Evaluation of Human Immune Responses to Influenza Virus Vaccination in Healthy Volunteers

Study Overview
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Brief Summary
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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.

Detailed Description
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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.

Official Title
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Evaluation of Human Immune Responses to Influenza Virus Vaccination in Healthy Volunteers

Conditions
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Influenza, Influenza Immunisation

Intervention / Treatment
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* Biological: Influenza Virus Vaccine Inactivated


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Male or female subjects between 18 and 49 years of age, inclusive. Subjects capable of providing written informed consent prior to initiation of any study procedures. Subjects able to understand and comply with planned study procedures and be available for all study visits. 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: Hemoglobin: Male- 12.9-16.1 gm/dL, Female- 11.4-14.4 gm/dL White blood cells (WBC): Male- 4.2-9.2 upper limit (uL), Female- 4-10 upper limit (uL) Platelet count: 150-400 upper limit (uL) Chemistries: Kidney function: Glomerular filtration rate (GFR) > / = 60 mL/min/1.73 m^2; Liver enzymes: Albumin > / = 3.5 g/dL; alanine aminotransferase (ALT) <66 U/L; aspartate aminotransferase (AST) <62 U/L Subjects who have not received the seasonal influenza vaccine in the current flu season and are not suspected to have had an influenza infection in the current flu season. Female subjects of child bearing potential must have a negative urine pregnancy test at the screening visit, enrollment visit and all subsequent study visits longer than 14 days since the last pregnancy test. Exclusion Criteria: Known infection with human immunodeficiency virus (HIV), hepatitis C virus (HCV), or hepatitis B virus (HBV). This information will be obtained verbally from the patient. If female, active pregnancy or breast-feeding or plans to become pregnant during study participation. Chronic medical conditions that cause immunodeficiency or that require medications which could alter immune function such as immunosuppressants and immunoenhancers. Have any medical disease or condition that, in the opinion of the site principal investigator or appropriate sub-investigator, is a contraindication to study participation. This includes any chronic medical disease or condition, defined as persisting 3 months (defined as 90 days) or longer, that would place the subject at an unacceptable risk of injury, render the subject unable to meet the requirements of the protocol, or may interfere with the evaluation of responses or the subject's successful completion of this study Have an acute illness, as determined by the site principal investigator or appropriate sub-investigator, within 72 hours prior to study vaccination. An acute illness which is nearly resolved with only minor residual symptoms remaining is allowable if, in the opinion of the site principal investigator or appropriate sub-investigator, the residual symptoms will not interfere with the ability to assess safety parameters as required by the protocol. Persons taking anticoagulants, long-term aspirin therapy, or long-term systemic steroids (greater than 3 months in the past 12 months and any within 30 days). Have known hypersensitivity or allergy to eggs, egg or chicken protein, or other components of the study vaccine; Have a known latex allergy; Have a history of severe reactions following previous immunization with licensed influenza virus vaccines. Have a history of Guillain-Barre syndrome. Subjects who had or are suspected to have had an influenza infection in the current influenza season. Subjects who, at screening, have abnormal vital signs and/or physical exam, including a temperature > / = 38.0 C, Systolic blood pressure < / = 90 or > / = 160 mmHg, pulse < / = 60 or > 110 beats per minute, new rash, signs of infection. Subjects who have already received the seasonal influenza vaccine in the current influenza vaccination season.

Ages Eligible for Study
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Minimum Age: 18 Years
Maximum Age: 49 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Prevention
Intervention Model: Single Group Assignment
Masking: None (Open Label)


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Experimental: Seasonal Flu Vaccine/Healthy Adult Volunteers<br>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<br>* 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.<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| 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. | 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. | Day 0 | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| 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. | 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. | 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. | 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. | 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. | 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. | 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. | 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. | 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. | 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. | 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. | 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. | 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. | 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. | 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. | 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. | 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. | 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. | 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. | 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. | 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. | Day 7 | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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ex-vivo, Healthy Volunteers, Immune Response, Influenza, plasmablasts, Vaccine