Diana L. Noah

Effects of Adjuvants on the Safety and Immunogenicity of an Avian Influenza H5N1 Vaccine in Adults

BACKGROUND Influenza A H5N1 viruses pose a significant threat to human health. METHODS We conducted a multicenter, randomized, double-blind study in 394 healthy adults. Subjects were randomly assigned to receive 2 intramuscular doses of either saline placebo; influenza A/Vietnam/1203/2004(H5N1) vaccine alone at 45, 30, or 15 microg per dose; vaccine at 15 or 7.5 microg per dose with MF59; or vaccine at 30, 15, or 7.5 microg per dose with aluminum hydroxide. Subjects were followed up for safety and blood samples were obtained to determine antibody responses. RESULTS The vaccine formulations were well tolerated but local adverse effects were common; the incidence of these effects increased in a dose-dependent manner and was increased by the addition of adjuvants. The addition of MF59 increased the antibody response, whereas the addition of aluminum hydroxide did not. The highest antibody responses were seen in the group that received 15 microg of vaccine per dose with MF59, in which 63% of subjects achieved the predetermined endpoint (hemagglutination-inhibition titer > or =40) 28 days after the second dose, compared with 29% in the group that received the highest dose (45 microg per dose) of vaccine alone. CONCLUSIONS A 2-dose regimen of subvirion influenza A (H5N1) vaccine was well tolerated. The antibody responses to 15 microg of A/H5 vaccine with MF59 were higher than the responses to 45 microg of vaccine alone. TRIAL REGISTRATION ClincalTrials.gov identifier: http://www.clinicaltrials.gov/ct2/show/NCT00280033?term= NCT00280033&rank=1 NCT00280033 .

Immune Responses of Healthy Subjects to a Single Dose of Intramuscular Inactivated Influenza A/Vietnam/1203/2004 (H5N1) Vaccine after Priming with an Antigenic Variant

BACKGROUND We administered a single dose of influenza A/Vietnam/1203/2004 (H5N1, clade 1) vaccine to subjects who had received 2 doses of influenza A/Hong Kong/156/1997 (H5N1, clade 0) vaccine in 1998. METHODS Thirty-seven subjects previously vaccinated with a baculovirus-expressed recombinant hemagglutinin A/Hong Kong/156/1997 vaccine in 1998 received a single intramuscular dose of 90 microg of inactivated subvirion A/Vietnam/1203/2004 vaccine in 2006. Serum antibody was measured before vaccination and 28 and 56 days after vaccination. Antibody responses were compared with those measured after one or two 90-microg doses in H5-naive subjects. RESULTS On day 28 after a single dose, the geometric mean titer (GMT) of hemagglutination-inhibition antibody in primed subjects was 64.0 (95% confidence interval [CI], 37.8-108.5), with 68% responding (4-fold increase in antibody level to a titer of >or=1:40). In contrast, H5-naive subjects who received two 90-microg doses had a day 56 (28 days after the second dose) GMT of 27.7 (95% CI, 20.3-38.0), with only 43% responding. CONCLUSIONS This study suggests that priming can result in immune responses to a single dose of an antigenically variant strain of H5N1 influenza virus and could be a useful strategy for pandemic control. TRIAL REGISTRATION ClinicalTrials.gov identifier: NCT00240903.

Safety and Immunogenicity of an Inactivated Influenza A/H5N1 Vaccine Given with or without Aluminum Hydroxide to Healthy Adults: Results of a Phase I–II Randomized Clinical Trial

BACKGROUND Dose-sparing strategies are being explored for vaccines against pandemic influenza. We evaluated the dose-sparing potential of aluminum hydroxide (AlOH) adjuvant. METHODS A total of 600 healthy subjects (age, 18-49 years) were randomized to receive 2 vaccinations 1 month apart with subvirion inactivated influenza A/H5N1 vaccine containing 7.5, 15, or 45 microg of hemagglutinin (HA), with or without 600 microg of aluminum hydroxide (AlOH), or 3.75 microg of HA, with or without 300 microg of AlOH. Serum specimens were obtained for antibody assays before and 1 month after each vaccination. RESULTS All formulations were safe. Injection site discomfort was more frequent in groups given vaccines with AlOH. Dose-related increases in antibody responses were noted after both vaccinations (P< .001) geometric mean titers of hemagglutination inhibition antibody in vaccines with and without AlOH, respectively, were 5.4 and 5.4 for subjects who received 3.75 microg of HA, 7.7 and 5.3 for those who received 7.5 microg of HA, 8.1 and 8.5 for those who received 15 microg of HA, and 14.8 and 12 for those who received 45 microg of HA. A > or =4-fold increase in titer was observed in 2% and 2% of subjects who received 3.75 microg of HA with or without AlOH, respectively; in 14% and 0% who received 7 microg of HA; in 14% and 13% who received 15 microg of HA; and in 33% and 25% who received 45 microg of HA. Addition of AlOH enhanced responses only for subjects who received 7.5 microg of HA, but responses in subjects who received 7.5 microg of HA without AlOH were unexpectedly low. CONCLUSION Overall, a meaningful beneficial effect of AlOH adjuvant was not observed. TRIAL REGISTRATION ClinicalTrials.gov identifier: NCT00296634 .

Serological Responses to an Avian Influenza A/H7N9 Vaccine Mixed at the Point-of-Use With MF59 Adjuvant: A Randomized Clinical Trial

IMPORTANCE Human infections with avian influenza A/H7N9 have resulted in high morbidity and mortality in China. OBJECTIVE To compare safety and immunogenicity of different doses of influenza A/Shanghai/2/13 (H7N9) vaccine mixed with or without the MF59 adjuvant. DESIGN, SETTING, AND PARTICIPANTS Multicenter, randomized, double-blind, phase 2 trial at 4 US sites enrolled 700 adults aged 19 to 64 years beginning in September 2013; 6-month follow-up was completed in May 2014. INTERVENTIONS The H7N9 inactivated virus vaccine was administered intramuscularly on days 0 and 21 at nominal doses of 3.75, 7.5, 15, or 45 µg of hemagglutinin (actual doses approximately 50% higher) with or without the MF59 adjuvant. A total 99, 100, or 101 participants were randomized to each group (7 groups; N = 700). MAIN OUTCOMES AND MEASURES Proportions achieving day 42 antibody titer of 40 or greater or seroconversion (a minimum 4-fold increase to titer ≥40) with the hemagglutination inhibition assay; vaccine-related serious adverse events through month 13; and solicited postvaccination symptoms through day 7. RESULTS Hemagglutination inhibition antibodies were minimal after participants received an unadjuvanted vaccine. After receiving 2 doses of H7N9 vaccine at a dosage of 3.75 µg plus the MF59 adjuvant, day 42 seroconversion occurred in 58 participants (59%; 95% CI, 48%-68%). The peak seroconversion occurred at day 29 in 62 participants (62%; 95% CI, 52%-72%). The day 42 geometric mean titer was 33.0 (95% CI, 24.7-44.1). Higher antigen doses were not associated with increased response. For the neutralizing antibody assays, after receiving 3.75 µg of H7N9 vaccine plus the MF59 adjuvant, day 42 seroconversion occurred in 81 participants (82%; 95% CI, 73%-89%). The day 42 geometric mean titer was 81.4 (95% CI, 66.6-99.5). There was no statistically significant difference in day 42 hemagglutination inhibition seroconversion after mixing adjuvant with either the first or both 15 µg doses (n = 34 [35%; 95% CI, 25%-45%] vs n = 47 [47%; 95% CI, 37%-58%], respectively; P = .10). Recent receipt of seasonal influenza vaccination and older age were associated with attenuated response. No vaccine-related serious adverse events occurred. Solicited postvaccination symptoms were generally mild with more local symptoms seen in participants who received the adjuvant. CONCLUSIONS AND RELEVANCE Point-of-use mixing and administration of 2 doses of H7N9 vaccine at the lowest tested antigen dose with MF59 adjuvant produced seroconversion in 59% of participants. Although these findings indicate potential value in this approach, the study is limited by the absence of antibody data beyond 42 days and the absence of clinical outcomes. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT01938742.

Immunogenicity of an Inactivated Monovalent 2009 H1N1 Influenza Vaccine in Pregnant Women

BACKGROUND Although pregnant women are at increased risk of severe illness following influenza infection, there is relatively little information on the immunogenicity of influenza vaccines administered during pregnancy. METHODS We conducted a clinical trial that enrolled 120 pregnant women in which participants were randomly assigned to receive an inactivated 2009 H1N1 influenza vaccine containing either 25 μg or 49 μg of hemagglutinin (HA) in a 2-dose series with a 21-day period between administration of the first and second doses. RESULTS Following the first vaccination, HA inhibition (HAI) titers of ≥1:40 were detected in 93% (95% confidence interval [CI], 82%-98%) of subjects who received the 25-μg dose and 97% (95% CI, 88%-100%) of subjects receiving the 49-μg dose. In cord blood samples, HAI titers of ≥1:40 were found in 87% (95% CI, 73%-96%) of samples from the 25-μg dose group and in 89% (95% CI, 76%-96%) from the 49-μg dose group. Microneutralization titers tended to be higher than HAI titers, but the patterns of response were similar. CONCLUSIONS In pregnant women, 1 dose of an inactivated 2009 H1N1 influenza vaccine containing 25 μg of HA elicited an antibody response typically associated with protection against influenza infection. Efficient transplacental transfer of antibody was also documented.

Influenza virus M2 protein inhibits epithelial sodium channels by increasing reactive oxygen species

The mechanisms by which replicating influenza viruses decrease the expression and function of amiloride‐sensitive epithelial sodium channels (ENaCs) have not been elucidated. We show that expression of M2, a transmembrane influenza protein, decreases ENaC membrane levels and amiloride‐sensitive currents in both Xenopus oocytes, injected with human α‐, β‐, and γ‐ENaCs, and human airway cells (H441 and A549), which express native ENaCs. Deletion of a 10‐aa region within the M2 C terminus prevented 70% of this effect. The M2 ENaC down‐regulation occurred at normal pH and was prevented by MG‐132, a proteasome and lysosome inhibitor. M2 had no effect on Liddle ENaCs, which have decreased affinity for Nedd4‐2. H441 and A549 cells transfected with M2 showed higher levels of reactive oxygen species, as shown by the activation of redox‐sensitive dyes. Pretreatment with glutathione ester, which increases intracellular reduced thiol concentrations, or protein kinase C (PKC) inhibitors prevented the deleterious effects of M2 on ENaCs. The data suggest that M2 protein increases steady‐state concentrations of reactive oxygen intermediates that simulate PKC and decrease ENaCs by enhancing endocytosis and its subsequent destruction by the proteasome. These novel findings suggest a mechanism for the influenza‐induced rhinorrhea and life‐threatening alveolar edema in humans.—Lazrak, A., Iles, K. E., Liu, G. Noah, D. L., Noah, J. W., Matalon, S. Influenza virus M2 protein inhibits epithelial sodium channels by increasing reactive oxygen species. FASEB J. 23, 3829–3842 (2009). www.fasebj.org

Qualification of the Hemagglutination Inhibition Assay in Support of Pandemic Influenza Vaccine Licensure

ABSTRACT Continued outbreaks of highly pathogenic avian influenza over the past decade have spurred global efforts to develop antivirals and vaccines. As part of vaccine development, standard methods are needed for determining serum antibody titers in response to vaccination. Hemagglutination inhibition (HAI) assays are appropriate for assessing the immunogenicity of pandemic influenza vaccines in support of license approval. We demonstrate that a rigorous qualification of the HAI assay for H5N1 influenza virus, evaluating for precision, intermediate precision, linearity, range, specificity, and robustness, satisfies the intent of regulatory guidance for assay validation despite the lack of availability of specific reference standard antigens and antisera.

Evaluation of the Safety and Immunogenicity of a Booster (Third) Dose of Inactivated Subvirion H5N1 Influenza Vaccine in Humans

UNLABELLED Previously, we evaluated 2 doses of H5N1 influenza vaccine in persons 18-64 years of age (placebo and 7.5-, 15-, 45-, or 90-microg doses), separated by 28 days. In this study, 337 participants received a third dose, 6 months thereafter. Microneutralization (MN) and hemagglutination-inhibition geometric mean titers (GMTs) of antibody declined before the third dose. Twenty-eight days after the third dose, 78%, 67%, 43%, and 31% of recipients in the 90-, 45-, 15-, and 7.5-mug-dose groups had a MN GMT > or =1:40, respectively. Five months later, MN GMTs were significantly greater than those after the second dose. ( TRIAL REGISTRATION Clinical Trials.gov identifier NCT00240968 .).

Safety and immunogenicity of a subvirion inactivated influenza A/H5N1 vaccine with or without aluminum hydroxide among healthy elderly adults.

A total of 600 healthy adults > or =65 years were randomized to receive 2 vaccinations 1 month apart of a subvirion avian influenza A/H5N1 vaccine containing 3.75, 7.5, 15, or 45microg of hemagglutinin (HA) with or without aluminum hydroxide (AlOH). All formulations were safe. Groups given the vaccine with AlOH had more injection site discomfort. Dose-related increases in antibody responses were noted after the second vaccination. Antibody responses to the vaccine were not enhanced by AlOH at any HA dose level. A microneutralization titer > or =40 was observed in 36% and 40% of subjects who received 45microg of HA with or without AlOH, respectively.

Effect of Varying Doses of a Monovalent H7N9 Influenza Vaccine With and Without AS03 and MF59 Adjuvants on Immune Response A Randomized Clinical Trial

IMPORTANCE Human infections with the avian influenza A(H7N9) virus were first reported in China in 2013 and continue to occur. Hemagglutinin H7 administered alone is a poor immunogen necessitating evaluation of adjuvanted H7N9 vaccines. OBJECTIVE To evaluate the immunogenicity and safety of an inactivated H7N9 vaccine with and without AS03 adjuvant, as well as mixed vaccine schedules that included sequential administration of AS03- and MF59-containing formulations and of adjuvanted and unadjuvanted formulations. DESIGN, SETTING, AND PARTICIPANTS Double-blind, phase 2 trial at 5 US sites enrolled 980 adults aged 19 through 64 years from September 2013 through November 2013; safety follow-up was completed in January 2015. INTERVENTIONS The H7N9 vaccine was given on days 0 and 21 at nominal doses of 3.75 µg, 7.5 µg, 15 µg, and 45 µg of hemagglutinin with or without AS03 or MF59 adjuvant mixed on site. MAIN OUTCOMES AND MEASURES Proportions achieving a hemagglutination inhibition antibody (HIA) titer of 40 or higher at 21 days after the second vaccination; vaccine-related serious adverse events through 12 months after the first vaccination; and solicited signs and symptoms after vaccination through day 7. RESULTS Two doses of vaccine were required to induce detectable antibody titers in most participants. After 2 doses of an H7N9 formulation containing 15 µg of hemagglutinin given without adjuvant, with AS03 adjuvant, or with MF59 adjuvant, the proportion achieving an HIA titer of 40 or higher was 2% (95% CI, 0%-7%) without adjuvant (n = 94), 84% (95% CI, 76%-91%) with AS03 adjuvant (n = 96), and 57% (95% CI, 47%-68%) with MF59 adjuvant (n = 92) (P < .001 for comparison of the AS03 and MF59 schedules). The 2 schedules alternating AS03-and MF59-adjuvanted formulations led to lower geometric mean titers (GMTs) of (41.5 [95% CI, 31.7-54.4]; n = 92) and (58.6 [95% CI, 44.3-77.6]; n = 96) than the group induced by 2 AS03-adjuvanted formulations (n = 96) (103.4 [95% CI, 78.7-135.9]; P < .001) but higher GMTs than 2 doses of MF59-adjuvanted formulation (n = 94) (29.0 [95% CI, 22.4-37.6]; P < .001). CONCLUSIONS AND RELEVANCE The AS03 and MF59 adjuvants augmented the immune responses to 2 doses of an inactivated H7N9 influenza vaccine, with AS03-adjuvanted formulations inducing the highest titers. This study of 2 adjuvants used in influenza vaccine formulations with adjuvant mixed on site provides immunogenicity information that may be informative to influenza pandemic preparedness programs. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT01942265.