performed using serial dilutions of plasma incubated on Maxisorp ELISA plates coated with 50 nanograms per well of H1N1 hemagglutinin protein from strain A/Michigan/45/2015. Plasma samples were drawn from donors who received the 2017-18 or 2018-19 influenza vaccine. Bound serum IgG was detected using 1:1000 diluted goat anti-human IgG-HRP from Jackson Immunoresearch and plates were developed using OPD substrate and developed for 7 minutes. The ELISA endpoint titer was defined as the reciprocal dilution of plasma that produced a background-subtracted OD490 value of 0.3, corresponding to 10% of maximum binding. Endpoint titers were determined by interpolation from the serial dilution curve results using Graphpad Prism software.
| H1 HA ELISA endpoint reciprocal dilution | Seasonal Flu Vaccine/Healthy Adult Volunteers |
|---|---|
| Endpoint IgG Titers for Serum Antibody Responses Directed Against the Full Length A/H1N1 Hemagglutinin (HA) Protein | 121284 (66218 to 190689) |
ELISAs were performed using serial dilutions of plasma incubated on Maxisorp ELISA plates coated with 50 nanograms per well of a chimeric HA protein containing the head domain from an H6 influenza virus and the stem domain from A/California/07/2009. Plasma samples were drawn from donors who received the 2017-18 or 2018-19 influenza vaccine. Bound serum IgG was detected using 1:1000 diluted goat anti-human IgG-HRP from Jackson Immunoresearch and plates were developed using OPD substrate and developed for 7 minutes. The ELISA endpoint titer was defined as the reciprocal dilution of plasma that produced a background-subtracted OD490 value of 0.3, corresponding to 10% of maximum binding. Endpoint titers were determined by interpolation from the serial dilution curve results using Graphpad Prism software.
| stem ELISA endpoint reciprocal dilution | Seasonal Flu Vaccine/Healthy Adult Volunteers |
|---|---|
| Endpoint IgG Titers for Serum Antibody Responses Directed Against the H1N1 Hemagglutinin (HA) Stem Region | 11140 (7032 to 22360) |
ELISAs were performed using serial dilutions of plasma incubated on Maxisorp ELISA plates coated with 50 nanograms per well of a chimeric HA protein containing the head domain from an H6 influenza virus and the stem domain from A/California/07/2009. Plasma samples were drawn from donors who received the 2017-18 or 2018-19 influenza vaccine. Bound serum IgG was detected using 1:1000 diluted goat anti-human IgG-HRP from Jackson Immunoresearch and plates were developed using OPD substrate and developed for 7 minutes. The ELISA endpoint titer was defined as the reciprocal dilution of plasma that produced a background-subtracted OD490 value of 0.3, corresponding to 10% of maximum binding. Endpoint titers were determined by interpolation from the serial dilution curve results using Graphpad Prism software.
| stem ELISA endpoint reciprocal dilution | Seasonal Flu Vaccine/Healthy Adult Volunteers |
|---|---|
| Endpoint IgG Titers for Serum Antibody Responses Directed Against the H1N1 Hemagglutinin (HA) Stem Region | 16694 (7818 to 32048) |
ELISAs were performed using serial dilutions of plasma incubated on Maxisorp ELISA plates coated with 50 nanograms per well of a chimeric HA protein containing the head domain from an H6 influenza virus and the stem domain from A/California/07/2009. Plasma samples were drawn from donors who received the 2017-18 or 2018-19 influenza vaccine. Bound serum IgG was detected using 1:1000 diluted goat anti-human IgG-HRP from Jackson Immunoresearch and plates were developed using OPD substrate and developed for 7 minutes. The ELISA endpoint titer was defined as the reciprocal dilution of plasma that produced a background-subtracted OD490 value of 0.3, corresponding to 10% of maximum binding. Endpoint titers were determined by interpolation from the serial dilution curve results using Graphpad Prism software.
| stem ELISA endpoint reciprocal dilution | Seasonal Flu Vaccine/Healthy Adult Volunteers |
|---|---|
| Endpoint IgG Titers for Serum Antibody Responses Directed Against the H1N1 Hemagglutinin (HA) Stem Region. | 29905 (18411 to 42782) |
Monoclonal antibodies were cloned from sorted H1 hemagglutinin-binding cells expressing high levels of the CD38 protein (CD38hi) plasmablasts isolated from a single donor on day 7 post vaccination.
| number of mAbs cloned | Seasonal Flu Vaccine/Healthy Adult Volunteers |
|---|---|
| Expression of Human Influenza-specific Monoclonal Antibodies From Plasmablasts | 6 |
Peripheral blood mononuclear cells from volunteers who received the 2017-18 or 2018-19 influenza vaccine were stimulated with R-848/interleukin (IL)-2 to induce polyclonal plasmablast differentiation. Total H1-specific memory B cells were detected using hemagglutinin protein from A/Michigan/45/2015 as an ELISPOT capture antigen and IgG detection. Frequencies were expressed as a percentage of the spots observed using total IgG capture antibody. Head specific memory B cell frequencies were calculated by subtracting the percentage of stem-specific IgG memory B cells from the percentage of total H1-specific cells.
| % of IgG memory B cells | Seasonal Flu Vaccine/Healthy Adult Volunteers |
|---|---|
| Frequency of Hemagglutinin (HA) Head-specific IgG Secreting Memory B Cells Per Total IgG Secreting Cells | 0.039 (0.027 to 0.063) |
Peripheral blood mononuclear cells from volunteers who received the 2017-18 or 2018-19 influenza vaccine were stimulated with R-848/IL-2 to induce polyclonal plasmablast differentiation. Total H1-specific memory B cells were detected using hemagglutinin protein from A/Michigan/45/2015 as an ELISPOT capture antigen and IgG detection. Frequencies were expressed as a percentage of the spots observed using total IgG capture antibody. Head specific memory B cell frequencies were calculated by subtracting the percentage of stem-specific IgG memory B cells from the percentage of total H1-specific cells.
| % of IgG memory B cells | Seasonal Flu Vaccine/Healthy Adult Volunteers |
|---|---|
| Frequency of Hemagglutinin (HA) Head-specific IgG Secreting Memory B Cells Per Total IgG Secreting Cells | 0.17 (0.084 to 0.32) |
Peripheral blood mononuclear cells from volunteers who received the 2017-18 or 2018-19 influenza vaccine were stimulated with R-848/IL-2 to induce polyclonal plasmablast differentiation. Total H1-specific memory B cells were detected using hemagglutinin protein from A/Michigan/45/2015 as an ELISPOT capture antigen and IgG detection. Frequencies were expressed as a percentage of the spots observed using total IgG capture antibody. Head specific memory B cell frequencies were calculated by subtracting the percentage of stem-specific IgG memory B cells from the percentage of total H1-specific cells.
| % of IgG memory B cells | Seasonal Flu Vaccine/Healthy Adult Volunteers |
|---|---|
| Frequency of Hemagglutinin (HA) Head-specific IgG Secreting Memory B Cells Per Total IgG Secreting Cells | 0.63 (0.27 to 1.50) |
Peripheral blood mononuclear cells from volunteers who received the 2017-18 or 2018-19 influenza vaccine were stimulated with R-848/IL-2 to induce polyclonal plasmablast differentiation. Stem-specific memory B cells were detected using a chimeric hemagglutinin containing an H6 influenza head domain and the stem domain from A/California/07/2009 as an ELISPOT capture antigen. Frequencies were expressed as a percentage of the spots observed using total IgG capture antibody.
| % of IgG memory B cells | Seasonal Flu Vaccine/Healthy Adult Volunteers |
|---|---|
| Frequency of Hemagglutinin (HA) Stem-specific IgG Secreting Memory B Cells Per Total IgG Secreting Cells | 0.023 (0.0094 to 0.041) |
Peripheral blood mononuclear cells from volunteers who received the 2017-18 or 2018-19 influenza vaccine were stimulated with R-848/IL-2 to induce polyclonal plasmablast differentiation. Stem-specific memory B cells were detected using a chimeric hemagglutinin containing an H6 influenza head domain and the stem domain from A/California/07/2009 as an ELISPOT capture antigen. Frequencies were expressed as a percentage of the spots observed using total IgG capture antibody.
| % of IgG memory B cells | Seasonal Flu Vaccine/Healthy Adult Volunteers |
|---|---|
| Frequency of Hemagglutinin (HA) Stem-specific IgG Secreting Memory B Cells Per Total IgG Secreting Cells | 0.088 (0.056 to 0.13) |
Peripheral blood mononuclear cells from volunteers who received the 2017-18 or 2018-19 influenza vaccine were stimulated with R-848/IL-2 to induce polyclonal plasmablast differentiation. Stem-specific memory B cells were detected using a chimeric hemagglutinin containing an H6 influenza head domain and the stem domain from A/California/07/2009 as an ELISPOT capture antigen. Frequencies were expressed as a percentage of the spots observed using total IgG capture antibody.
| % of IgG memory B cells | Seasonal Flu Vaccine/Healthy Adult Volunteers |
|---|---|
| Frequency of Hemagglutinin (HA) Stem-specific IgG Secreting Memory B Cells Per Total IgG Secreting Cells | 0.17 (0.091 to 0.29) |
Madin-Darby canine kidney (MDCK) cells in 96-well plates were infected in duplicate for 5 hours with H1N1 influenza virus (strain A/Michigan/45/2015) that had been pre-incubated for 15 minutes at 37 degrees with influenza monoclonal antibodies. Pre-incubations were performed using serial 2-fold dilutions of each monoclonal antibody, beginning at a concentration of 30 micrograms per milliliter. Virus was used at a dose sufficient to yield 5% infection in the absence of antibody. Infection was assessed after 5 hours in methanol-permeabilized cells by staining with an influenza nucleoprotein-specific mouse antibody (HB65), then phycoerythrin (PE)-conjugated anti-mouse IgG, followed by identification of PE-positive cells by flow cytometry. The 50% neutralizing titer reported is equivalent to the lowest monoclonal antibody concentration that resulted in a greater than or equal to 2-fold reduction in viral infection relative to the no-antibody control.
| 50% neutralizing conc. (micrograms/ml) | Seasonal Flu Vaccine/Healthy Adult Volunteers |
|---|---|
| Function of Human Influenza-specific Monoclonal Antibodies Assessed by Virus Neutralization Assay | 0.94 (0.58 to 1.9) |
Influenza-specific mAbs were assessed for HA binding by ELISA using recombinant HA protein from H1N1 strain A/California/07/2009. 50 ng of HA were coated per well in Maxisorp ELISA plates and serial dilutions of mAb were added. The concentration of each mAb that gave 50% maximal binding (EC50) was determined.
| ELISA EC50 titer in micrograms per ml | Seasonal Flu Vaccine/Healthy Adult Volunteers |
|---|---|
| Function of Human Influenza-specific Monoclonal Antibodies Assessed by Enzyme-Linked Immunosorbent Assay (ELISA) | 0.13 (.087 to .274) |
The HAI titer for each antibody was determined using the World Health Organization (WHO) standard protocol using H1N1 strain A/Michigan/45/2015.
| HAI titer (micrograms per millliliter) | Seasonal Flu Vaccine/Healthy Adult Volunteers |
|---|---|
| Function of Human Influenza-specific Monoclonal Antibodies Assessed by Hemagglutination Inhibition (HAI) Assay | 7.5 (5 to 10) |
ELISA titers were measured against the matched seasonal quadrivalent inactivated influenza vaccine received by each subject using a secondary antibody specific for human IgG1. The endpoint ELISA titer was defined as the reciprocal dilution of plasma that gave an optical density (OD) reading of 0.2, which corresponded to \~3 standard deviations above the background OD for ELISA wells incubated without plasma.
| Reciprocal endpoint plasma dilution | Seasonal Flu Vaccine/Healthy Adult Volunteers |
|---|---|
| Quadrivalent Influenza Vaccine Immunoglobulin Gamma-1 (IgG1) ELISA Endpoint Titer | 1877 (1435 to 2489) |
ELISA titers were measured against the matched seasonal quadrivalent inactivated influenza vaccine received by each subject using a secondary antibody specific for human IgG1. The endpoint ELISA titer was defined as the reciprocal dilution of plasma that gave an optical density (OD) reading of 0.2, which corresponded to \~3 standard deviations above the background OD for ELISA wells incubated without plasma.
| Reciprocal endpoint plasma dilution | Seasonal Flu Vaccine/Healthy Adult Volunteers |
|---|---|
| Quadrivalent Influenza Vaccine IgG1 ELISA Endpoint Titer | 3026 (2453 to 3722) |
ELISA titers were measured against the matched seasonal quadrivalent inactivated influenza vaccine received by each subject using a secondary antibody specific for human IgG1. The endpoint ELISA titer was defined as the reciprocal dilution of plasma that gave an optical density (OD) reading of 0.2, which corresponded to \~3 standard deviations above the background OD for ELISA wells incubated without plasma.
| reciprocal endpoint titer | Seasonal Flu Vaccine/Healthy Adult Volunteers |
|---|---|
| Quadrivalent Influenza Vaccine IgG1 ELISA Endpoint Titer | 3744 (3018 to 4356) |
Plasmablasts (PB) were enumerated directly from frozen PBMC without stimulation. Total H1-specific cells were detected using hemagglutinin protein from A/Michigan/45/2015 as an ELISPOT capture antigen and IgG detection. Frequencies were expressed as the number of spots observed per million peripheral blood mononuclear cells. Head specific PB frequencies were calculated by subtracting the number of stem-specific PB per million peripheral blood mononuclear cells from the number of total H1-specific PB per million cells.
| Head-specific IgG PB per million PBMC | Seasonal Flu Vaccine/Healthy Adult Volunteers |
|---|---|
| Plasmablast Responses Against Hemagglutinin (HA) Head Antigens Assessed by Direct ex Vivo Enzyme-linked Immunospot (ELISPOT) | 1096 (635 to 2060) |
Plasmablasts (PB) were enumerated directly from frozen PBMC without stimulation. Stem-specific cells were detected using a chimeric hemagglutinin containing an H6 influenza head domain and the stem domain from A/California/07/2009 as an ELISPOT capture antigen and IgG detection. Frequencies were expressed as the number of spots observed per million peripheral blood mononuclear cells.
| Stem-specific IgG PB per million PBMC | Seasonal Flu Vaccine/Healthy Adult Volunteers |
|---|---|
| Plasmablast Responses Against Hemagglutinin (HA) Stem Antigens Assessed by Direct ex Vivo Enzyme-linked Immunospot (ELISPOT) | 47.6 (15.5 to 98.5) |
Collected over 6 months Adverse event data were collected from Day 0 (enrollment) to Day 180 (the end of follow-up). Adverse event data were collected at each of the follow-up visits (Day 7, Day 14, Day 28, Day 90, and Day 180).. Non-serious events are listed at a 2% frequency threshold.
| Group | Deaths | Serious | Other |
|---|---|---|---|
| Seasonal Flu Vaccine/Healthy Adult Volunteers | 0/60 (0%) | 0/60 (0%) | 9/60 (15%) |
| Event | Seasonal Flu Vaccine/Healthy Adult Volunteers |
|---|---|
| InfluenzaGeneral disorders | 3/60 |
| Sore throat (pharyngitis)Infections and infestations | 2/60 |
| Ear painEar and labyrinth disorders | 1/60 |
| Urinary tract infectionInfections and infestations | 1/60 |
| Foot painGeneral disorders | 1/60 |
| Bell's PalsyNervous system disorders | 1/60 |
Baseline characteristics were measured for all 60 participants. All participants in this study received 0.5 ml of seasonal inactivated influenza vaccine (IIV), administered intramuscularly (IM) on Day 0 of each study season.
| Age, Categorical(Participants) | Seasonal Flu Vaccine/Healthy Adult Volunteers |
|---|---|
| <=18 years | 0 |
| Between 18 and 65 years | 60 |
| >=65 years | 0 |
| Age, Continuous(years) | Seasonal Flu Vaccine/Healthy Adult Volunteers |
|---|---|
| Mean | 28.5 (18 to 49) |
| Sex: Female, Male(Participants) | Seasonal Flu Vaccine/Healthy Adult Volunteers |
|---|---|
| Female | 33 |
| Male | 27 |
| Ethnicity (NIH/OMB)(Participants) | Seasonal Flu Vaccine/Healthy Adult Volunteers |
|---|---|
| Hispanic or Latino | 4 |
| Not Hispanic or Latino | 55 |
| Unknown or Not Reported | 1 |
| Race (NIH/OMB)(Participants) | Seasonal Flu Vaccine/Healthy Adult Volunteers |
|---|---|
| American Indian or Alaska Native | 0 |
| Asian | 22 |
| Native Hawaiian or Other Pacific Islander | 0 |
| Black or African American | 3 |
| White | 31 |
| More than one race | 1 |
| Unknown or Not Reported | 3 |
| Region of Enrollment(Participants) | Seasonal Flu Vaccine/Healthy Adult Volunteers |
|---|---|
| United States | 60 |
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National Institute of Allergy and Infectious Diseases (NIAID)