Abstract
Background
Safe, effective, and easy to deploy adjuvants are needed for influenza prepandemic preparedness. Based on recent reports, we hypothesized that preapplication of topical imiquimod followed by intradermal (ID) vaccination with monovalent inactivated influenza A/H5N1 vaccine (MIV A/H5N1) results in improved serologic responses.
Methods
We randomized 50 healthy adults in a 1:1 ratio to receive topical imiquimod (group 1) or control cream (group 2) followed by ID injection of 9 µg of the hemagglutinin MIV A/H5N1 in 2 doses, 21 days apart. Subjects were followed for safety and serologic responses as measured by the hemagglutination inhibition (HAI) and microneutralization (MN) assays.
Results
Solicited and unsolicited adverse events were comparable between groups 1 and 2, and were mostly mild to moderate in severity. At 21 days after dose 2, the geometric mean titers (GMTs) of HAI antibodies against the vaccine strain were 16.2 and 24.3 in groups 1 and 2, respectively. The MN antibody GMTs were 9.3 and 10.7 in groups 1 and 2, respectively. There were no significant differences in antibody levels between groups at study time points.
Conclusions
Topical imiquimod administration combined with ID MIV A/H5N1 was safe but did not result in improved serologic responses to the vaccine.
Clinical Trials Registration. NCT03472976.
Keywords: topical, adjuvant, influenza, vaccine
Using a controlled, randomized approach, we investigated if topical imiquimod applied prior to intradermal prepandemic influenza A/H5N1 vaccine has an adjuvant effect. We found that topical imiquimod was safe but provided no adjuvant effect compared to control cream.
There have been 4 influenza virus pandemics since 1918, caused by emergent strains to which the population had no to little immunity [1]. These pandemics are difficult to predict and result in significant morbidity and mortality [2]. In recent decades, global surveillance systems have identified novel influenza strains with pandemic potential, including influenza A/H7N9, A/H7N7, A/H5N1, and A/H9N2 [3]. Influenza vaccines targeting antigens from many novel influenza strains are poorly immunogenic. For example, 2 doses of a 90-μg hemagglutinin (HA) of the influenza A/H5N1 vaccine were needed to stimulate a putatively protective level of antibodies in approximately 57% of healthy adults [4]. In a study evaluating 2 doses of nonadjuvanted inactivated influenza A/H7N7 vaccine administered as 7.5, 15, 45, or 90 μg HA per dose, 0%–25% of subjects developed any measurable antibody responses [5]. The addition of an adjuvant such as MF59 or AS03 to the inactivated novel influenza vaccine leads to significant improvement in vaccine immunogenicity, with the potential of antigen sparing which can stretch the vaccine supply [6, 7].
Toll-like receptor 7 (TLR7) is heavily expressed in plasmacytoid dendritic cells [8]. When used with various antigens as an adjuvant, a TLR7 agonist induces a T helper 1 (Th1)-biased immune response as well as increased antibody production in animal models [9, 10]. Recent publications have proposed the use of topical TLR7 adjuvant with seasonal inactivated influenza vaccines (IIVs). In one study, elderly patients (median age, 73 years) received intradermal (ID) seasonal trivalent IIV (IIV3; 15 μg HA/strain) with or without topical imiquimod (a TLR7 agonist), or IIV3 intramuscularly (IM) without imiquimod. Subjects in the topical imiquimod plus IIV3 arm achieved significantly higher and earlier hemagglutination inhibiting (HAI) antibody titers and seroconversion rates against all vaccine strains, and these differences were maintained at 1 year postvaccination [11]. In a second randomized trial, Hung et al found that topical imiquimod with ID seasonal IIV3 in young adults resulted in similar rapid seroconversion rates on day 7 compared to seasonal IIV3 given IM or ID with a control cream. The topical imiquimod plus IIV3 ID also elicited robust, seroprotective responses to several drifted variants of H1, H3, and B influenza viruses [12].
A safe, effective, and easily deployable method to improve the immunogenicity of novel antigen influenza vaccines would significantly improve pandemic influenza preparedness. We hypothesized that topical imiquimod used as an adjuvant with monovalent inactivated influenza A/H5N1 vaccine (MIV A/H5N1) administered ID will result in dose sparing and improved serologic responses to this poorly immunogenic HA variant.
METHODS
Study Design
This was a single-site, phase 1, randomized, observer-blind, placebo-controlled trial in healthy adult men and nonpregnant women between the ages of 18 and 49 years. We evaluated the safety, reactogenicity, and immunogenicity of MIV A/H5N1 administered ID with topical imiquimod or control cream as a 2-dose regimen given on study days 1 and 22. Subjects were randomized in a 1:1 ratio to receive the vaccine with Aldara (group 1) or with a control cream (group 2). Eligibility criteria are provided at clinicaltrials.gov, NCT03472976.
Ethical Considerations
The study was approved by the Institutional Review Board at Baylor College of Medicine. All subjects provided written consent to study participation prior to any study procedure.
Study Products
The study vaccine was MIV A/H5N1, manufactured by Sanofi Pasteur and licensed by the Food and Drug Administration (FDA) in 2007 for the prevention of influenza A/H5N1. It was supplied in 5-mL multidose vials. The vaccine was formulated to contain 90 μg HA of influenza A/Vietnam/1203/2004 (H5N1, clade 1) per 1 mL. Study vaccine was administered ID in a volume of 0.1 mL containing 9 μg HA, a dose that our group evaluated previously and found to be poorly immunogenic [13]. Aldara cream (imiquimod 5%; Valeant Pharmaceutical International) was supplied in single-use packets, each of which contained 250 mg of the cream, equivalent to 12.5 mg of imiquimod. The content of 1 packet was applied to the participant’s arm prior to vaccination in subjects randomized to the imiquimod arm, while 250 mg of Aqueous Cream B.P. (control cream supplied in a 500-g jar; Pinewood Healthcare) was applied in a similar fashion for those randomized to receive the control cream.
Intradermal Vaccine Delivery Device
To minimize operator-dependent variability in ID vaccine delivery, we utilized MicronJet600 (NanoPass Technologies), which is a small plastic device that has been used previously to immunize subjects ID against influenza and that yielded safety and immunogenicity results comparable to IM delivery [14].
Study Procedures
Following randomization, imiquimod or control cream was applied over a 4 × 4 cm2 area of the deltoid. After a wait period of 5–15 minutes to allow the cream to be absorbed/vanish from the skin, 0.1 mL of the vaccine was administered ID at the center of the deltoid using the MicronJet600 device. Subjects recorded prespecified injection site and systemic reactions on a memory aid for 8 days after vaccination (vaccination day was day 1). Adverse events (AE) were collected for 28 days after study vaccination and graded as mild (grade 1) if they did not interfere with subjects’ daily activities and required no treatment, moderate (grade 2) if they caused some interference with subjects’ daily activities or required therapeutic measures, and severe (grade 3) if they interrupted the subjects’ daily activities or incapacitated him/her. Serious adverse events (SAEs), medically attended adverse events (MAAEs), new onset chronic medical conditions (NOCMCs), and potentially immune-mediated medical conditions (PIMMCs) data were collected for 6 months after study vaccination. SAEs were defined as Guillain-Barré syndrome or any AE that resulted in death, life threatening event, hospitalization or prolongation of hospitalization, persistent or significant incapacity, birth defect/congenital anomaly, or AE that required a medical intervention to prevent death, threat to life or hospitalization. Venous blood was collected on days 1 (prevaccine), 22, 29, 43, and 202 for HAI and neutralizing antibody (MN Ab) assays.
Immunogenicity Laboratory Assays
HAI and MN Ab assays against the homologous influenza A/Vietnam/1203/2004 (H5N1) were performed at a single central laboratory (Southern Research, Birmingham, AL), as previously described [15]. Serum samples were tested in duplicate and the geometric mean titer (GMT) of replicate results was used for analysis. The initial dilution was defined as 1:10 per US FDA recommendations; serum samples without activity were scored as 5. Seroconversion was defined as either a prevaccination antibody titer < 10 and a postvaccination antibody titer ≥ 40 or a prevaccination antibody titer ≥ 10 and a minimum 4-fold rise in postvaccination antibody titer. In addition to assessing the immunogenicity of the vaccination regimens against the vaccine strain, we measured the HAI and MN antibody levels against influenza A/H5 variants A/Gyrfalcon/Washington/41088-6/2014 (H5N8), A/Hubei/1/2010 (H5N1), A/Indonesia/05/05 (H5N1), and A/Anhui/01/2005 (H5N1).
Sample Size and Statistical Considerations
The sample size of 50 participants (25 per group) was selected to provide preliminary estimates of the safety and immunogenicity of 2 doses of vaccine with and without imiquimod. The planned sample size provided >90% power to detect safety events that would occur in 1 in 10 subjects as well as provide additional evidence of the safety of using imiquimod as an adjuvant for influenza vaccination. Safety summaries utilize all available data from any participant who received a study vaccination. Descriptive analyses were used for participant demographic and safety data. Categorical variables were summarized using percentages with corresponding 95% confidence interval (CI) for safety data only, and continuous variables were summarized by mean and standard deviation, unless otherwise specified. Immunogenicity summaries and analyses are presented for the modified intent-to-treat population, defined as any participant who received a study vaccination and had a prevaccination and at least 1 postvaccination antibody titer. Fisher exact test was used to assess the independence of the proportion of subjects achieving seroconversion and the presence of imiquimod at day 43, and associations between the GMT of HAI or MN antibodies and the presence of imiquimod at day 43 were assessed using Wilcoxon rank sum test. Significance is considered at a level of α = .05, without adjusting for multiple comparisons.
RESULTS
Between 19 June 2018 and 14 August 2018, 63 persons were screened and 50 were enrolled. Twenty-five participants were randomized to group 1, of whom 22/25 received both doses of the vaccine; and 25 participants were randomized to group 2, all of whom received both doses of the vaccine (Figure 1). The 3 subjects who did not receive the second dose in group 1 met 1 of the protocol prespecified exclusion criteria: 1 subject due to intercurrent illness, 1 subject initiated new medication for depression, and 1 subject had a potential exposure to herpes B virus for which he needed medical evaluation and treatment. Baseline demographic characteristics were comparable between the 2 study groups. Overall, the mean age was 27.2 years, 56% were female, 30% were Hispanic/Latinx, 38% white, 28% Asian, and 20% African American. The majority of subjects (56%) had received seasonal influenza vaccines for the preceding 2 seasons (Table 1).
Figure 1.
CONSORT diagram: disposition of study participants by group.
Table 1.
Clinical and Demographics Characteristics at Enrollment by Study Group
| Characteristics | Arm 1, 9 μg A/H5N1 + Aldara (n = 25) | Arm 2, 9 μg A/H5N1 + Control Cream (n = 25) | All Subjects (n = 50) |
|---|---|---|---|
| Age, median (range) | 25 (18–34) | 25 (18–45) | 25 (18–45) |
| BMI, median (range) | 24.1 (17.3–42.6) | 27.3 (18.6–41.3) | 25.2 (17.3–42.6) |
| Sex | |||
| Male | 12 (48) | 10 (40) | 22 (44) |
| Female | 13 (52) | 15 (60) | 28 (56) |
| Ethnicity, Hispanic or Latinx | 9 (36) | 6 (24) | 15 (30) |
| Race | |||
| American Indian or Alaska Native | 0 (0) | 1 (4) | 1 (2) |
| Asian | 6 (24) | 8 (32) | 14 (28) |
| Black or African American | 4 (16) | 6 (24) | 10 (20) |
| White | 12 (48) | 7 (28) | 19 (38) |
| Multiple | 1 (4) | 1 (4) | 2 (4) |
| Unknown | 2 (8) | 2 (8) | 4 (8) |
| Prior seasonal influenza vaccination | |||
| Neither 2016–2017, 2017–2018 | 2 (8) | 6 (24) | 8 (16) |
| 2016–2017 Only | 3 (12) | 3 (12) | 6 (12) |
| 2017–2018 Only | 3 (12) | 5 (20) | 8 (16) |
| Both 2016–2017, 2017–2018 | 17 (68) | 11 (44) | 28 (56) |
Data are No. (%) except where indicated.
Safety
There were no SAEs or deaths reported during the study period. At least 1 solicited injection site symptom was reported by all 50 study participants after any vaccination, with no differences between first and second dose. In group 1, the most common reported injection site symptom was redness (25/25) and induration/swelling (25/25). With the exception of 1 subject who reported a large area of redness, all injection-site symptoms were mild to moderate in severity. In group 2, the most common injection site symptom was induration/swelling. With the exception of 4 subjects who reported a large area of redness, all injection site symptoms were mild to moderate in severity in group 2.
Solicited systemic symptoms were reported by 13/25 (52%) subjects in group 1 and 11/25 (44%) in group 2 after any vaccination, with no differences between first and second dose. Fatigue was the most common solicited systemic symptom in group 1 (44% of subjects) and in group 2 (36% of subjects). All solicited systemic symptoms were mild to moderate in severity with the exception of 1 report of severe fatigue in a subject in group 1, reported on day 2, lasting for 1 day.
Unsolicited AEs were reported by 18/25 subjects in group 1, and 16/25 subjects in group 2. All unsolicited AEs were graded as mild to moderate in severity.
There were 5 MAAEs; all were mild to moderate in severity and deemed unrelated to the study vaccine: inner ear infection, pharyngitis, exacerbation of seasonal allergies, herpes B virus exposure, and psoriasis. The psoriasis event was also considered a PIMMC and NOCMC.
Immunogenicity
At baseline, the GMTs of HAI antibodies against the vaccine strain A/Vietnam/1203/2004 were low in groups 1 and 2 (5.8 and 5.4, respectively). Modest and similar increases in serum HAI antibody levels were observed in both groups: at 21 days after the second dose of MIV A/H5N1 plus imiquimod and A/H5N1 plus control cream the GMTs of HAI antibodies were 16.2 and 24.3 respectively. The proportion of subjects with the putatively protective HAI antibodies titer of 40 or more and the proportion of subjects with seroconversion were low and comparable between the 2 groups at 21 and 202 days after dose 2 (Table 2 and Figure 2). MN antibodies were not detected in subjects in either group at baseline, and at 21 days after dose 2 the neutralizing antibody GMTs only increased modestly to 9.7 in group 1 and 10.7 in group 2. The proportions of subjects with MN antibody titers of 40 or more and the proportions of subjects with MN antibody seroconversion were low and comparable 21 and 202 days after dose 2 (Table 2 and Figure 2).
Table 2.
Proportion of Subjects With Seroprotection, Seroconversion and Geometric Mean Titers of Hemagglutination Inhibition and Neutralizing Antibodies in the 2 Study Groups
|
Measure |
Arm 1: 9 μg A/H5N1 + Aldara (n = 25) | Arm 2: 9 μg A/H5N1 + Control Cream (n = 25) |
|---|---|---|
| Day 1, baseline | ||
| HAI antibodies | ||
| No. | 25 | 25 |
| GMT (95% CI) | 5.8 (4.7–7.2) | 5.4 (4.9–5.9) |
| Titer ≥ 1:40, % (95% CI) | 4 (0–20) | 0 (0–14) |
| MN antibodies | ||
| No. | 25 | 25 |
| GMT (95% CI) | 5.0 (–) | 5.0 (–) |
| Titer ≥ 1:40, % (95% CI) | 0 (0–14) | 0 (0–14) |
| Day 22, dose 2 | ||
| HAI antibodies | ||
| No. | 25 | 24 |
| GMT (95% CI) | 13.4 (7.6–23.6) | 18.6 (11.0–31.3) |
| Titer ≥ 1:40, % (95% CI) | 16 (5–36) | 25 (10–47) |
| Seroconversion, % (95% CI) | 16 (5–36) | 25 (10–47) |
| MN antibodies | ||
| No. | 25 | 24 |
| GMT (95% CI) | 7.4 (4.3–12.7) | 9.0 (5.7–14.4) |
| Titer ≥ 1:40, % (95% CI) | 8 (1–26) | 13 (3–32) |
| Seroconversion, % (95% CI) | 8 (1–26) | 13 (3–32) |
| Day 29, 7 days after dose 2 | ||
| HAI antibodies | ||
| No. | 25 | 25 |
| GMT (95% CI) | 16.5 (9.6–28.3) | 25.7 (15.6–42.2) |
| Titer ≥ 1:40, % (95% CI) | 24 (9–45) | 44 (24–65) |
| Seroconversion, % (95% CI) | 20 (7–41) | 40 (21–61) |
| MN antibodies | ||
| No. | 25 | 25 |
| GMT (95% CI) | 9.3 (5.1–17.1) | 10.1 (6.2–16.5) |
| Titer ≥ 1:40, % (95% CI) | 12 (3–31) | 16 (5–36) |
| Seroconversion, % (95% CI) | 12 (3–31) | 16 (5–36) |
| Day 43, 21 days after dose 2 | ||
| HAI antibodies | ||
| No. | 25 | 25 |
| GMT (95% CI) | 16.2 (9.8–26.8) | 24.3 (15.3–38.5) |
| Titer ≥ 1:40, % (95% CI) | 24 (9–45) | 44 (24–65) |
| Seroconversion, % (95% CI) | 20 (7–41) | 40 (21–61) |
| MN antibodies | ||
| No. | 25 | 25 |
| GMT (95% CI) | 9.7 (5.4–17.4) | 10.7 (6.6–17.4) |
| Titer ≥ 1:40, % (95% CI) | 12 (3–31) | 16 (5–36) |
| Seroconversion, % (95% CI) | 12 (3–31) | 16 (5–36) |
| Day 202, 180 days after dose 2 | ||
| HAI antibodies | ||
| No. | 19 | 23 |
| GMT (95% CI) | 8.3 (5.5–12.6) | 10.5 (7.1–15.5) |
| Titer ≥ 1:40, % (95% CI) | 5 (0–26) | 9 (1–28) |
| Seroconversion, % (95% CI) | 5 (0–26) | 9 (1–28) |
| MN antibodies | ||
| No. | 19 | 23 |
| GMT (95% CI) | 7.5 (4.1–13.5) | 6.8 (4.7–9.6) |
| Titer ≥ 1:40, % (95% CI) | 5 (0–26) | 9 (1–28) |
| Seroconversion, % (95% CI) | 5 (0–26) | 9 (1–28) |
Abbreviations: CI, confidence interval; GMT, geometric mean titer; HAI, hemagglutination inhibition; MN, microneutralization.
Figure 2.
A, Geometric mean titers of hemagglutination inhibition antibodies to the vaccine influenza A/H5N1 strain with corresponding 95% confidence intervals at all study time points. B, Geometric mean titers of neutralizing antibody to the vaccine influenza A/H5N1 strain with corresponding 95% confidence intervals, collected at all study time points.
There were no statistically significant differences between the 2 groups in the breadth of the immune responses as measured by the GMTs of HAI or MN antibodies against influenza A/H5 variants at 21 days after dose 2. For groups 1 and 2, the respective GMTs of HAI antibodies were 5.1 and 6.5 against influenza A/Gyrfalcon/Washington/41088-6/2014 (H5N8), 18.2 and 26.0 against influenza A/Hubei/1/2010 (H5N1), 6.9 and 7.3 against A/Indonesia/05/05 (H5N1), and 5.7 and 5.2 against A/Anhui/01/2005 (H5N1) (Figure 3A and 3B). The MN GMTs against influenza A/H5 were also low and comparable between the 2 study groups postvaccination.
Figure 3.
A, Geometric mean titers of hemagglutination inhibition antibodies to antigenically distinct influenza A/H5N1 strains with corresponding 95% confidence intervals, collected at baseline and 21 days after second vaccination. B, Geometric mean titers of neutralizing antibodies to 4 antigenically distinct influenza strains with corresponding 95% confidence intervals, collected at baseline and 21 days after second vaccination.
Discussion
We tested the safety and the adjuvant effect of coadministering topical imiquimod with novel antigen MIV A/H5N1 given ID to healthy subjects. We found that topical imiquimod was well tolerated and safe, but administration prior to ID vaccination was not associated with improved serologic responses to a novel avian influenza vaccine.
Our findings are in stark contrast to published data from 3 clinical trials. In one study, Hung et al demonstrated that administering the seasonal IIV3 with topical imiquimod to older persons with comorbidities resulted in significant improvement in the GMTs of HAI antibodies to the 3 vaccine strains as compared to coadministration with topical aqueous cream: at 21 days after vaccination, the GMTs were 207.5 versus 99.3 against influenza A/H1N1, 147.9 versus 129.7 against influenza A/H3N2, and 213.8 versus 91.2 against influenza B (Brisbane), respectively [11]. Hung et al published data demonstrating a similar adjuvant effect of topical imiquimod with ID seasonal IIV3 in young adults [12]. The difference in the findings between our study and the 2 studies could be explained by the difference in the nature of the antigens utilized in the studies. Adult individuals are immunologically primed against seasonal influenza antigens, but are naive to the HA of influenza A/H5N1, as demonstrated by the low baseline antibody titers. It is possible that topical TLR7 agonist provides an adjuvant effect to antigens to which individuals are primed, but not to novel antigens such A/H5N1. But a more recent publication did not fully support this hypothesis. Hung et al randomized patients on hemodialysis 1:1:1 to receive ID Sci-B-Vac (recombinant hepatitis B vaccine expressing pre-S1, pre-S2, and S protein) with topical imiquimod or with topical aqueous control cream, or IM Sci-B-Vac with topical aqueous cream [16]. The patients had no evidence of previous infection with hepatitis B virus, and 57.4% of the patients were nonresponders to previous hepatitis B vaccination. At 52 weeks, the seroprotection rates were 96.9%, 74.2%, and 48.4%, respectively. Another potential explanation for the difference between our findings and those of Hung et al is methodological. We tried to minimize this variability by using the same imiquimod dose, timing of application, and route of vaccination as the ones used in the published reports. The GMTs of HAI antibody response to unadjuvanted 9 µg of MIV A/H5N1 in our study were low but comparable to those found following ID administration of this vaccine in doses ranging between 3 and 38.7 µg HA [13].
We found no publications describing a lack of an adjuvant effect of topical TLR7 on coadministered vaccines. However, there at least 3 completed studies examining this question on clinicaltrials.gov (NCT01737580 and NCT00175435) that were not published as of the time of drafting this manuscript. A publication bias cannot be ruled out.
In animal models where the adjuvant effect of imiquimod and other TLR7/8 agonists were examined, the TLR7/8 agonist coadministration with vaccines was accomplished via coadmixing of the vaccine with the TLR7/8 agonist or conjugation with the vaccine antigen, as opposed to topical preapplication of the TLR7/8 followed by ID vaccine administration. No published data are available on the utility of the use of topical TLR7/8 agonists as vaccine adjuvants in animal models. When coadmixed with PCV13, the addition of TLR7/8 agonists resulted in improved serotype-specific antibody titers and opsonophagocytosis in young rhesus macaques [17]. Vaccination with imiquimod coadmixed with MIV A(H1N1)pdm09 resulted in improved MN antibody titers and survival rates in mice following challenge [18]. Vaccination of nonhuman primates with HIV Gag antigen conjugated to 3M-012 (TLR7/8 agonist) results in increased Th1 and antibody responses compared to vaccination with antigen alone [19]. In these reports, the TLR7/8 agonist and vaccine mixture were administered parenterally to the animals. However, parenterally administered TLR7/8 agonists have been shown to be poorly tolerated in humans, with influenza-like symptoms being the main side effects [20–22]. New-generation TLR7/8 agonists that can be parenterally administered in humans as vaccine adjuvant are now part of an active field of research [23].
In summary we were unable to demonstrate an adjuvant effect of topical imiquimod when applied prior to ID MIV influenza A/H5N1 in healthy adults. Taken together, our data and the absence of suggestive preclinical data call into question the utility of imiquimod as a topical adjuvant for vaccination against infectious agents.
Notes
Acknowledgments. We are thankful to our study subjects, the Safety Monitoring Committee (Drs Stephen Greenberg, Janet McElhaney, and Holly Janes), the Division of Microbiology and Infectious Diseases (Robin Mason, Rhonda Pikaart-Tautges, and Drs Francisco Jose Leyva, Mohamed Elsafy, and Chris Roberts), the Independent Safety Monitors (Drs Jose Serpa-Alvarez and Galant Au Chan), and the Baylor College of Medicine Vaccine and Treatment Evaluation Unit clinic and laboratory teams.
Financial support. This work was supported by the National Institutes of Health (grant numbers HHSN272201300015I and HHSN272201500002C).
Potential conflicts of interest. All authors: no reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.
Contributor Information
Hana M El Sahly, Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, USA; Department of Medicine, Baylor College of Medicine, Houston, Texas, USA.
Robert L Atmar, Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, USA; Department of Medicine, Baylor College of Medicine, Houston, Texas, USA.
Eli Sendra, The Emmes Company, Rockville, Maryland, USA.
Ashley Wegel, The Emmes Company, Rockville, Maryland, USA.
Wendy A Keitel, Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, USA; Department of Medicine, Baylor College of Medicine, Houston, Texas, USA.
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