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Abstract
Seasonal influenza continues to cause significant morbidity and mortality, particularly for the elderly and immunocompromised. Current licensed influenza vaccines provide only partial protection even for immunocompetent hosts. Vaccine adjuvants can improve the magnitude and breadth of immune responses and there is considerable interest in identifying new adjuvants that can improve immune responses to seasonal influenza vaccines. This phase I, randomized, double-blind trial evaluated the safety and immunogenicity of one dose of 2018/2019 quadrivalent influenza vaccine (either Fluzone® or Flublok®) administered intramuscularly with or without one of two adjuvants (AF03 or Advax-CpG55.2). A total of 241 healthy adults aged 18–45 years were enrolled and randomized to 1 of 6 groups. Groups 1–3 received one dose of Fluzone® QIV 2018/2019 administered alone or with AF03 or Advax-CpG55.2 and Groups 4–6 received one dose of Flublok® QIV 2018/2019 alone or with one of these two adjuvants. All participants received Fluzone® or Flublok® QIV 2019/2020 ninety days later. Primary objectives were to evaluate safety and reactogenicity along with changes in hemagglutinin inhibition (HAI), neuraminidase inhibition (NAI) and neutralizing antibodies to 2018/2019 seasonal influenza antigens, comparing Day 1 and Day 29 titers. Secondary objectives evaluated the impact of adjuvants on immune responses after subsequent doses of unadjuvanted seasonal influenza vaccine and immunologic responses to heterologous influenza H1 and H3 antigens. Overall, the adjuvanted vaccines were safe and generated robust immune responses against both homologous and heterologous strains. Similar responses were seen across all six study arms. Both adjuvants were associated with qualitatively improved immune responses against some strains at varying timepoints, but results were inconsistent. There were no substantial differences in safety or reactogenicity identified between the study groups and all vaccine formulations were well tolerated. In this highly immunologically-experienced cohort, neither AF03 nor Advax-CpG55.2 demonstrated notable benefit when added to the seasonal influenza vaccine. (
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Currently licensed influenza vaccines have varying efficacy from year to year. Meta-analyses examining controlled trials comparing seasonal influenza vaccines to no vaccine for preventing laboratory-confirmed influenza illness have shown remarkable variability in efficacy between different seasons, ranging from 10 %–76 %, with improved efficacy seen when the vaccine strains matched the circulating strains [ 1, 2]. Others have evaluated the effectiveness of seasonal influenza vaccines in preventing severe influenza illness associated with hospitalization, and found a pooled efficacy of 51 % in adults aged 18–64 and 37 % in adults 65 years of age or older [ 3]. Since seasonal influenza vaccines have such inconsistent efficacy, there is considerable interest in exploring new adjuvants to improve the efficacy particularly in the elderly and other vulnerable populations. Adjuvants could have beneficial effects in combination with inactivated or recombinant protein influenza vaccines, although tolerability and safety would first need to be established [ 4, 5].
The selection of an optimal adjuvant for a particular antigen often requires trial and error. Even modest changes to adjuvant formulations can markedly alter immune responses, and different adjuvants work better for certain antigens [ 6, 7]. For example, the older aluminum-containing adjuvants induce strong humoral immunity but are less effective at inducing cellular immunity needed to fight intracellular pathogens [ 7].
Adjuvants in development or in use include a large variety of compounds such as aluminum salts, oil emulsions, saponins, immune-stimulating complexes, liposomes, microparticles, nonionic block copolymers, polysaccharides, CpG oligonucleotides, small molecule TLR agonists, and cytokines and bacterial derivatives [ 8–13]. Many of the adjuvants in use today were developed empirically, and their mechanisms of action are only now being researched, long after their approval for use in humans [ 4, 7, 14]. As older attenuated and inactivated virus vaccines are replaced with vaccines based on highly purified antigens, it is becoming clear that the strong immunogenicity exhibited by certain older vaccines (such as the live vaccinia virus smallpox vaccine, live attenuated yellow fever vaccine, and live Sabin polio vaccine) was at least partly the result of endogenous adjuvants [ 6, 15]. Subunit vaccines or vaccines based on recombinant proteins hold the promise of increased safety, but contain little or no endogenous adjuvant material, leading to the potential need for addition of adjuvants to drive a sufficiently robust immune response [ 5, 6, 15].
This study sought to evaluate two newer adjuvants, specifically the squalene-based oil-in-water emulsion AF03 [ 16]; and Advax-CpG55.2, a combination of polysaccharide particules based on delta inulin and trade-marked as “Advax” [ 17] combined with CpG55.2, a cytosine-phospho-guanosine motif (CpG) containing oligonucleotide with a phosphorothioate backbone. Squalene-based emulsions such as AF03 are thought to enhance immune response by both increasing the availability of antigen to antigen-presenting cells and by inducing a local inflammatory state [ 4], while CpG works via activation of toll-like receptor-9 [ 15]. Delta inulin was recently identified to be an agonist of dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN); it is considered a “non-inflammatory” adjuvant that enhances humoral and cellular immune responses with few inflammatory side effects [ 17, 18]. Both AF03 and Advax adjuvants have had extensive pre-clinical evaluation of immune responses with seasonal influenza antigens and there are previous human studies evaluating these adjuvants in combination with pandemic avian influenza vaccines [ 19–23]. This trial represents the first time that the Advax-CpG55.2 combination adjuvant was tested in humans. Subsequent to this study, Advax-CpG55.2 was used as a component of the SpikoGen® protein subunit COVID-19 vaccine (also known as COVAX-19), which received emergency use authorization in Iran in October 2021 [ 24, 25].
This study was designed to evaluate the effects of the adjuvants described above (AF03 and Advax-CpG55.2) in humans when used in combination with two different commercially available influenza vaccines. Fluzone® is a split virus vaccine that is produced from influenza viruses grown in embryonated chicken eggs or cell lines that then undergo chemical disruption and purification to produce the antigens included in the vaccine. As a result, Fluzone® vaccines include not only the dominant hemagglutinin protein (HA) from influenza, but also smaller quantities of neuraminidase protein (NA). In contrast, Flublok® contains only recombinant HA proteins (rHA) produced in an insect cell line using a baculovirus vector encoding the influenza HA gene. Therefore, in addition to the comparison between adjuvant effects on vaccines based on insect cell-derived recombinant HA and egg-derived HA antigens, the study design also allowed the evaluation of adjuvant effects on the NA portion of the Fluzone® vaccine (not present in Flublok®).
A prior similarly-designed study in mice utilizing the 2017/2018 Fluzone® and Flublok® commercial vaccines with or without the AF03 adjuvant showed that the AF03 groups had an increase in HA-specific antibody titers for all four homologous seasonal influenza strains [ 26]. The study also demonstrated higher neuraminidase inhibition activity in the Fluzone® plus AF03 group. Mice receiving either of the seasonal vaccines combined with the AF03 adjuvant in the study by Ustyugova et al had a significantly higher increase in antibodies against an extensive panel of HA antigens than groups not receiving the adjuvant, suggesting the potential for AF03 to trigger a strong heterologous immune response following seasonal influenza vaccination. If a similar increase in the magnitude and/or breadth of immune response is seen with addition of either AF03 or Advax-CpG55.2 in humans, this might translate into increased influenza vaccine efficacy.
2 Methods2.1 Study Design, participants, and objectives
This was a randomized, double blinded, phase I clinical trial. Five hundred and twelve participants were screened, with enrollment of 241 males and non-pregnant females, 18–45 years of age, inclusive, who were in good health and met all eligibility criteria. Detailed eligibility criteria are provided at www.ClinicalTrials.gov ( NCT03945825). The study was conducted at eight centers in the United States from June 2019 to September 2020. The study protocol and informed consent were approved by the National Institute of Allergy and Infectious Diseases and by each local institutional review board. The study was conducted in accordance with the guidelines from the International Conference on Harmonization Good Clinical Practice Guidelines and the Declaration of Helsinki. After a discussion about the nature and possible consequences of the study, all participants gave written informed consent prior to any study procedures.
Participants were stratified by prior receipt of licensed, seasonal influenza vaccine (defined as receipt of at least one of the 2017/2018 and/or 2018/2019 influenza vaccines) and within each stratum were randomly assigned to 1 of 6 treatment arms to receive a single dose of one of the two seasonal 2018/2019 quadrivalent influenza vaccine (QIV) vaccine formulations with or without one of the two adjuvants on Day 1. On approximately Day 90, each participant received a single dose of the seasonal 2019/2020 QIV. This ensured that participants received the most current available influenza vaccine per standard of care, and also allowed for the evaluation of the potential impact of these adjuvants on immune responses after a subsequent dose of unadjuvanted seasonal influenza vaccine. Groups 1, 2, and 3 received Fluzone® QIV at both vaccination time points while Groups 4, 5, and 6 received Flublok® QIV at both.
The primary safety objectives of the study were to assess the incidence of unsolicited adverse events and solicited reactogenicity events following vaccination with 2018/2019 Fluzone® or Flublok® with or without AF03 or Advax-CpG55.2 adjuvant. The primary immunogenicity objectives were to assess serum hemagglutination inhibition (HAI) antibody responses, serum neuraminidase inhibition antibody (NAI) responses, and influenza neutralizing antibody titer responses (Neut) against 2018/2019 QIV strains at Day 29 after receipt of vaccine as compared to baseline. As a Phase I, first-in-human study, it was not powered to be able to identify statistically significant differences in immune responses between groups. Full details of the planned primary and secondary outcome measures are available at www.clinicaltrials.gov.
2.2 Study vaccine and adjuvantsVaccines were obtained from Sanofi (Paris, France) in preservative-free, single-dose, pre-filled syringes. 2018/2019 Fluzone® QIV [Lot # UT6375MA] was formulated to contain 60 μg of HA per 0.5 mL dose with 15 μg of HA from each of the following four influenza virus strains recommended for the 2018/2019 influenza season: A/Michigan/45/2015 X-275 (H1N1); A/Singapore/INFIMH-16-009/2016 IVR-186 (H3N2); B/Maryland/15/2016 BX-69A (B/Colorado/06/2017-like; Victoria lineage), and B/Phuket/3073/2013 (Yamagata lineage). 2018/2019 Flublok® [Lot # QFAA1830] was formulated to contain 180 μg of HA per 0.5 mL dose, with 45 μg of HA derived from each of four influenza virus strains recommended for the 2018/2019 season as noted above. The 2019/2020 influenza strains recommended for use in Fluzone® [Lot # UJ210AA] and Flublok® [Lot # QFAA1916] commercial quadrivalent vaccines were: A/Brisbane/02/2018 (H1N1), A/Kansas/14/2017 (H3N2), B/Colorado/06/2017-like (Victoria lineage), and B/Phuket/3073/2013-like (Yamagata lineage).
The AF03 adjuvant [Lot # S4500F01] was manufactured by Sanofi and supplied as a sterile, preservative-free injectable suspension containing 5 % squalene. Each 0.75 mL dose of study vaccine admixed with AF03 contained approximately 12.5 mg of squalene. Advax-CpG55.2 was a combination adjuvant formulation comprised of two components, Advax [Lot # BP036] (manufactured by Sypharma Pty Ltd. for Vaxine, Adelaide, Australia) and the CpG55.2 oligonucleotide [Lot # BP040] (manufactured by Nikko Denka Avecia and Sypharma Pty Ltd. for Vaxine). These two components were mixed with quadrivalent influenza vaccines on the day of administration with each 1.0 mL vaccine dose containing approximately 15 mg of Advax and 0.15 mg of CpG55.2.
2.3 Evaluation of safety and reactogenicityReactogenicity was measured by the occurrence of solicited injection site and systemic reactions from the time of the first study vaccination through Day 8 (vaccination day was designated Day 1). Unsolicited non-serious adverse events (AEs) were collected from Day 1 through Day 29 post-vaccination. AE severity was classified as mild if there was no interference with daily activities, moderate if it caused some interference with daily activities or a low level of inconvenience, and severe if there was interruption of normal daily activities and/or systemic drug therapy or other treatment was required.
Information about serious adverse events (SAEs) and adverse events of special interest (AESIs) defined as tearing, dry mouth, or dry eyes, was collected from the time of first study vaccination through approximately 12 months. The secondary safety endpoints of medically attended AEs (MAAEs), including new onset chronic medical conditions (NOCMCs) and potentially immune mediated medical conditions (PIMMCs), were also collected through 12 months after the first study vaccination. Clinical safety laboratory evaluations were performed at screening, immediately prior to the first study vaccination and around Day 8 post-vaccination.
2.4 Immunogenicity assaysPrimary and secondary immunogenicity objectives were evaluated via serological assays to measure hemagglutination inhibition antibody titers (HAI), neutralizing antibody titers (Neut), and neuraminidase inhibition antibody titers (NAI) at multiple time points following study vaccination. Samples were provided to Sanofi in a blinded fashion, with assay results then uploaded to The Emmes Company, LLC, for analysis and reporting. Specifics of these assay methods have been previously described in detail [ 27, 28]. A multiplex ELISA assay was utilized to measure hemagglutinin binding antibody titers as described previously [ 26, 29, 30]. Antibody responses were determined for the quadrivalent vaccine strains as well as against heterologous influenza strains which included both historical and contemporary vaccine strains (see Fig. 4 legend).
2.5 Statistical analysisSafety summaries are presented overall and grouped by study arm. When calculating the incidence of solicited events, each participant is counted once at the highest severity following the applicable vaccination. Logistic regression models were used to model the relationships between study vaccination and the incidence of local and systemic events post-vaccination.
The study was designed to obtain sufficient data for meaningful estimates of immune responses, not for formal testing of a specific null hypothesis. Thus, immunogenicity analyses are primarily descriptive in nature, and results were assessed by qualitative evaluation of various statistical estimates alongside the bounds of the corresponding confidence intervals. Immune responses were summarized by study arm at each time point. Immunogenicity data summaries and analysis for primary and secondary endpoints were conducted for the modified Intent-to-Treat (mITT) population. Sensitivity analyses for those endpoints, as well as exploratory analyses, were performed in the Per Protocol (PP) population. The mITT group included all randomized participants who received the first vaccine dose and had at least one post-vaccine immunogenicity test with a valid result reported. The PP population excluded participants in the mITT group who were found ineligible at baseline or had no baseline (Day 1) immunogenicity blood samples; data from visits that were substantially out of window, or which occurred subsequent to major protocol deviations, were also excluded from PP population analyses. mITT analyses were conducted as-randomized, and PP analyses were conducted as-treated. Ratios of geometric mean titers (GMTs) along with their 95 % CIs between adjuvanted and unadjuvanted groups were presented at each timepoint. Exact confidence intervals using the Clopper Pearson method are presented for proportional endpoints. Confidence intervals for GMTs, geometric mean titers fold-rises (GMFRs; fold-rise computed per-participant as the ratio of post baseline titer to baseline titer), and GMT ratios (GMTRs; the ratio between adjuvanted group and unadjuvanted reference group GMTs at a given time point) were calculated based on the t-distribution. Missing data were minimal, and no imputation was performed for missing values.
3 Results3.1 Participants
Fig. 1 provides full details of participant enrollment, randomization to the six groups, and final disposition. A total of 241 participants were randomized and 239 received the assigned first study vaccination; one participant was assigned FluBlok® + AF03 and terminated the study prior to first vaccination, and another assigned to FluBlok® + AF03 received FluBlok® without adjuvant due to an error at the clinical site. 95.4 % of participants received the second study vaccination at approximately Day 90 and 93.4 % completed the study. Overall, the majority of participants were female (63 %), non-Hispanic (90 %), and White (73 %). Ethnicity and race were similarly distributed across study arms. Sex was similarly distributed across the study arms, other than a slightly higher proportion of males in the Fluzone® group 1. The mean age for all participants was 29.5 years (range: 18 to 45, median: 27) ( Table 1 ). The mean BMI for all participants was 26.2 (range: 16.9 to 54, median: 25.2). Both age and BMI were similar across study arms.
3.2 Immune response3.2.1 Antibody responses against 2018/2019 QIV strains
HAI, NAI, and Neut results are presented for the mITT population. Results from sensitivity analyses in the PP population were very similar to those in mITT, across assays, strains, and time points. Given the small sample size in each group, statistical power was limited, and qualitative evaluations are described.
There were substantial serum HAI titers measured at baseline (pre-vaccination) across all study groups for all 2018/2019 vaccine strains, demonstrating that the study population was immunologically experienced to influenza (GMTs and 95 % CIs shown in Fig. 2 ). Neut titers showed a similar pattern (Supplemental Fig. 1). Increases in HAI responses from baseline were observed by Day 8, with geometric mean fold rise (GMFR) of at least 2.3 for all groups, and lower bounds for the corresponding confidence intervals greater than 1. Neut responses showed a similar pattern (Supplemental Fig. 1). Responses tended to increase further at Day 29 (the primary immunogenicity timepoint), but only the Neut responses against A/Singapore/INFIMH-16-0019/2016 IVR-186 (H3N2) had a significant increase at Day 29 compared to Day 8, with no overlap in confidence intervals for any of the six groups between those time points (Supplemental Fig. 1). There were no clear differences between groups in the patterns of HAI and Neut responses against 2018/2019 QIV strains. Qualitatively, the highest titers were observed in Group 4 (Flublok®), and the highest increases in terms of GMFR were observed in group 5 (Flublok® plus AF03) [ Fig. 2 and Supplemental Fig. 1].
Baseline NAI antibody GMTs against all four 2018/2019 influenza strains were high in all study groups (group GMTs ranged from 63 to 257, results not shown). Administration of the 2018/2019 Fluzone® product with or without adjuvants boosted NAI titers approximately 2-fold on Day 29 (group GMFRs of 1.6–2.6; Fig. 3 ). There were no evident increases in the GMFRs associated with either adjuvant, across the four 2018/2019 strains.
3.2.2 Adjuvant effects on immunological responses to future influenza vaccineParticipants received a second dose of vaccine using unadjuvanted current season 2019/2020 QIV Fluzone® (Groups 1–3) or 2019/2020 Flublok® (Groups 4–6) on Day 90. This allowed the evaluation of future effects that the adjuvants might have on responsiveness to subsequent doses of seasonal influenza vaccine. The 2019/20 study vaccination did not substantially increase responses to the original 2018/2019 influenza antigens at Day 118 when compared to Day 90 (samples taken prior to the second study vaccination). The antibody titers at Day 90 specific to the 2019/20 vaccine antigens were high, with only marginal increases in GMTs noted at Day 118 to most of the antigens. However, HAI GMFRs to A/Kansas/14/2017 (H3N2 component in 2019/2020 QIV) ranged from 3.3 in Group 2 (Fluzone® plus AF03) to 7.1-fold in Group 1 (Fluzone®), as visualized in Fig. 4 . Inclusion of an adjuvant in the preceding season's vaccine did not appear to alter the response to the subsequent unadjuvanted seasonal influenza vaccine.
3.2.3 Impact of participant-reported historical seasonal influenza vaccines on immunological responsesPrevious vaccination status was assessed as a potential study confounder. Stratification by vaccine history was performed during randomization, and previous vaccination history also was assessed in subgroup analyses for HAI, Neut, and NAI results in the PP population. For all the analyses, 80 % of participants had received previous influenza vaccines in one or both of the two prior seasons. Thus, the unvaccinated subgroup sizes were extremely small, ranging from 5 to 10 individuals per study arm. For HAI and Neut results against 2018/19 quadrivalent influenza vaccine strains, participants without seasonal flu vaccination in the previous two seasons (2017–18 or 2018–19, prior to study start) frequently had qualitatively lower titers prior to the first study vaccination and showed a greater GMFR increase from baseline after receiving the assigned 2018/19 study vaccination. As expected, the small sample sizes for unvaccinated subgroups led to reduced precision to evaluate potential differences. This qualitative pattern was not observed for responses to the 2019/20 QIV strains after participants received the 2019–20 vaccine.
3.2.4 Immunological cross-protection against heterologous influenza antigensTo evaluate whether the adjuvants affected cross-neutralization against heterologous influenza strains, HAI and Neut assays were performed against five heterologous H3 strains (results not shown), and HA IgG binding assays against a larger panel of influenza vaccine strains, with the analysis done in the PP population. Fig. 5 demonstrates that increases in GMTs of HA binding antibodies were seen in almost all cases starting at Day 8 with only a slight further increase at Day 29. Qualitatively, titers appeared to increase more in the Flublok® groups compared to the Fluzone® groups though the differences were modest and not assessed statistically. Following the unadjuvanted 2019/2020 vaccinations (Day 90), antibody titers again increased in all groups and responses tended to be qualitatively higher for the Flublok® groups (Groups 4, 5, and 6), particularly the Flublok® + AF03 group (Group 5). NAI responses against heterologous N1 and N2 antigens were significantly boosted in the Fluzone® groups by Day 8, then gradually declined but remained above baseline at Day 90 (before the second study vaccination), after which another increase in responses was observed (results not shown). All Fluzone® groups (Groups 1, 2, and 3) had qualitatively similar patterns, while the Flublok® groups did not show evidence of increased NAI responses at Day 29 (the only day post-vaccination where samples were tested for Flublok® groups).
3.2.5 Durability of immune responsesHAI responses remained strong through Day 365 (275 days after the 2019/20 study vaccination) with well over 90 % of participants in each group retaining a titer of 1:40 against each 2018/2019 QIV strain (results not shown).
3.3 Safety and ReactogenicitySolicited local injection site and systemic reactogenicity events were evaluated for 8 days following the first vaccination using a memory aid. As shown in Table 2 , severe solicited AEs were rare and there was no statistical evidence of differences between study groups, as based on overlapping CI's. Two participants [5 %] in Group 1, 1 participant [3 %] in Group 2, 4 participants [10 %] in Group 3, 1 participant [2 %] in Group 4, 0 participants in Group 5, and 2 participants [5 %] in Group 6 reported severe symptoms. Mild to moderate systemic reactions of fatigue and headache were commonly reported, but there were no major trends between groups; fatigue was reported in 29-43 % and headache reported in 25-42 % of participants per group. Local injection site tenderness was common and occurred in 71–95 % of participants per group. Qualitatively, the groups receiving adjuvanted vaccine reported more mild or moderate local pain at the injection site.
Twenty-nine percent of participants reported a total of 96 unsolicited AEs during the 28 days following the first vaccination. The most frequently reported unsolicited AEs were diarrhea (6 participants [3 %]) and upper respiratory tract infection (4 participants [3 %]). No SAEs were reported within the 29 days following the first vaccination; two SAEs involving hospitalization were reported during the remainder of the study. One participant (Group 6) reported chronic left knee pain and one participant (Group 2) reported Clostridium difficile colitis following antibiotic exposure. Both SAEs were moderate in severity and were determined to be unrelated to study treatment.
No AESIs or PIMMCs were reported in this study. Three participants reported NOCMCs during the study. One participant experienced vitamin D deficiency, one participant experienced heart palpitations (mild in severity), and one participant experienced fatigue; none were considered related to the study product. Two pregnancies occurred during this study. One pregnancy occurred in Group 5 (estimated study day of conception: Day 97) and resulted in the live birth of a healthy infant. The other pregnancy occurred in Group 3 (estimated study day of conception: Day 116) and resulted in spontaneous abortion/miscarriage. Overall, there were no notable differences in safety or reactogenicity identified between the six study groups and the vaccines were well tolerated.
4 DiscussionThere is an urgent unmet need for strategies to improve the immunogenicity and effectiveness of seasonal influenza vaccines. This study evaluated the safety, reactogenicity and immunogenicity of one dose of commercially available 2018/2019 QIV (either Fluzone® or Flublok®) administered intramuscularly with or without one of two adjuvants (AF03 or Advax-CpG55.2). A previous study with similar design in murine models showed promising results [ 26]. The current study evaluated the traditional HAI and Neut responses among groups, and also explored additional immunological parameters. Since antibodies targeting NA may represent an independent correlate of protection against influenza infection [ 31, 32], we also assessed the NAI responses in each group receiving Fluzone®, which contains NA protein. The longitudinal kinetics and durability of HAI, Neut, and NAI responses were evaluated for one year following vaccination.
Each of the vaccine candidates were safe and well-tolerated. Overall, immunological analyses, which included HAI, Neut as well as HA antibody binding assays against a large number of homologous and heterologous strains, showed similar patterns over time and across study arms, with robust responses but few substantial differences between study arms. Similar to the findings in mice by Ustyugova et. al [ 26], the addition of AF03 showed a nonsignificant trend towards higher antibody levels against some strains, suggesting an increase in magnitude of antibody response to heterologous influenza strains. It was not clear from these results whether the addition of Advax-CpG55.2 had any impact on antibody responses.
This study had several limitations. With a relatively small sample size of 240 participants across six study groups, it was not designed to test statistical differences between groups. Cellular immune responses were not characterized in depth. Additionally, this was a highly immunologically experienced population with high baseline titers to homologous and heterologous H1N1, H3N2 and B influenza strains. This high baseline immunity may have overshadowed effects that might otherwise have been seen with addition of the AF03 and Advax-CpG55.2 adjuvants in influenza-naïve participants. Adjuvants have their strongest effects when being used to prime an initial immune response, rather than when being used to boost an already existing immune response [ 33]. It is therefore possible that these same adjuvants would have a more profound effect when combined with avian or other non-seasonal influenza virus antigens to which more individuals are naïve, or if used for seasonal immunization of young children completely naïve to influenza infection. There was some evidence of greater responses to 2018/19 strains among the participants who hadn't received seasonal influenza vaccination in the previous two seasons, but the numbers of naïve participants were too small for statistical evaluation of those results.
Influenza vaccines now commonly utilize highly purified or recombinant HA proteins, making the antigen component of the vaccines safer but in some cases less immunogenic. This may contribute to their inconsistent and lower than ideal real-world effectiveness in preventing influenza disease and hospitalization [ 1, 3]. Adjuvants have shown the capacity to improve immune responses to influenza vaccines, and although the results of this study provide reassuring human safety data for both the AF03 and Advax-CpG55.2 adjuvants, there is still the potential for safety risks including local, systemic, and immunological toxicities that might not have been identified in this small phase 1 study. We are now entering an era of “rational vaccine design,” where detailed knowledge of the mechanism of action of various adjuvants can allow for targeting immune pathways best suited to vaccine protection against specific pathogens [ 6]. The current study did not show significant differences between the immunogenicity elicited by the two adjuvants, which work through different immune mechanisms. Both licensed QIVs were well tolerated and effective when combined with either AF03 or Advax-CpG55.2. This trial provided the first human safety data on the novel Advax-CpG55.2 combination adjuvant, with no adverse safety signals seen.
FundingThis work was supported by funding provided by the Division of Microbiology and Infectious Diseases at the National Institutes of Health under contracts HHSN272201300020I (University of Iowa), HHSN272201300015I (Baylor College of Medicine), HHSN272201300021I (Saint Louis University), HHSN272201300016I (Cincinnati Children's Medical Center), HHSN272201300017I (Duke University), HHSN272201300023I (Vanderbilt University Medical Center), HHSN272201300018I (Emory University), HHSN272201300022I (University of Maryland School of Medicine Center for Vaccine Development) and 75N93021C00012 (Emmes Corporation). Development of Advax-CpG55.2 adjuvant was supported by funding from Vaxine Pty Ltd. and National Institute of Allergy and Infectious Diseases of the National Institutes of Health under Contract HHS-N272201400053C, HHSN272201800044C, and HHSN272201800024C.
Authorship StatementAll authors attest they meet the ICMJE criteria for authorship.
CRediT authorship contribution statementTheresa E. Hegmann: Writing – review & editing, Writing – original draft, Visualization, Validation, Investigation. Emmanuel B. Walter: Writing – review & editing, Supervision, Resources, Project administration, Investigation, Funding acquisition. Michael J. Smith: Writing – review & editing, Investigation, Conceptualization. James Campbell: Writing – review & editing, Investigation. Hana M. El Sahly: Writing – review & editing, Supervision, Project administration, Investigation, Funding acquisition, Conceptualization. Jennifer A. Whitaker: Writing – review & editing, Investigation. C. Buddy Creech: Writing – review & editing, Methodology, Investigation, Conceptualization. Irina V. Ustyugova: Writing – review & editing, Validation, Investigation, Data curation. Ana P. Goncalvez: Writing – review & editing, Supervision, Methodology, Conceptualization. Aseem Pandey: Writing – review & editing, Validation, Methodology, Investigation, Data curation. Timothy Alefantis: Supervision, Project administration, Funding acquisition, Conceptualization. Saranya Sridhar: Writing – review & editing, Validation, Methodology, Funding acquisition, Conceptualization. Yoshikazu Honda-Okubo: Writing – review & editing, Resources. Nikolai Petrovsky: Writing – review & editing, Methodology, Conceptualization. Sharon E. Frey: Writing – review & editing, Investigation. Getahun Abate: Writing – review & editing, Investigation. Grant Paulsen: Writing – review & editing, Supervision, Methodology, Investigation. Evan J. Anderson: Writing – review & editing, Supervision, Resources, Methodology, Data curation, Conceptualization. Christina A. Rostad: Writing – review & editing, Investigation. Nadine Rouphael: Writing – review & editing, Investigation. Mamodikoe Makhene: Writing – review & editing, Supervision, Project administration, Methodology. Paul C. Roberts: Writing – review & editing, Visualization, Supervision, Project administration, Methodology, Funding acquisition, Conceptualization. Bonifride Tuyishimire: Visualization, Validation, Supervision, Software, Project administration, Methodology, Formal analysis, Conceptualization. Christopher Bryant: Writing – review & editing, Writing – original draft, Validation, Supervision, Project administration, Formal analysis. Patricia Winokur: Writing – review & editing, Writing – original draft, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Conceptualization.
Declaration of competing interestThe authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Emmanuel B. Walter reports a relationship with Pfizer Inc. that includes: consulting or advisory and funding grants. Emmanuel B. Walter reports a relationship with Moderna Inc. that includes: funding grants. Emmanuel B. Walter reports a relationship with Seqirus USA Inc. that includes: funding grants. Emmanuel B. Walter reports a relationship with Clinetic that includes: funding grants. Emmanuel B. Walter reports a relationship with Najit Technologies Inc. that includes: funding grants. Emmanuel B. Walter reports a relationship with Vaxcyte Inc. that includes: consulting or advisory. Emmanuel B. Walter reports a relationship with ILiAD Biotechnologies LLC that includes: consulting or advisory. Emmanuel B. Walter served on a Data Safety Monitoring Board for Shionogi. Michael Smith reports a relationship with Pfizer that includes: funding grants. Michael Smith reports a relationship with Sanofi Pasteur Inc. that includes: funding grants. C. Buddy Creech reports a relationship with Sanofi Pasteur Inc. that includes: consulting or advisory. C. Buddy Creech reports a relationship with Moderna Inc. that includes: consulting or advisory. C. Buddy Creech reports a relationship with GlaxoSmithKline Inc. that includes: consulting or advisory. Irina V. Ustyugova reports financial support, administrative support, and equipment, drugs, or supplies were provided by Sanofi Vaccines. Irina V. Ustyugova reports a relationship with Sanofi Vaccines that includes: employment and equity or stocks. Ana P. Goncalvez reports financial support, administrative support, and equipment, drugs, or supplies were provided by Sanofi Vaccines. Ana P. Goncalvez reports a relationship with Sanofi Vaccines that includes: employment and equity or stocks. Aseem Pandey reports financial support, administrative support, and equipment, drugs, or supplies were provided by Sanofi Vaccines. Aseem Pandey reports a relationship with Sanofi Vaccines that includes: employment and equity or stocks. Timothy Alefantis reports financial support, administrative support, and equipment, drugs, or supplies were provided by Sanofi. Timothy Alefantis reports a relationship with Sanofi Pasteur that includes: employment and equity or stocks. Timothy Alefantis reports a relationship with Icosavax, Inc. that includes: employment and equity or stocks. Timothy Alefantis is formerly an employee of Sanofi Pasteur and Icosavax. Saranya Sridhar reports a relationship with Sanofi that includes: equity or stocks. Saranya Sridhar has patent pending to Sanofi. Yoshikazu Honda-Okubo reports financial support was provided by Vaxine Pty Ltd. Yoshikazu Honda-Okubo reports a relationship with Vaxine Pty Ltd. that includes: employment. Yoshikazu Honda-Okubo has patent #P2014–515234 issued to Vaxine Pty Ltd. Nikolai Petrovsky reports equipment, drugs, or supplies was provided by Vaxine Pty Ltd. Nikolai Petrovsky reports a relationship with Vaxine Pty Ltd. that includes: board membership and employment. Nikolai Petrovsky has patent on a vaccine adjuvant composition comprising inulin particles issued to Vaxine Pty Ltd. Evan Anderson reports a relationship with Moderna Inc. that includes: employment and equity or stocks. Evan Anderson is currently employed by Moderna. Christina Rostad reports financial support was provided by Pfizer Inc. Christina Rostad reports financial support was provided by Moderna Inc. Christina Rostad reports financial support was provided by Merck & Co Inc. Christina Rostad reports financial support was provided by Sanofi. Christina Rostad reports financial support was provided by Janssen Pharmaceuticals Inc. Christina Rostad has patents on Chimeric RSV, Immunogenic Compositions, and Methods of Use with royalties paid to Meissa Vaccines, Inc. Christina Rostad has patents on RSV Live-Attenuated Vaccine Candidates with Deleted G-Protein Mucin Domains issued to Emory University. Nadine Rouphael reports a relationship with Emory University School of Medicine that includes: employment. Emory receives funds for NR to conduct research from Sanofi, Lilly, Merck, Quidel, Immorna, Vaccine Company and Pfizer. NR served on selected advisory boards for Sanofi, Seqirus, Pfizer and Moderna and is a paid clinical trials safety consultant for ICON, CyanVac, Imunon and EMMES. Bonifride Tuyishimire reports financial support was provided by The Emmes Company LLC. Christopher Bryant reports financial support was provided by The Emmes Company LLC. Patricia Winokur reports a relationship with Emmes Corporation that includes: board membership. Patricia Winokur reports a relationship with Blue Lake Biotechnology that includes: board membership. Dr. Winokur has received research funding from Pfizer. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
AcknowledgmentsWe are thankful for the study participants, the Respiratory Diseases Branch at the National Institutes of Allergy and Infectious Diseases (Melinda Tibbals, Tena Knudsen, Kathy Ormanoski, Robin Mason, Rhonda Pikaart-Tautges, Ahsen Khan), and the Emmes Company (Ashley Wegel, Bruno DosSantos, Shu Yang). This study would not have been possible without the support of the Iowa study team (Jack Stapleton, MD, Jeff Meier, MD, Laura Stulken, PA, Deb Pfab, RN, AJ Carr, Michelle Rodenburg); the SLU study team (Linda Eggemeyer-Sharpe, RN, BSN study coordinator, Dan Hoft, MD, PhD, Abeer Al Majali, MD, Helay Amini, PharmD, Azra Blazevic, DVM, MPH, Tamar Blevins, MS, Chase Colbert, Soumya Chatterjee, MD, Kathleen Chirco, RN, BSN, Sabrina DiPiazza, RN, BSN, MA, Heather Douds, MSNS, BA, BSN, RN, Carol Duane, RN, PhD, Eric Eggemeyer, BA, Sarah George, MD, Geoffrey Gorse, MD, Irene Graham, MD, Michelle Harris, PharmD, Daniel Hoft, MD, PhD, Kate Liefer, RN, BSN, Melissa Loyet, RN, Keith Meyer, BS, Krystal Meza, BS, Camerra Miller, BS, Karla Mosby, RN, Theresa Munsell, RN, Amanda Nethington, M, Huan Ning, MD, Nicole Purcell, Joan Siegner, RN, BSN, MA, Susan Stewart, RN, Janice Tennant, RN, BSN, MPH, Kiana Wilder, BA, Mei Xia, MD, PhD, Yinyi Yu, BS); the staff and faculty of the Emory Hope Clinic and Emory Childrens Center- Vaccine Research Clinic; and the University of Maryland study team (Karen Kotloff, Melissa Billington, Alyson Kwon, Lisa Chrisley, Mardi Reymann, Myounghee Lee, Brenda Dorsey, Regina Harley, Parula Bernal, Justin Ortiz, Linda Wadsworth, and Colleen Boyce). We also wish to acknowledge the support of the University of Maryland, Baltimore, Institute for Clinical & Translational Research (ICTR), the General Clinical Research Center, and the National Center for Advancing Translational Sciences (NCATS) Clinical Translational Science Award (CTSA) grant number UL1TR003098. Our thanks also to the Sanofi team for their expertise in performing the immunogenicity assays utilized by this study.
Appendix A Supplementary dataSupplementary material Supplemental Fig. 1. Geometric Mean Titers of Neutralizing Antibody Against 2018/2019 QIV strains by Study Day and Study Arm, Modified Intent-to-Treat Population. Supplementary material
Appendix A Supplementary dataSupplementary data to this article can be found online at https://doi.org/10.1016/j.vaccine.2025.126991.
| | | | | | | | |||||||||
| | | | | | | | | | | | | | | | |
| Sex | Male | 19 | 46 | 11 | 30 | 12 | 30 | 14 | 34 | 16 | 41 | 17 | 40 | 89 | 37 |
| Female | 22 | 54 | 26 | 70 | 28 | 70 | 27 | 66 | 23 | 59 | 26 | 60 | 152 | 63 | |
| BMI | <30 | 32 | 78 | 30 | 81 | 34 | 85 | 31 | 76 | 31 | 79 | 35 | 81 | 193 | 80 |
| ≥ 30 | 9 | 22 | 7 | 19 | 6 | 15 | 10 | 24 | 8 | 21 | 8 | 19 | 48 | 20 | |
| Ethnicity | Not Hispanic or Latino | 40 | 98 | 33 | 89 | 36 | 90 | 34 | 83 | 34 | 87 | 40 | 93 | 217 | 90 |
| Hispanic or Latino | 1 | 2 | 4 | 11 | 3 | 8 | 7 | 17 | 5 | 13 | 3 | 7 | 23 | 10 | |
| Not Reported | – | – | – | – | 1 | 3 | – | – | – | – | – | – | 1 | <1 | |
| Unknown | – | – | – | – | – | – | – | – | – | – | – | – | – | – | |
| Race | American Indian or
Alaska Native | – | – | – | – | – | – | 1 | 2 | – | – | – | – | 1 | <1 |
| Asian | 1 | 2 | 2 | 5 | 4 | 10 | 5 | 12 | 5 | 13 | 5 | 12 | 22 | 9 | |
| Native Hawaiian or
Other Pacific Islander | – | – | – | – | – | – | – | – | – | – | – | – | – | – | |
| Black or African
American | 7 | 17 | 4 | 11 | 2 | 5 | 7 | 17 | 8 | 21 | 2 | 5 | 30 | 12 | |
| White | 33 | 80 | 28 | 76 | 32 | 80 | 26 | 63 | 24 | 62 | 34 | 79 | 177 | 73 | |
| Multi-Racial | – | – | 2 | 5 | 2 | 5 | 1 | 2 | 2 | 5 | 2 | 5 | 9 | 4 | |
| Unknown | – | – | 1 | 3 | – | – | 1 | 2 | – | – | – | – | 2 | <1 | |
| Prior Seasonal
Influenza Vaccination | Neither 2017–2018 or
2018–2019 | 5 | 12 | 7 | 19 | 8 | 20 | 8 | 20 | 9 | 23 | 7 | 16 | 44 | 18 |
| Either 2017–2018 or
2018–2019 | 36 | 88 | 29 | 78 | 32 | 80 | 32 | 78 | 30 | 77 | 36 | 84 | 195 | 81 | |
| Unknown | – | – | 1 | 3 | – | – | 1 | 2 | – | – | – | – | 2 | <1 | |
| | | | | | | ||||||||
| | | | | | | | | | | | | | |
| Any Symptom | None | 5 | 12 (4.1, 26.2) | 1 | 3 (0.1, 14.2) | 0 | 0 (0, 8.8) | 8 | 19 (8.6, 34.1) | 3 | 8 (1.7, 21.9) | 0 | 0 (0, 8.2) |
| Mild | 26 | 63 (46.9, 77.9) | 21 | 57 (39.5, 72.9) | 23 | 58 (40.9, 73) | 28 | 67 (50.5, 80.4) | 27 | 73 (55.9, 86.2) | 30 | 70 (53.9, 82.8) | |
| Moderate | 8 | 20 (8.8, 34.9) | 14 | 38 (22.5, 55.2) | 13 | 33 (18.6, 49.1) | 5 | 12 (4, 25.6) | 7 | 19 (8, 35.2) | 11 | 26 (13.5, 41.2) | |
| Severe | 2 | 5 (0.6, 16.5) | 1 | 3 (0.1, 14.2) | 4 | 10 (2.8, 23.7) | 1 | 2 (0.1, 12.6) | 0 | 0 (0, 9.5) | 2 | 5 (0.6, 15.8) | |
| Any Systemic
Symptom | None | 17 | 41 (26.3, 57.9) | 12 | 32 (18, 49.8) | 17 | 43 (27, 59.1) | 19 | 45 (29.8, 61.3) | 19 | 51 (34.4, 68.1) | 17 | 40 (25, 55.6) |
| Mild | 20 | 49 (32.9, 64.9) | 15 | 41 (24.8, 57.9) | 19 | 48 (31.5, 63.9) | 19 | 45 (29.8, 61.3) | 14 | 38 (22.5, 55.2) | 20 | 47 (31.2, 62.3) | |
| Moderate | 4 | 10 (2.7, 23.1) | 10 | 27 (13.8, 44.1) | 3 | 8 (1.6, 20.4) | 4 | 10 (2.7, 22.6) | 4 | 11 (3, 25.4) | 6 | 14 (5.3, 27.9) | |
| Severe | 0 | 0 (0, 8.6) | 0 | 0 (0, 9.5) | 1 | 3 (0.1, 13.2) | 0 | 0 (0, 8.4) | 0 | 0 (0, 9.5) | 0 | 0 (0, 8.2) | |
| Fever | None | 41 | 100 (91.4, 100) | 36 | 97 (85.8, 99.9) | 40 | 100 (91.2, 100) | 41 | 98 (87.4, 99.9) | 37 | 100 (90.5, 100) | 43 | 100 (91.8, 100) |
| Mild | 0 | 0 (0, 8.6) | 0 | 0 (0, 9.5) | 0 | 0 (0, 8.8) | 1 | 2 (0.1, 12.6) | 0 | 0 (0, 9.5) | 0 | 0 (0, 8.2) | |
| Moderate | 0 | 0 (0, 8.6) | 1 | 3 (0.1, 14.2) | 0 | 0 (0, 8.8) | 0 | 0 (0, 8.4) | 0 | 0 (0, 9.5) | 0 | 0 (0, 8.2) | |
| Severe | 0 | 0 (0, 8.6) | 0 | 0 (0, 9.5) | 0 | 0 (0, 8.8) | 0 | 0 (0, 8.4) | 0 | 0 (0, 9.5) | 0 | 0 (0, 8.2) | |
| Feverishness | None | 39 | 95 (83.5, 99.4) | 33 | 89 (74.6, 97) | 34 | 85 (70.2, 94.3) | 37 | 88 (74.4, 96) | 32 | 86 (71.2, 95.5) | 39 | 91 (77.9, 97.4) |
| Mild | 2 | 5 (0.6, 16.5) | 1 | 3 (0.1, 14.2) | 6 | 15 (5.7, 29.8) | 4 | 10 (2.7, 22.6) | 4 | 11 (3, 25.4) | 4 | 9 (2.6, 22.1) | |
| Moderate | 0 | 0 (0, 8.6) | 3 | 8 (1.7, 21.9) | 0 | 0 (0, 8.8) | 1 | 2 (0.1, 12.6) | 1 | 3 (0.1, 14.2) | 0 | 0 (0, 8.2) | |
| Severe | 0 | 0 (0, 8.6) | 0 | 0 (0, 9.5) | 0 | 0 (0, 8.8) | 0 | 0 (0, 8.4) | 0 | 0 (0, 9.5) | 0 | 0 (0, 8.2) | |
| Fatigue | None | 29 | 71 (54.5, 83.9) | 21 | 57 (39.5, 72.9) | 26 | 65 (48.3, 79.4) | 29 | 69 (52.9, 82.4) | 23 | 62 (44.8, 77.5) | 28 | 65 (49.1, 79) |
| Mild | 9 | 22 (10.6, 37.6) | 15 | 41 (24.8, 57.9) | 11 | 28 (14.6, 43.9) | 11 | 26 (13.9, 42) | 12 | 32 (18, 49.8) | 13 | 30 (17.2, 46.1) | |
| Moderate | 3 | 7 (1.5, 19.9) | 1 | 3 (0.1, 14.2) | 3 | 8 (1.6, 20.4) | 2 | 5 (0.6, 16.2) | 2 | 5 (0.7, 18.2) | 2 | 5 (0.6, 15.8) | |
| Severe | 0 | 0 (0, 8.6) | 0 | 0 (0, 9.5) | 0 | 0 (0, 8.8) | 0 | 0 (0, 8.4) | 0 | 0 (0, 9.5) | 0 | 0 (0, 8.2) | |
| Malaise | None | 32 | 78 (62.4, 89.4) | 26 | 70 (53, 84.1) | 32 | 80 (64.4, 90.9) | 36 | 86 (71.5, 94.6) | 29 | 78 (61.8, 90.2) | 38 | 88 (74.9, 96.1) |
| Mild | 8 | 20 (8.8, 34.9) | 8 | 22 (9.8, 38.2) | 8 | 20 (9.1, 35.6) | 6 | 14 (5.4, 28.5) | 7 | 19 (8, 35.2) | 4 | 9 (2.6, 22.1) | |
| Moderate | 1 | 2 (0.1, 12.9) | 3 | 8 (1.7, 21.9) | 0 | 0 (0, 8.8) | 0 | 0 (0, 8.4) | 1 | 3 (0.1, 14.2) | 1 | 2 (0.1, 12.3) | |
| Severe | 0 | 0 (0, 8.6) | 0 | 0 (0, 9.5) | 0 | 0 (0, 8.8) | 0 | 0 (0, 8.4) | 0 | 0 (0, 9.5) | 0 | 0 (0, 8.2) | |
| Myalgia | None | 30 | 73 (57.1, 85.8) | 22 | 59 (42.1, 75.2) | 28 | 70 (53.5, 83.4) | 36 | 86 (71.5, 94.6) | 29 | 78 (61.8, 90.2) | 32 | 74 (58.8, 86.5) |
| Mild | 11 | 27 (14.2, 42.9) | 11 | 30 (15.9, 47) | 11 | 28 (14.6, 43.9) | 4 | 10 (2.7, 22.6) | 7 | 19 (8, 35.2) | 9 | 21 (10, 36) | |
| Moderate | 0 | 0 (0, 8.6) | 4 | 11 (3, 25.4) | 1 | 3 (0.1, 13.2) | 2 | 5 (0.6, 16.2) | 1 | 3 (0.1, 14.2) | 2 | 5 (0.6, 15.8) | |
| Severe | 0 | 0 (0, 8.6) | 0 | 0 (0, 9.5) | 0 | 0 (0, 8.8) | 0 | 0 (0, 8.4) | 0 | 0 (0, 9.5) | 0 | 0 (0, 8.2) | |
| Arthralgia | None | 39 | 95 (83.5, 99.4) | 32 | 86 (71.2, 95.5) | 39 | 98 (86.8, 99.9) | 40 | 95 (83.8, 99.4) | 36 | 97 (85.8, 99.9) | 42 | 98 (87.7, 99.9) |
| Mild | 2 | 5 (0.6, 16.5) | 3 | 8 (1.7, 21.9) | 1 | 3 (0.1, 13.2) | 2 | 5 (0.6, 16.2) | 1 | 3 (0.1, 14.2) | 1 | 2 (0.1, 12.3) | |
| Moderate | 0 | 0 (0, 8.6) | 2 | 5 (0.7, 18.2) | 0 | 0 (0, 8.8) | 0 | 0 (0, 8.4) | 0 | 0 (0, 9.5) | 0 | 0 (0, 8.2) | |
| Severe | 0 | 0 (0, 8.6) | 0 | 0 (0, 9.5) | 0 | 0 (0, 8.8) | 0 | 0 (0, 8.4) | 0 | 0 (0, 9.5) | 0 | 0 (0, 8.2) | |
| Headache | None | 30 | 73 (57.1, 85.8) | 16 | 43 (27.1, 60.5) | 30 | 75 (58.8, 87.3) | 29 | 69 (52.9, 82.4) | 26 | 70 (53, 84.1) | 33 | 77 (61.4, 88.2) |
| Mild | 9 | 22 (10.6, 37.6) | 17 | 46 (29.5, 63.1) | 9 | 23 (10.8, 38.5) | 10 | 24 (12.1, 39.5) | 10 | 27 (13.8, 44.1) | 9 | 21 (10, 36) | |
| Moderate | 2 | 5 (0.6, 16.5) | 4 | 11 (3, 25.4) | 0 | 0 (0, 8.8) | 3 | 7 (1.5, 19.5) | 1 | 3 (0.1, 14.2) | 1 | 2 (0.1, 12.3) | |
| Severe | 0 | 0 (0, 8.6) | 0 | 0 (0, 9.5) | 1 | 3 (0.1, 13.2) | 0 | 0 (0, 8.4) | 0 | 0 (0, 9.5) | 0 | 0 (0, 8.2) | |
| Nausea | None | 39 | 95 (83.5, 99.4) | 34 | 92 (78.1, 98.3) | 37 | 93 (79.6, 98.4) | 37 | 88 (74.4, 96) | 32 | 86 (71.2, 95.5) | 37 | 86 (72.1, 94.7) |
| Mild | 2 | 5 (0.6, 16.5) | 0 | 0 (0, 9.5) | 3 | 8 (1.6, 20.4) | 4 | 10 (2.7, 22.6) | 3 | 8 (1.7, 21.9) | 5 | 12 (3.9, 25.1) | |
| Moderate | 0 | 0 (0, 8.6) | 3 | 8 (1.7, 21.9) | 0 | 0 (0, 8.8) | 1 | 2 (0.1, 12.6) | 2 | 5 (0.7, 18.2) | 1 | 2 (0.1, 12.3) | |
| Severe | 0 | 0 (0, 8.6) | 0 | 0 (0, 9.5) | 0 | 0 (0, 8.8) | 0 | 0 (0, 8.4) | 0 | 0 (0, 9.5) | 0 | 0 (0, 8.2) | |
| Any Local
Symptom | None | 6 | 15 (5.6, 29.2) | 1 | 3 (0.1, 14.2) | 0 | 0 (0, 8.8) | 10 | 24 (12.1, 39.5) | 4 | 11 (3, 25.4) | 0 | 0 (0, 8.2) |
| Mild | 29 | 71 (54.5, 83.9) | 26 | 70 (53, 84.1) | 24 | 60 (43.3, 75.1) | 29 | 69 (52.9, 82.4) | 27 | 73 (55.9, 86.2) | 32 | 74 (58.8, 86.5) | |
| Moderate | 4 | 10 (2.7, 23.1) | 9 | 24 (11.8, 41.2) | 12 | 30 (16.6, 46.5) | 2 | 5 (0.6, 16.2) | 6 | 16 (6.2, 32) | 9 | 21 (10, 36) | |
| Severe | 2 | 5 (0.6, 16.5) | 1 | 3 (0.1, 14.2) | 4 | 10 (2.8, 23.7) | 1 | 2 (0.1, 12.6) | 0 | 0 (0, 9.5) | 2 | 5 (0.6, 15.8) | |
| Pain | None | 29 | 71 (54.5, 83.9) | 15 | 41 (24.8, 57.9) | 14 | 35 (20.6, 51.7) | 35 | 83 (68.6, 93) | 16 | 43 (27.1, 60.5) | 16 | 37 (23, 53.3) |
| Mild | 10 | 24 (12.4, 40.3) | 18 | 49 (31.9, 65.6) | 21 | 53 (36.1, 68.5) | 7 | 17 (7, 31.4) | 18 | 49 (31.9, 65.6) | 24 | 56 (39.9, 70.9) | |
| Moderate | 2 | 5 (0.6, 16.5) | 4 | 11 (3, 25.4) | 5 | 13 (4.2, 26.8) | 0 | 0 (0, 8.4) | 3 | 8 (1.7, 21.9) | 3 | 7 (1.5, 19.1) | |
| Severe | 0 | 0 (0, 8.6) | 0 | 0 (0, 9.5) | 0 | 0 (0, 8.8) | 0 | 0 (0, 8.4) | 0 | 0 (0, 9.5) | 0 | 0 (0, 8.2) | |
| Tenderness | None | 12 | 29 (16.1, 45.5) | 2 | 5 (0.7, 18.2) | 2 | 5 (0.6, 16.9) | 12 | 29 (15.7, 44.6) | 7 | 19 (8, 35.2) | 2 | 5 (0.6, 15.8) |
| Mild | 28 | 68 (51.9, 81.9) | 31 | 84 (68, 93.8) | 30 | 75 (58.8, 87.3) | 29 | 69 (52.9, 82.4) | 29 | 78 (61.8, 90.2) | 36 | 84 (69.3, 93.2) | |
| Moderate | 1 | 2 (0.1, 12.9) | 4 | 11 (3, 25.4) | 8 | 20 (9.1, 35.6) | 1 | 2 (0.1, 12.6) | 1 | 3 (0.1, 14.2) | 5 | 12 (3.9, 25.1) | |
| Severe | 0 | 0 (0, 8.6) | 0 | 0 (0, 9.5) | 0 | 0 (0, 8.8) | 0 | 0 (0, 8.4) | 0 | 0 (0, 9.5) | 0 | 0 (0, 8.2) | |
| Pruritus | None | 40 | 98 (87.1, 99.9) | 36 | 97 (85.8, 99.9) | 39 | 98 (86.8, 99.9) | 41 | 98 (87.4, 99.9) | 36 | 97 (85.8, 99.9) | 40 | 93 (80.9, 98.5) |
| Mild | 1 | 2 (0.1, 12.9) | 1 | 3 (0.1, 14.2) | 1 | 3 (0.1, 13.2) | 1 | 2 (0.1, 12.6) | 1 | 3 (0.1, 14.2) | 3 | 7 (1.5, 19.1) | |
| Moderate | 0 | 0 (0, 8.6) | 0 | 0 (0, 9.5) | 0 | 0 (0, 8.8) | 0 | 0 (0, 8.4) | 0 | 0 (0, 9.5) | 0 | 0 (0, 8.2) | |
| Severe | 0 | 0 (0, 8.6) | 0 | 0 (0, 9.5) | 0 | 0 (0, 8.8) | 0 | 0 (0, 8.4) | 0 | 0 (0, 9.5) | 0 | 0 (0, 8.2) | |
| Ecchymosis | None | 39 | 95 (83.5, 99.4) | 34 | 92 (78.1, 98.3) | 39 | 98 (86.8, 99.9) | 40 | 95 (83.8, 99.4) | 37 | 100 (90.5, 100) | 39 | 91 (77.9, 97.4) |
| Mild | 2 | 5 (0.6, 16.5) | 3 | 8 (1.7, 21.9) | 1 | 3 (0.1, 13.2) | 2 | 5 (0.6, 16.2) | 0 | 0 (0, 9.5) | 4 | 9 (2.6, 22.1) | |
| Moderate | 0 | 0 (0, 8.6) | 0 | 0 (0, 9.5) | 0 | 0 (0, 8.8) | 0 | 0 (0, 8.4) | 0 | 0 (0, 9.5) | 0 | 0 (0, 8.2) | |
| Severe | 0 | 0 (0, 8.6) | 0 | 0 (0, 9.5) | 0 | 0 (0, 8.8) | 0 | 0 (0, 8.4) | 0 | 0 (0, 9.5) | 0 | 0 (0, 8.2) | |
| Ecchymosis
(measurement) | None | 39 | 95 (83.5, 99.4) | 34 | 92 (78.1, 98.3) | 39 | 98 (86.8, 99.9) | 40 | 95 (83.8, 99.4) | 37 | 100 (90.5, 100) | 39 | 91 (77.9, 97.4) |
| Mild | 1 | 2 (0.1, 12.9) | 1 | 3 (0.1, 14.2) | 1 | 3 (0.1, 13.2) | 2 | 5 (0.6, 16.2) | 0 | 0 (0, 9.5) | 4 | 9 (2.6, 22.1) | |
| Moderate | 1 | 2 (0.1, 12.9) | 2 | 5 (0.7, 18.2) | 0 | 0 (0, 8.8) | 0 | 0 (0, 8.4) | 0 | 0 (0, 9.5) | 0 | 0 (0, 8.2) | |
| Severe | 0 | 0 (0, 8.6) | 0 | 0 (0, 9.5) | 0 | 0 (0, 8.8) | 0 | 0 (0, 8.4) | 0 | 0 (0, 9.5) | 0 | 0 (0, 8.2) | |
| Erythema | None | 33 | 80 (65.1, 91.2) | 28 | 76 (58.8, 88.2) | 24 | 60 (43.3, 75.1) | 36 | 86 (71.5, 94.6) | 30 | 81 (64.8, 92) | 31 | 72 (56.3, 84.7) |
| Mild | 8 | 20 (8.8, 34.9) | 8 | 22 (9.8, 38.2) | 16 | 40 (24.9, 56.7) | 6 | 14 (5.4, 28.5) | 7 | 19 (8, 35.2) | 12 | 28 (15.3, 43.7) | |
| Moderate | 0 | 0 (0, 8.6) | 1 | 3 (0.1, 14.2) | 0 | 0 (0, 8.8) | 0 | 0 (0, 8.4) | 0 | 0 (0, 9.5) | 0 | 0 (0, 8.2) | |
| Severe | 0 | 0 (0, 8.6) | 0 | 0 (0, 9.5) | 0 | 0 (0, 8.8) | 0 | 0 (0, 8.4) | 0 | 0 (0, 9.5) | 0 | 0 (0, 8.2) | |
| Erythema
(measurement) | None | 33 | 80 (65.1, 91.2) | 28 | 76 (58.8, 88.2) | 24 | 60 (43.3, 75.1) | 36 | 86 (71.5, 94.6) | 30 | 81 (64.8, 92) | 31 | 72 (56.3, 84.7) |
| Mild | 7 | 17 (7.2, 32.1) | 7 | 19 (8, 35.2) | 12 | 30 (16.6, 46.5) | 6 | 14 (5.4, 28.5) | 5 | 14 (4.5, 28.8) | 7 | 16 (6.8, 30.7) | |
| Moderate | 0 | 0 (0, 8.6) | 1 | 3 (0.1, 14.2) | 2 | 5 (0.6, 16.9) | 0 | 0 (0, 8.4) | 2 | 5 (0.7, 18.2) | 4 | 9 (2.6, 22.1) | |
| Severe | 1 | 2 (0.1, 12.9) | 1 | 3 (0.1, 14.2) | 2 | 5 (0.6, 16.9) | 0 | 0 (0, 8.4) | 0 | 0 (0, 9.5) | 1 | 2 (0.1, 12.3) | |
| Induration
/Swelling | None | 29 | 71 (54.5, 83.9) | 33 | 89 (74.6, 97) | 27 | 68 (50.9, 81.4) | 36 | 86 (71.5, 94.6) | 31 | 84 (68, 93.8) | 30 | 70 (53.9, 82.8) |
| Mild | 10 | 24 (12.4, 40.3) | 4 | 11 (3, 25.4) | 12 | 30 (16.6, 46.5) | 6 | 14 (5.4, 28.5) | 6 | 16 (6.2, 32) | 13 | 30 (17.2, 46.1) | |
| Moderate | 2 | 5 (0.6, 16.5) | 0 | 0 (0, 9.5) | 1 | 3 (0.1, 13.2) | 0 | 0 (0, 8.4) | 0 | 0 (0, 9.5) | 0 | 0 (0, 8.2) | |
| Severe | 0 | 0 (0, 8.6) | 0 | 0 (0, 9.5) | 0 | 0 (0, 8.8) | 0 | 0 (0, 8.4) | 0 | 0 (0, 9.5) | 0 | 0 (0, 8.2) | |
| Induration
/Swelling (measurement) | None | 29 | 71 (54.5, 83.9) | 33 | 89 (74.6, 97) | 27 | 68 (50.9, 81.4) | 36 | 86 (71.5, 94.6) | 31 | 84 (68, 93.8) | 30 | 70 (53.9, 82.8) |
| Mild | 9 | 22 (10.6, 37.6) | 3 | 8 (1.7, 21.9) | 4 | 10 (2.8, 23.7) | 4 | 10 (2.7, 22.6) | 4 | 11 (3, 25.4) | 9 | 21 (10, 36) | |
| Moderate | 1 | 2 (0.1, 12.9) | 1 | 3 (0.1, 14.2) | 6 | 15 (5.7, 29.8) | 1 | 2 (0.1, 12.6) | 2 | 5 (0.7, 18.2) | 2 | 5 (0.6, 15.8) | |
| Severe | 2 | 5 (0.6, 16.5) | 0 | 0 (0, 9.5) | 3 | 8 (1.6, 20.4) | 1 | 2 (0.1, 12.6) | 0 | 0 (0, 9.5) | 2 | 5 (0.6, 15.8) | |
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