Abstract
Background:
The continuing evolution of influenza viruses poses a challenge to vaccine prevention, highlighting the need for a universal influenza vaccine. We evaluated the safety and immunogenicity of one such candidate, Multimeric-001 (M-001), when used as a priming vaccine prior to administration of quadrivalent inactivated influenza vaccine (IIV4).
Methods:
Healthy adults 18 to 49 years of age were enrolled in a phase 2 randomized, double-blind placebo-controlled trial. Participants received two doses of either 1.0-mg M-001 or saline placebo (60 per study arm) on Days 1 and 22 followed by a single dose of IIV4 on about Day 172. Safety, reactogenicity, cellular immune responses and influenza hemagglutination inhibition (HAI) and microneutralization (MN) were assessed.
Results:
The M-001 vaccine was safe and had an acceptable reactogenicity profile. Injection site tenderness (39% post-dose 1, 29% post-dose 2) was the most common reaction after M-001 administration. Polyfunctional CD4+ T cell responses (perforin-negative, CD107α-negative, TNFα+, IFN-γ+, with or without IL-2) to the pool of M-001 peptides increased significantly from baseline to two weeks after the second dose of M-001, and this increase persisted through Day 172. However, there was no enhancement of HAI or MN antibody responses among M-001 recipients following IIV4 administration.
Conclusions:
M-001 administration induced a subset of polyfunctional CD4+ T cells that persisted through 6 months of follow-up, but it did not improve HAI or MN antibody responses to IIV4. (clinicaltrials.gov NCT03058692)
Keywords: Influenza vaccine, M-001, hemagglutination inhibition, neutralization, CD4+ T cells, CD8+ T cells
Background
Seasonal and pandemic influenza are a continual threat to human populations. The rapid and ongoing evolution of influenza viruses poses challenges to the development of vaccines for disease prevention. Efforts to develop universal vaccines that target multiple influenza subtypes are being pursued [1]. Multimeric-001 (M-001) is a candidate universal vaccine that is a recombinant protein consisting of three copies each of nine conserved linear epitopes from the hemagglutinin (four B and one T helper [Th] epitope), nucleoprotein (two cytotoxic T cell [CTL] and one Th epitope) and matrix-1 protein (one peptide containing both B and CTL epitopes) present in the large majority of influenza viruses [2, 3]. Early clinical data using adjuvanted and/or non-adjuvanted M-001 suggested that M-001 could both enhance cell-mediated immune responses and prime for enhanced antibody responses following subsequent immunization with licensed inactivated influenza vaccines (IIVs) [3, 4].
The goals of this study were to evaluate the safety, reactogenicity and immunogenicity of a M-001 two-dose prime followed by a quadrivalent inactivated influenza vaccine (IIV4) boost strategy and to assess the effects of M-001 vaccination on cellular and humoral immunity to seasonal influenza strains. We also evaluated the hypothesis that immunization with M-001 would provide priming immunity against subsequent immunization with IIV4.
Material and Methods
Study design
This phase 2, randomized, double-blind, placebo-controlled clinical trial was conducted at three NIH-funded Vaccine Treatment and Evaluation Units: Baylor College of Medicine (BCM) in Houston, TX; Cincinnati Children’s Hospital Medical Center in Cincinnati, OH; and the University of Iowa in Iowa City, IA. Eligible participants were healthy male and non-pregnant, non-breastfeeding female adults 18–49 years of age. See clinicaltrials.gov (NCT03058692) for full inclusion/exclusion criteria. The protocol was approved by the ethics committees at each participating institution, and written informed consent was obtained from participants prior to the performance of other study procedures.
Study products
M-001 (lot DP010) was obtained under contract from the manufacturer, BiondVax (Nes Ziona, Israel), and it was produced under Good Manufacturing Practice as a recombinant protein expressed in E coli. It is formulated at a concentration of 2.5 mg/mL in 20 mM citrate buffer with 0.2 M arginine (pH 6). Each 1.0 mg M-001 dose was administered intramuscularly into a deltoid muscle in a 0.4 mL volume.
Fluzone® Quadrivalent influenza vaccine (2018–2019 formulation, lot UT6261JA; Sanofi Pasteur) was administered as a 0.5 mL dose in the deltoid muscle. Strains included in the vaccine were A/Michigan/45/2015 X-275 (H1N1), A/Singapore/INFIMH-16–0019/2016 IVR-186 (H3N2), B/Maryland/15/2016 BX-69A (B Victoria lineage), and B/Phuket/3073/2013 (B Yamagata lineage).
Clinical procedures
Eligible participants were assigned randomly to 1 of 2 treatment arms (60 participants per arm) to receive two doses of the M-001 vaccine or placebo (saline) followed by a single dose of IIV4, all in the same deltoid (selected by the participant at enrollment). Group A received two doses of M-001 on Days 1 and 22, followed by a single dose of seasonal IIV4 on approximately Day 172 (protocol-specified window for this visit ranged from Day 142 to Day 202). Group B received saline placebo on Days 1 and 22, followed by a single dose of IIV4 on approximately Day 172.
Reactogenicity was measured by the occurrence of solicited injection site and systemic reactions from the time of each study vaccination with M-001 (or placebo) through 7 days after the vaccination. Unsolicited adverse events (AEs) were collected from the time of each M-001 vaccination through approximately 21 days after each study vaccination. AEs were graded as mild (grade 1) if they did not interfere with participants’ daily activities and required no treatment, moderate (grade 2) if they caused some interference with participants’ daily activities or required therapeutic measures, and severe (grade 3) if they interrupted the participants’ daily activities or incapacitated the participant. Serious adverse events (SAEs) were collected from the time of the first study vaccination through approximately 6 months after the second M-001 (or placebo) study vaccination. Clinical laboratory evaluations for safety were performed on venous blood collected approximately 8 days after each M-001 (or placebo) vaccination (Days 9 and 30). Blood samples for immunogenicity analyses were also collected prior to each vaccination and at different times after vaccination (see below).
Laboratory methods
Flow cytometry
The percentages of CD4+ and CD8+ T cell subsets expressing perforin, CD107a, interferon gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), and interleukin 2 (IL-2), alone or in combination, after stimulation with M-001 component peptides in peripheral blood mononuclear cells (PBMCs) collected at baseline (Day 1) and 14 days after the second dose of M-001 were determined via flow cytometry studies. The assays were performed at BCM on all participants for whom samples were available. Previously cryopreserved PBMCs were thawed, assessed for viability using trypan blue, rested overnight in RPMI 1640 (HyClone) + 10% fetal calf serum (R10 media) at 37°C, 5% CO2, and stimulated for 6 hours with R10 media alone (negative control), universal influenza peptide pool (6 μg /mL) (positive control) (Tables S1 and S2 [5], or vaccine peptide library (4 μg /mL) (M001) (Table S3) containing anti-CD28 and anti-CD49d (costimulatory agents for peptide pool stimulation) in the presence of brefeldin A, monensin, and anti-CD107a antibody.
After stimulation, cells were washed in calcium- and magnesium-free phosphate buffered saline (PBS) and then incubated with a viability dye. Next, cells were incubated with human FcR block followed by extracellular antibody cocktails (Table S4). After washing, cells were permeabilized using TONBO Fix/perm buffer according to the manufacturer’s instructions. Following permeabilization, cells were washed in 1X permeabilization wash buffer followed by incubation with intracellular antibody cocktails. Cells were then washed once in TONBO permeabilization wash buffer and once in FACS buffer and the samples were fixed in 1% paraformaldehyde. Data were acquired using a Fortessa flow cytometer and were analyzed using FlowJo version 10.1.
The above-described methods were used to assess the persistence of responses using samples from a random convenience sample of participants, collected at baseline, 14 days after the first and last vaccinations (Days 15 and 186), approximately 21 days (Day 43) and approximately 56 days (Day 78) following receipt of the second M-001 (or placebo) dose, and immediately prior to and approximately 28 days after the IIV4 dose (Days 172 and 200).
Hemagglutination inhibition (HAI) antibody assay
HAI antibody assays were performed at Saint Louis University using previously described methods [6, 7]. Sera were assayed against the 2018–2019 vaccine virus strains A/Michigan/45/2015 X-275 (H1N1), A/Singapore/INFIMH-16–0019/2016 IVR-186 (H3N2), B/Colorado/6/2017 (B Victoria Lineage), and B/Phuket/3073/2013 (B Yamagata lineage) that were provided by International Reagent Resource, a program established by Centers for Disease Control and Prevention (CDC, Atlanta, GA). To eliminate concerns related to insufficient binding of turkey red blood cells, the H3N2 strain was also tested in the presence of 20nM oseltamivir (Millipore Sigma, St. Louis, MO) and a 1% guinea pig red blood cell suspension [8].
Microneutralization antibody assay
MN assays were performed at Saint Louis University [9–12]. The same strains used for HAI were used in the MN assays and were obtained from the International Reagent Resource.
M-001 antibody ELISA
An indirect ELISA to measure IgG antibody levels against M-001 was performed at BiondVax as described previously [4].
Statistical methods
The primary safety objectives were to assess vaccine safety as measured by the occurrence of vaccine-related SAEs through ~6 months after the second M-001 vaccination and solicited injection site and systemic reactogenicity and clinical safety laboratory AEs for 7 days following each M-001 injection. The primary immunogenicity objective was to assess T cell responses to the M-001 component peptides following two doses of M-001. Secondary safety objectives included assessment of the occurrence of all SAEs through the end of the study and the occurrence of all unsolicited non-serious AEs from the time of first study vaccination through 21 days after each M-001 vaccination. Secondary immunogenicity endpoints included the assessment of HAI and MN antibody responses to the 2018–2019 IIV4 vaccine viruses. Exploratory immunogenicity endpoints included the assessment of IgG responses to the M-001 vaccine after receipt of placebo or one or two doses of M-001 vaccine and following receipt of IIV4, the assessment of T cell responses to M-001 component peptides after one dose of M-001 and following IIV4 administration, and an assessment of the longevity of T cell responses to M-001 peptides, seasonal influenza virus antigens and pandemic influenza virus antigens. The study was not designed to detect any specific differences between groups in the primary immunogenicity or safety endpoints.
Safety analyses were conducted in the Safety Population, which consisted of all vaccinated participants. Immunogenicity analyses were conducted in the modified Intent-to-Treat (MITT) population and/or the Per Protocol (PP) Population. The MITT population included all participants who received at least one study vaccination and contributed at least one post-vaccination blood sample for which valid results were reported. The PP population included the participants in the MITT population with exclusions of data subsequent to protocol deviations such as failing to receive subsequent doses, out of the protocol-defined window for visits or vaccinations, or receipt of non-study vaccination. The PP population was used for sensitivity analysis of primary immunogenicity endpoints and exclusively used for secondary and exploratory analyses.
AEs were classified by MedDRA® System Organ Class (SOC) and Preferred Term (PT) and summarized by study arm post-each M-001 vaccination and post-either M-001 vaccination, along with 95% exact Clopper-Pearson confidence intervals (CIs) for the probability of each event. Solicited reactogenicity events were summarized similarly, by maximum severity reported post-each M-001 dose and post-either dose.
For HAI and MN titers, responses censored below the lower limit of detection (LOD) for the assays (10 for each assay) were imputed as LOD/2. Individual titers were computed as the geometric mean of technical replicate results. Titers were descriptively analyzed for each group by computing geometric mean titers (GMTs) and associated 95% CIs based on back-transforming estimates from the t-distribution on the log-scale. HAI and MN responses were also assessed by the percentage of participants reporting a titer of at least 40 at each time point and the percentage of participants reporting a minimum 4-fold rise from baseline (seroresponse), along with corresponding exact 95% Clopper-Pearson CIs. Cell phenotypes were described via the percentages of CD4+ and CD8+ T cells expressing various combinations of cytokines with or without stimulation within a given gate, where Fluorescence Minus One gates were set to 0 percent positive cells. Mean percentages were computed for each group, each cell type, each cell stimulation, and each time point, and 95% bootstrap CIs were calculated based on 10,000 empirical bootstrap samples. Mann-Whitney U tests were applied to compare distributions of each cell type between vaccination groups to aid interpretation by filtering out cell types with no evidence of being induced by the study vaccine. Ratios of group means to baseline were calculated at Day 36 and 95% bootstrap CIs were computed from the same bootstrap samples used to determine CIs for the individual group means, again to simplify interpretation across numerous cell types.
Analyses were primarily conducted according to a pre-specified statistical analysis plan, but the specific methodology described above for analysis of the T cell phenotyping data was implemented post hoc in order to account for intricacies in the data that led to technical issues when attempting to apply the originally-planned methods.
Results
Demographics
One hundred forty-two persons were screened to enroll 120 participants in the study; 61 and 59 received the first dose of M-001 and placebo, respectively, and 55 in each group received the assigned second dose (Figure S1). IIV4 was administered to 54 and 53 participants in the M-001 and placebo groups, respectively. Most enrolled participants were female, and the mean age, race and ethnicity distributions were similar between treatment groups (Table 1). Most participants had received influenza vaccine within two years of enrollment.
Table 1.
Demographics* of study participants by enrollment group.
| Group A (M-001) | Group B (Placebo) | |
|---|---|---|
| Number enrolled | 61 | 59 |
| Sex | ||
| Number females (%) | 42 (69) | 33 (56) |
| Race | ||
| Number Asian (%) | 8 (13) | 5 (8) |
| Number Black (%) | 3 (5) | 3 (5) |
| Number White (%) | 49 (80) | 50 (85) |
| Number Multi-racial (%) | 1 (2) | 1 (2) |
| Ethnicity | ||
| Number Hispanic (%) | 5 (8) | 6 (10) |
| Mean Age (SD) | 32.6 (7.9) | 33.5 (7.8) |
| BMI | ||
| Mean (SD) | 28.5 (7.2) | 27.2 (5.8) |
| Median BMI (Range) | 26.8 (17.4–52.0) | 25.9 (19.1–42.1) |
| Number with BMI ≥30 (%) | 19 (31) | 15 (25) |
| Previous seasonal influenza vaccine | ||
| 2016–17 only | 4 (7) | 2 (3) |
| 2017–18 only | 2(3) | 2 (3) |
| Both prior years | 49 (80) | 48 (81) |
| Neither prior year | 5 (8) | 4 (7) |
| Unknown | 1 (2) | 3 (5) |
Information on smoking not systematically collected
Safety
The M-001 vaccine was safe and generally well tolerated. Injection site reactions of pain and tenderness were significantly more common in the M-001 recipients compared to placebo recipients (Figure 1). These reactions were predominantly mild, short in duration (1 to 4 days), and occurred with similar frequency after each M-001 dose. Other solicited injection site reactions (redness and swelling) and solicited systemic reactions occurred with similar frequencies between groups. Similarly, solicited systemic reactions were also predominantly mild and of short duration. Fever was rare, detected in vaccinees. One severe solicited reaction (fever and malaise) associated with an exudative pharyngitis illness beginning on study day 5 after the first vaccination was reported by a placebo recipient. One M-001 recipient developed itching the day after the first vaccination and subsequently developed a rash. No other etiology was identified, and the rash was deemed related to study product. The participant received no further vaccinations. Evaluation of safety labs did not reveal any concerns.
Figure 1.
Maximum severity of solicited injection site and systemic reactions by study group and vaccine number.
One SAE classified as severe and deemed unrelated to study product (deafness after planned surgery for an acoustic neuroma) occurred in a M-001 recipient. One placebo recipient had a pre-existing anemia of moderate severity identified in the safety laboratory studies performed at enrollment and was withdrawn from the study when repeat bloodwork collected at day 9 showed persistence of the moderate grade anemia.
Immunogenicity
Primary endpoint
The mean percentage of CD4+ T cells (CD45+, CD3+, CD4+) expressing IFN-γ, TNFα, IL-2, perforin or CD107α following stimulation with a pool of the individual influenza peptides contained in the M-001 construct did not change significantly from baseline to two weeks after the second dose of M-001 (Table 2), although the number of CD45+, CD3+, CD4+, IFN-γ+ T cells tended to be higher in the M-001 vaccine group at Day 36 (Mann-Whitney p=0.023). However, mean polyfunctional CD4+ T cell responses (perforin-negative, CD107α-negative, TNFα+, IFN-γ+, with or without IL-2) to the pool of M-001 peptides did increase significantly from baseline to two weeks after the second dose of M-001. No increases in the abundance of other measured markers on CD4+ T cells or in subsets of CD8+ T cells were observed (Tables S7–S10). Unstimulated cells and cellular responses to a universal influenza peptide pool were similar between vaccine groups at baseline and at study day 36.
Table 2.
CD4 T cell responses following stimulation with M-001 peptide pool two weeks after second dose (study day 36)
| M-001 (N=52) | Placebo (N=53) | P value (Mann Whitney U test) | |||||
|---|---|---|---|---|---|---|---|
| Marker | n * | Mean % (95% CI)+ | Ratio of Mean to Day 1 (95% CI) | n * | Mean % (95% CI) + | Ratio of Mean to Day 1 (95% CI) | |
| CD3+, CD4+, CD45+ | |||||||
| Perforin | 52 | 0.80 (0.49, 1.17) | 0.68 (0.36, 1.25) | 53 | 0.74 (0.42, 1.14) | 0.95 (0.48, 1.86) | 0.63 |
| CD107α | 52 | 0.24 (0.19, 0.30) | 0.94 (0.66, 1.32) | 53 | 0.27 (0.21, 0.33) | 1.01 (0.70, 1.44) | 0.56 |
| IL-2 | 52 | 0.072 (0.055, 0.089) | 1.05 (0.71, 1.59) | 53 | 0.068 (0.052, 0.086) | 1.07 (0.76, 1.50) | 0.69 |
| TNFα | 52 | 0.44 (0.32, 0.61) | 1.01 (0.66, 1.57) | 53 | 0.41 (0.31, 0.54) | 0.79 (0.51, 1.23) | 0.78 |
| IFN γ | 52 | 0.050 (0.040, 0.061) | 1.19 (0.86, 1.63) | 53 | 0.045 (0.030, 0.069) | 1.34 (0.84, 2.19) | 0.023 |
| CD3+, CD4+, CD45+, perforin-, CD107α- | |||||||
| IFN γ+, TNFα−, IL2− | 51 | 0.018 (0.014, 0.022) | 0.75 (0.48, 1.16) | 51 | 0.027 (0.013, 0.050) | 1.82 (0.86, 3.63) | 0.56 |
| IFN γ−, TNFα+, IL2− | 52 | 0.38 (0.26, 0.53) | 0.98 (0.62, 1.53) | 53 | 0.38 (0.28, 0.49) | 0.78 (0.50, 1.25) | 0.77 |
| IFN γ−, TNFα−, IL2+ | 51 | 0.036 (0.024, 0.049) | 0.82 (0.48, 1.38) | 48 | 0.046 (0.032, 0.062) | 1.16 (0.74, 1.85) | 0.48 |
| IFN γ+, TNFα+, IL2− | 49 | 0.016 (0.012, 0.021) | 1.68 (1.15, 2.45) | 43 | 0.0075 (0.0056, 0.0096) | 0.85 (0.56, 1.31) | <0.001 |
| IFN γ+, TNFα−, IL-2+ | 8 | 0.00034 (0.00014, 0.00057) | 2.02 (0.63, 9.61) | 5 | 0.00032 (0.000068, 0.00064) | 0.72 (0.14, 2.06) | 0.53 |
| IFN γ−, TNFα+, IL-2+ | 48 | 0.017 (0.012, 0.022) | 1.45 (0.93, 2.33) | 44 | 0.012 (0.0080, 0.016) | 0.95 (0.60, 1.43) | 0.015 |
| IFN γ+, TNFα+, IL-2+ | 45 | 0.010 (0.0064, 0.0160) | 3.11 (1.79, 5.45) | 32 | 0.0029 (0.0019, 0.0039) | 0.78 (0.45, 1.40) | <0.001 |
| IFN γ−, TNFα−, IL-2− | 52 | 98.5 (98.1, 98.8) | 1.00 (1.00, 1.01) | 53 | 98.5 (98.1, 98.8) | 1.00 (1.00, 1.01) | 0.85 |
n=number with cell type present
The denominator for all percentages analyzed was the CD4+ cells, as gated by CD3+, CD4+, and CD45+. The numerator reflects the string listed in the Marker column.
Notes: All combinations of Perforin, CD107α, IL-2, TNFα, and IFN-γ were analyzed, and a subset of those results are shown here. Analysis conducted in the Per Protocol Population.
HAI and MN antibody responses
HAI and MN antibody titers did not increase after M-001 vaccination (Tables 3 and S6). Antibody levels, as measured by seroresponse frequencies, geometric mean antibody titers and percentage of individuals with antibody titers of 40 or higher, increased modestly after IIV4 administration and were similar between the M-001 and placebo groups.
Table 3.
HAI antibody responses by enrollment group and study day*
| Group A (M-001) | Group B (Placebo) | |||||||
|---|---|---|---|---|---|---|---|---|
| Day 1 | Day 43 | Day 172 | Day 200 | Day 1 | Day 43 | Day 172 | Day 200 | |
| (n=60) | (n=53) | (n=53) | (n=52) | (n=59) | (n=53) | (n=52) | (n=50) | |
| A/Michigan/4 5/2015 (H1N1) | ||||||||
| GMT (95% CI) | 84.8 (63.1, 113.9) | 80.0 (57.7, 110.9) | 80.0 (59.3, 108.0) | 117.8 (87.5, 158.5) | 75.4 (56.9, 99.9) | 67.5 (50.3, 90.6) | 63.8 (46.8, 86.9) | 114.7 (85.8, 153.4) |
| Titer ≥40 (95% CI) | 80 (68, 89) | 79 (66, 89) | 81 (66, 89) | 94 (84, 99) | 81 (69, 90) | 79 (66, 89) | 79 (65, 89) | 88 (76, 96) |
| Seroresponse vs Day 172 (95% CI) | --- | --- | --- | 15 (7, 28) | --- | --- | --- | 16 (7, 29) |
| A/Singapore/I NFIMH-16-0019/2016 (H3N2) | ||||||||
| GMT (95% CI) | 25.2 (19.0, 33.5) | 23.6 (17.2, 32.2) | 24.2 (17.6, 33.2) | 54.7 (40.2, 74.5) | 23.9 (17.8, 32.0) | 23.2 (16.7, 32.3) | 22.3 (16.2, 30.5) | 47.2 (33.6, 66.5) |
| Titer ≥40 (95% CI) | 43 (31, 57) | 40 (26, 54) | 40 (26, 54) | 67 (53, 80) | 34 (22, 47) | 36 (23, 50) | 31 (19, 45) | 60 (45, 74) |
| Seroresponse vs Day 172 (95% CI) | --- | --- | --- | 25 (14, 39) | --- | --- | --- | 20 (10, 34) |
| B/Colorado/6/2017 (Victoria lineage) | ||||||||
| GMT (95% CI) | 82.8 (70.6, 97.1) | 77.9 (63.5, 95.7) | 77.9 (64.6, 94.0) | 114.7 (92.8, 141.6) | 87.9 (72.0, 107.3) | 85.4 (68.6, 106.4) | 78.9 (62.5, 99.8) | 111.6 (91.4, 136.3) |
| Titer ≥40 (95% CI) | 98 (91, 100) | 96 (87, 100) | 94 (84, 99) | 98 (90, 100) | 93 (84, 98) | 92 (82, 98) | 88 (77, 96) | 96 (86, 100) |
| Seroresponse vs Day 172 (95% CI) | --- | --- | --- | 13 (7, 26) | --- | --- | --- | 6 (1, 16) |
| B/Phuket/307 3/2013 (Yamagata lineage) | ||||||||
| GMT (95% CI) | 80.0 (66.2, 96.7) | 76.9 (61.5, 96.1) | 80.0 (65.6, 97.5) | 104.4 (80.8, 135.0) | 88.9 (72.5, 109.0) | 80.0 (64.5, 99.2) | 76.9 (60.8, 97.1) | 108.5 (84.9, 138.8) |
| Titer ≥40 (95% CI) | 93 (84, 98) | 94 (84, 99) | 94 (84, 99) | 98 (90,100) | 93 (84, 98) | 92 (82, 98) | 88 (77, 96) | 96 (86, 100) |
| Seroresponse vs Day 172 (95% CI) | --- | --- | --- | 11 (4, 23) | --- | --- | --- | 8 (2, 19) |
Days 1 and 172 are the days of initial M-001 vaccination and IIV4 administration, respectively
Notes: Confidence intervals for GMTs computed using the t-distribution for log GMTs; 95% exact Clopper-Pearson CIs computed for seroresponse. Analysis conducted in the Per Protocol Population.
M-001 ELISA antibody responses
ELISA antibody responses against M-001 were modest (Table 4), with four-fold increases in antibody levels being observed in 20% of M-001 recipients at study day 78 (56 days after the second dose of M-001). M-001 antibody levels did not correlate with HAI or MN titers across timepoints.
Table 4.
M-001 antibody responses by enrollment group and study day*
| Group A (M-001) | Group B (Placebo) | |||||
|---|---|---|---|---|---|---|
| Study day | n | GMT (95% CI) | Seroresponse vs Day 1 (95% CI) | n | GMT (95% CI) | Seroresponse vs Day 1 (95% CI) |
| Day 1* | 60 | 504 (390, 652) | --- | 59 | 417 (340, 510) | --- |
| Day 22 | 58 | 496 (380, 647) | 9 (3, 19) | 56 | 389 (304, 498) | 2 (0, 10) |
| Day 43 | 53 | 693 (524, 917) | 19 (9, 32) | 53 | 346 (277, 434) | 6 (1, 16) |
| Day 78 | 50 | 786 (579, 1068) | 20 (10, 34) | 46 | 391 (299, 511) | 2 (0, 12) |
| Day 172 | 53 | 606 (474, 775) | 15 (7, 28) | 52 | 442 (336, 581) | 12 (4, 23) |
| Day 200 | 52 | 702 (556, 887) | 17 (8, 30) | 50 | 371 (283, 485) | 6 (1, 16) |
Days 1 and 172 are the days of initial M-001 vaccination and IIV4 administration, respectively
Notes: Confidence intervals for GMTs computed using the t-distribution for log GMTs; 95% exact Clopper-Pearson CIs computed for seroresponse. Analysis conducted in the Per Protocol Population.
Duration of cellular immune responses
Polyfunctional CD4+ T cell responses (perforin-negative, CD107α-negative, TNFα-positive, IFN-γ-positive, with or without IL-2) following stimulation with the pool of M-001 peptides were detected at Day 15 (after one dose of M-001) and peaked, on average, at Day 43 (3 weeks after the second dose of M-001) in the subset of participants in whom longitudinal analyses were performed (Table 5). Although the relative increase in the mean percentage of IFN-γ+, TNFα+, IL-2- CD4+ T cells was not different compared to baseline by Day 78, they remained elevated compared to placebo recipients through day 172, and mean IFN-γ+, TNFα+, IL-2+ CD4+ T cell responses remained elevated relative to baseline at all timepoints.
Table 5.
Persistence of certain CD4+ T cell subset responses following stimulation with M-001 peptide pool
| M-001 (N=44) | Placebo (N=45) | P value (Mann Whitney U test) | |||||
|---|---|---|---|---|---|---|---|
| Study Day | n/N* | Mean % (95% CI) | Ratio of Mean to Day 1 (95% CI) | n/N* | Mean % (95% CI) | Ratio of Mean to Day 1 (95% CI) | |
| IFN γ+, TNFα+, IL2- | |||||||
| 15 | 42/44 | 0.012 (0.009, 0.014) | 1.25 (0.89, 1.77) | 39/45 | 0.0056 (0.0004, 0.0070) | 0.63 (0.42, 0.96) | <0.001 |
| 43 | 38/40 | 0.017 (0.010, 0.028) | 1.83 (1.02, 3.20) | 33/42 | 0.0063 (0.0042, 0.0087) | 0.71 (0.44, 1.15) | 0.003 |
| 78 | 34/35 | 0.012 (0.008, 0.016) | 1.23 (0.82, 1.85) | 35/37 | 0.0072 (0.0053, 0.0094) | 0.82 (0.53, 1.26) | 0.024 |
| 172 | 36/39 | 0.012 (0.009, 0.016) | 1.26 (0.84, 1.87) | 35/42 | 0.0071 (0.0049, 0.0095) | 0.80 (0.50, 1.27) | 0.009 |
| 186 | 34/36 | 0.012 (0.008, 0.016) | 1.25 (0.80, 1.93) | 32/39 | 0.0085 (0.0057, 0.0118) | 0.97 (0.45, 1.68) | 0.085 |
| 200 | 33/37 | 0.013 (0.0091, 0.018) | 1.40 (0.91, 2.12) | 36/42 | 0.0078 (0.0056, 0.0101) | 0.88 (0.57, 1.37) | 0.138 |
| IFN γ+, TNFα+, IL-2+ | |||||||
| 15 | 38/44 | 0.0071 (0.0050, 0.009) | 2.16 (1.40, 3.31) | 22/45 | 0.0020 (0.0012, 0.0031) | 0.56 (0.29, 1.04) | <0.001 |
| 43 | 36/40 | 0.0126 (0.0069, 0.022) | 3.84 (1.95, 7.26) | 23/42 | 0.0023 (0.0015, 0.0034) | 0.64 (0.35, 1.16) | <0.001 |
| 78 | 25/35 | 0.0082 (0.0034, 0.017) | 2.49 (0.96, 5.51) | 20/37 | 0.0021 (0.0013, 0.0030) | 0.58 (0.31, 1.05) | 0.014 |
| 172 | 31/39 | 0.0089 (0.0052, 0.015) | 2.71 (1.45, 4.96) | 25/42 | 0.0024 (0.0016, 0.0032) | 0.66 (0.38, 1.15) | <0.001 |
| 186 | 24/36 | 0.0088 (0.0044, 0.015) | 2.69 (1.28, 4.95) | 20/39 | 0.0033 (0.0018, 0.0049) | 0.89 (0.45, 1.68) | 0.125 |
| 200 | 28/37 | 0.0079 (0.0035, 0.015) | 2.40 (1.02, 5.14) | 26/42 | 0.0026 (0.0016, 0.0038) | 0.71 (0.39, 1.31) | 0.029 |
n/N: n=number with cell subset present; N=number samples tested at that timepoint
Discussion
A number of different strategies are being pursued to develop a universal influenza vaccine that will provide protection against emerging seasonal and pandemic influenza virus strains [13]. M-001, a candidate polypeptide vaccine containing conserved T cell epitopes, has been reported previously to enhance humoral and cellular influenza-specific responses when used as a priming regimen for IIV [3].
We evaluated whether we could measure enhanced T cell-specific responses and whether such responses were associated with improved responses following administration of IIV4 ~5 months after the second dose of M-001. While a small set of M-001 polyfunctional T cell responses were observed following M-001 administration, enhanced HAI or MN antibody responses were not identified for any of the four vaccine antigens evaluated.
The potential benefit of the increased number of polyfunctional CD4+ T cells that recognize epitopes in M-001 is unclear. When used as a standalone vaccine in a clinical trial, M-001 did not protect from influenza-like illness or influenza infection (https://clinicaltrials.gov/ct2/show/results/NCT03450915). Previous reports have indicated that M-001 can prime for cellular or humoral responses to influenza antigens [2–4]. We did not observe increased CD4+ or CD8+ responsiveness following stimulation with a panel of influenza peptides that represent shared epitopes recognized across the human population [5], although four of the epitopes were wholly or partially included in the panel. Our results contrast with those of Atsmon et al. [3], who reported an increase in IFN-γ-producing CD4+ and CD8+ T cells following stimulation with influenza A antigens (hemagglutinin, nucleoprotein, live attenuated vaccine virus) among persons administered two doses of M-001. Our study examined a larger number of participants who received two doses of M-001 than the earlier study (58 vs 10). Similarly, we observed no enhancement in antibody responses among those vaccinated with IIV, in contrast to results from earlier trials [3, 14]. One difference in our study was that the IIV was delivered 5 months later compared to 3 weeks later in earlier trials. Based on earlier studies, one proposed use of M-001 is to combine it with a pandemic vaccine [2]. However, we did not see evidence of M-001 priming the HAI or MN antibody responses against seasonal influenza antigens, although high baseline antibody levels for 3 of the antigens may have diminished the ability to identify seroresponses.
The strategy to develop an epitope-based peptide vaccine as a universal influenza vaccine candidate may still be reasonable. Flu-v is a combination of four synthetic peptides representing conserved epitopes from the influenza nucleoprotein and matrix (M1 and M2) proteins [15]. Coadministration of the adjuvant Montanide ISA-51 enhanced T cell and antibody responses to the vaccine antigens [16]. In a controlled human infection model, vaccine recipients had a lower symptom score than did placebo recipients [17]. The optimal peptides to include in an epitope-based vaccine remain to be determined, but an adjuvant may be required to optimize immunogenicity.
In summary, we found that the administration of unadjuvanted M-001 led to the expansion of a subset of polyfunctional CD4+ T cells that persisted through 6 months of follow-up. However, we found no evidence that M-001 primed for the development of HAI or MN antibody responses after the delivery of an IIV4.
Supplementary Material
Acknowledgements:
We are thankful for the study participants, the Data and Safety Monitoring Board members (David Rimland, MD, Pia S Pannaraj, MD, MPH, Janet E McElhaney, MD, Maria Knoll, PhD), Respiratory Diseases Branch at the National Institutes of Allergy and Infectious Diseases (Tena Knudsen, BSN, Kathy Ormanoski, MS, Trinka Coster, MD, Soju Chang, MD, Rhonda Pikaart-Tautges, BSBA, BS, Hyung Koo, RN, BSN, Melinda Tibbals, RAC, CCRA), BCM study team (Tracey Lanford, RN, Coni Cheesman, PA-C, Barbara Annette Nagel, RN, Janet Brown PharmD, Lisreina Toro, MD, Kathy Bosworth, Rodrigo Gomez-Carlin), the CCHMC study team (Rebecca Brady, MD, Michelle Dickey, MS, APRN, Kristen Buschle, APRN), the Iowa study team (Jack Stapleton, MD, Kiran Gajurel, MD, Laura Stulken, PA, Dan Zhao, MA, MNHP, Nancy Wagner, RN, AJ Carr, Michelle Rodenburg, David Bush, MBA, Reshika Dhakal, MBBS, BPH), the SLU team (Tammy Blevins, MS, Yinyi Yu, BS) and Emmes Corporation (Ashley Wegel, CCRA, Abbie Bellamy, PhD, Carie Petrie, PhD, Ranjan Sitaula, PhD).
This work was supported by funding provided by the Division of Microbiology and Infectious Diseases at the National Institutes of Health under contract HHSN272201300015I (Baylor College of Medicine), HHSN272201300021I (Saint Louis University), HHSN272201300020I (University of Iowa), contract HHSN272201300016I (Cincinnati Children’s Medical Center), and 75N93021C00012 (Emmes Corporation). This project also was supported by the Cytometry and Cell Sorting Core at Baylor College of Medicine with funding from the CPRIT Core Facility Support Award (CPRIT-RP180672), the NIH (P30 CA125123 and S10 RR024574) and the expert assistance of Joel M. Sederstrom.
Footnotes
Declaration of Interests
PW reports consulting or advisory work and receives grant funding for Pfizer, Inc. and receives grant funding from Sanofi. TBY is an employee of BiondVax. RLA, DIB, SEF, LSA, CB, PCR, HME and WAK have nothing to declare.
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