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Conceived and designed the experiments: RBC. Performed the experiments: RBC SMP CLW DN. Analyzed the data: RBC. Wrote the paper: RBC.
Concern for a pandemic caused by a newly emerged avian influenza A virus has led to clinical trials with candidate vaccines as preparation for such an event. Most trials have involved vaccines for influenza A (H5N1), A (H7N7) or A (H9N2).
To evaluate dosage-related safety and immunogenicity of an inactivated influenza A (H7N7) vaccine in humans.
One hundred twenty-five healthy young adults were randomized to receive two doses intramuscularly of placebo or 7.5, 15, 45 or 90 µg of HA of an inactivated subunit influenza A (H7N7) vaccine (25 per group), four weeks apart. Reactogenicity was evaluated closely for one week and for any adverse effect for six months after each dose. Serum hemagglutination-inhibiting and neutralizing antibody responses were determined four weeks after each dose and at six months.
Reactogenicity evaluations indicated the vaccinations were well tolerated. Only one subject developed a ≥4-fold serum hemagglutination-inhibition (HAI) antibody response and a final titer of ≥1∶40 four weeks after dose two and only five subjects developed a neutralizing antibody rise and a final titer of ≥1∶40 in tests performed at a central laboratory. Four of the five were given the 45 or 90 µg HA dosage. A more sensitive HAI assay at the study site revealed a dose-response with increasing HA dosage but only 36% in the 90 µg HA group developed a ≥4-fold rise in antibody in this test and only one of these achieved a titer of ≥1∶32.
This inactivated subunit influenza A (H7N7) vaccine was safe but poorly immunogenic in humans.
ClinicalTrials.gov
The prevailing concept for the origin of new influenza A virus subtypes leading to pandemic influenza in humans is that a new virus with the ability to spread and cause illness contains an avian influenza virus hemagglutinin (HA) and/or neuraminidase glycoprotein (NA) acquired from an avian influenza virus
The present report is of a report of a clinical trial with an influenza A (H7N7) vaccine prepared by a manufacturer that used established methods for annual production of seasonal influenza vaccine. The objective was to evaluate dosage-related safety and immunogenicity. This vaccine proved to be safe but poorly immunogenic in humans. A second related report is of results comparing a number of the prototype inactivated vaccines containing avian HAs and NAs that have been evaluated in humans, including the A/H7N7 vaccine used in this report, in various in vitro tests in an effort to identify correlates that related to immunogenicity in humans other than the standard single radial immunodiffusion (SRID) assays of HA concentration.
The protocol for this trial and supporting CONSORT checklist are available as supporting information; see
Subjects were healthy male and nonpregnant females between the ages of 18 and 40 years. The study was reviewed and approved by the Institutional Review Board for Protection of Human Subjects in research at Baylor College of Medicine before commencing. The study was conducted in a clinic setting and all subjects gave written informed consent before any procedures were conducted. The Declaration of Helsinki principles were followed.
The tested vaccine is a monovalent inactivated influenza A (H7N7) vaccine produced by Sanofi Pasteur Inc. using their seasonal vaccine production methods. The virus used was a 6-2 reassortant generated in eggs. The virus donating the HA was A/Mallard/Netherlands/12/2000 (H7N3) and that donating the NA was A/Mallard/Netherlands/2/2000 (H10N7); both are low pathogenic avian influenza viruses. The six internal genes were donated by an influenza A (H1N1) strain, FDA-Resvir-12; the NP gene was from A/Johannesburg/82/96(H1N1) and the other five internal protein genes were from A/Puerto Rico/8/34 (H1N1)
The sample size (25 per group), was selected by the data coordinating center of the Microbiology Division of the National Institutes of Allergy and Infectious Diseases to provide a robust initial safety database of 125 vaccinees receiving the antigen while providing some information concerning the dose-response immunogenicity in a timely fashion. Immunogenicity results were intended to be sufficient to establish a basis for narrowing the dose selection for further studies in infants, children, and elderly populations. A power calculation was not performed for this Phase I study but for an assumed serious adverse event frequency of 0.1% to 20%, the probability of detecting one or more events for 25 to 100 subjects ranged from 2.47% to 100%.
The study was conducted in a randomized, double-blind fashion; randomization was conducted after enrollment at the data coordinating center of the sponsor. One hundred thirty-six subjects were screened and 125 were randomly assigned to groups of 25 each to receive 0.5 ml doses intramuscularly of placebo or 7.5 µg, 15 µg, or 45 µg of vaccine HA on days 0 and 28. A fifth group of 25 received one ml of the 45 µg per 0.5 ml dosage (total of 90 µg) at each vaccination. Vaccine dosages were based on single radial immunodiffusion assays (SRID) for HA
A central laboratory performed hemagglutination-inhibition (HAI) and neutralizing (neut) antibody assays on all serum specimens as described
The number of subjects in each vaccine group reporting a specific solicited local or systemic adverse effect (AE) that was mild, moderate or severe for seven days after each vaccination and the number and description of unsolicited AEs for 28 days after each vaccination were totaled. Occurrence of severe AEs (SAE) was monitored for six months and individually evaluated in detail for any potential vaccine relationship.
The number of subjects in each vaccine group developing a ≥4-fold serum antibody response and achieving a final titer of ≥1∶40 in HAI and neut assays was totaled 28 days after each vaccination and at 208 days. Also, the number (%) of subjects developing a ≥4-fold and ≥1∶32 final titer 28 days after dose 2 (day 56) in the more sensitive HAI assay was totaled. GMT calculations were not done.
Statistical analyses were conducted using SPSS version 17.0.
Safety, reactogenicity, and immunogenicity of inactivated influenza A/H7/N7 vaccine in healthy adults. No.: NCT00546585. Initiated March 10, 2008, completed February 10, 2009.
Study performance conformed to CONSORT guidelines and is reported conforming to Consort 10 guidelines (PLoS Medicine 2010, 7(3):1–7 e1000251).
A flow diagram of the randomized trial is shown in
One hundred twenty-five subjects were randomized; complete followup for 208 days was available on 122 (97.6%). Of the three who withdrew, one thought dose 1 had caused an acute respiratory illness and two withdrew for travel. Four others were not given dose 2 because of an SAE (see reactogenicity), and one of these had started a new medicine (exclusion criterion). The number of subjects in each vaccination group tested for antibody at each specified time is shown in the figure.
Demographics of the 125 enrolled subjects are shown in
ALL (No. = 125) | 7.5 mcg (No. = 25) | 15 mcg (No. = 25) | 45 mcg (No. = 25) | 90 mcg (No. = 25) | Placebo (No. = 25) | |
|
||||||
Male | 61 (49) | 10 (40) | 11 (44) | 14 (56) | 13 (52) | 13 (52) |
Female | 64 (51) | 15 (60) | 14 (56) | 11 (44) | 12 (48) | 12 (48) |
|
||||||
American Indian/Alaskan Native | 0 | 0 | 0 | 0 | 0 | 0 |
Asian | 14 (11) | 4 (16) | 2 (8) | 2 (8) | 4 (16) | 2 (8) |
Hawaiian/Pacific Islander | 0 | 0 | 0 | 0 | 0 | 0 |
Black/African American | 10 (8) | 2 (8) | 0 | 5 (20) | 1 (4) | 2 (8) |
White | 94 (75) | 19 (76) | 23 (92) | 15 (60) | 18 (72) | 19 (76) |
Multi-Racial | 6 (5) | 0 | 0 | 2 (8) | 2 (8) | 2 (8) |
Other/Unknown | 1 (1) | 0 | 0 | 1 (4) | 0 | 0 |
|
||||||
Mean (STD) | 28.1 (5.0) | 29.1 (5.5) | 29.6 (5.5) | 27.8 (4.9) | 28.2 (4.8) | 25.7 (3.7) |
Median | 26.4 | 28.8 | 28.4 | 26.1 | 28.0 | 25.2 |
Min,Max | (18, 40) | (22, 40) | (18, 40) | (21, 38) | (19, 38) | (20, 38) |
Male vs. nonmale frequency by dosage, chi-square 1.73, p = .785.
White vs. nonwhite frequency by dosage, chi-square 7.04, p = .134.
Age vs. dosage, Anova, p>.05.
Solicited adverse events in the seven days following each vaccination are summarized in
No. (%) with ≥One AE in Category | |||||||||
Dose 1 | Dose 2 | ||||||||
Adverse Events | Dosage |
No. |
Mild | Mod | Severe | No. |
Mild | Mod | Severe |
Systemic | 0 | 25 | 5 (20) | 3 (12) | 0 | 22 | 5 (23) | 1 (5) | 0 |
7.5 | 25 | 10 (40) | 1 (4) | 0 | 24 | 1 (4) | 1 (4) | 0 | |
15 | 25 | 9 (36) | 2 (8) | 0 | 24 | 5 (21) | 1 (4) | 0 | |
45 | 25 | 5 (20) | 2 (8) | 0 | 23 | 3 (13) | 0 (0) | 0 | |
90 | 25 | 4 (16) | 6 (24) | 0 | 25 | 4 (16) | 1 (4) | 0 | |
Local | 0 | 25 | 13 (52) | 0 (0) | 0 | 22 | 12 (55) | 0 (0) | 0 |
7.5 | 25 | 15 (60) | 0 (0) | 0 | 24 | 14 (58) | 0 (0) | 0 | |
15 | 25 | 11 (44) | 2 (8) | 0 | 24 | 17 (71) | 1 (4) | 0 | |
45 | 25 | 14 (56) | 1 (4) | 0 | 23 | 15 (65) | 3 (13) | 0 | |
90 | 25 | 14 (56) | 3 (12) | 0 | 25 | 13 (52) | 3 (12) | 0 |
As determined in single radial immunodiffusion assays.
Number subjects evaluated.
Four subjects were designated as an SAE during the study period. Two persons were hospitalized, one for moderately severe back pain considered related to a car accident two years earlier but the hospitalization was extended because of depression; both illnesses resolved without sequelae. The other hospitalization was for stress-related severe depression that improved during hospitalization and was considered resolved on discharge. A 40-year old female was noted to have eyelid edema when reporting for dose two and reported a new physician-prescribed medication; she also reported experiencing eyelid edema in the past. Follow-up evaluations led to a diagnosis of mixed connective tissue disease and special tests on her prevaccination and earlier available sera revealed evidence of a preexisting autoimmune disorder. The illness was considered moderately-severe. None of these three subjects were given the second vaccination. Both the site investigators and the independent safety monitoring committee considered these SAEs as not associated with vaccination. The fourth SAE was a mild transient decrease in hearing by the left ear with onset nine days after the initial vaccination that did not interfere with normal activities. A specific cause other than vaccination was not identified and treatment provided by ENT consultation did not lead to improvement. Hearing returned to normal in 84 days; the second vaccination was not done. Both the site investigators and the independent safety monitoring committee designated the AE as vaccination associated; the independent monitoring committee required an SAE designation because of “significant disability.”
Serum antibody response measurements at the central laboratory 28 days after each vaccination are shown in
Post Dose 1 | Post Dose 2 | |||||||||
No. (%) ≥4-Fold Increase | No. (%) ≥1∶40 | No. (%) ≥4-Fold Increase | No.(%) ≥1∶40 | |||||||
Dosage (µg HA) |
No. |
HAI |
Neut |
HAI |
Neut |
No. |
HAI |
Neut |
HAI |
Neut |
0 | 25 | 0 | 0 | 0 | 0 | 25 | 0 | 0 | 0 | 0 |
7.5 | 25 | 0 | 0 | 0 | 0 | 24 | 0 | 0 | 0 | 0 |
15 | 24 | 0 | 1 (4) | 0 | 1 (4) | 24 | 0 | 1 (4) | 0 | 1 (4) |
45 | 25 | 0 | 0 | 0 | 0 | 24 | 0 | 2 (8) | 0 | 2 (8) |
90 | 25 | 0 | 0 | 0 | 0 | 21 | 1 (5) | 2 (10) | 1 (5) | 2 (10) |
As determined at central laboratory; increase defined as ≥4-fold or <10 to 40.
As determined in single radial immunodiffusion assays.
Number of subjects in evaluation.
Hemagglutination-inhibition (HAI) and neutralizing (neut) antibody assays at central laboratory.
HAI Antibody Post Dose 2 | |||
Dosage/µg HA |
No. |
No. (%) ≥4 Fold Increase |
No. (%) ≥1∶32 |
0 | 25 | 0 | 0 |
7.5 | 21 | 2 (9.5) | 0 |
15 | 21 | 2 (9.5) | 1 (5) |
45 | 24 | 6 (25) | 0 |
90 | 22 | 8 (36) | 1 (5) |
Hemagglutination-inhibition antibody assays at primary study 'site.
As determined in single redial immunodiffusion assays.
Number of subjects in evaluation.
Increasing dosage induced increased % increase; logistic regression, p = .001.
The major finding of this study is that an inactivated influenza A (H7N7) subunit vaccine prepared using manufacturing methods approved for seasonal influenza vaccines was poorly immunogenic in healthy young adults despite their having been given two doses a month apart of dosages up to 90 µg of the HA as determined in SRID assays. The neut assay at the central laboratory appeared to be slightly more sensitive than the HAI assay whereas the HAI assay appeared more sensitive than the neut assay at a laboratory with considerable experience with avian influenza virus serology (data not shown). Nevertheless, the serologic findings at the central laboratory were essentially confirmed. The more sensitive HAI assay performed at the study site provided a more complete assessment of immunogenicity. That test indicated that immunogenic HA was present in the vaccine although the immunogenicity was still considerably lower than what was expected for the dosages administered. The major limitation of these findings is that the sample size was not selected to produce definitive immunogenicity data on any of the dosages tested. However, the desired guidance for any further testing was obtained.
Influenza A (H7N7) is proposed as a priority for vaccine development for potential use in humans along with H5N1 and H9N2 because of human infections that have been detected with these viruses
Considerable effort has been expended on vaccine development with H5N1 viruses for potential use in humans
The present report and the two summarized clinical trials, in combination with the good immunogenicity in animal immunizations, indicate that a satisfactory H7 vaccine can be made for humans. Because of the potential need for an H7N7 vaccine for humans, developing a vaccine for potential use in humans seems indicated. To aid in this goal, we have directed research toward gaining some understanding of why this H7N7 vaccine of high HA dosage was so poorly immunogenic in humans. These studies are reported in a companion report on in vitro correlates of immunogenicity of avian influenza virus vaccines in humans.
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Statistical analyses: Robert L. Atmar, M.D.
Manuscript preparation: I. Darlene Kirk, CCRP.
Data summaries: EMMES Corporation, Heather Hill.
Study design and vaccine contribution: National Institute of Allergy and Infectious Diseases, Roland Levandowski.