Subjects and Study Design
We conducted a single-center, phase I/II, randomized, double-blinded, placebo-controlled, clinical trial to assess the safety and immunogenicity of intradermal and intramuscular immunization with a similar dosage (38.7 μg) of an inactivated subvirion influenza A/H5N1 vaccine. Study subjects were healthy men and nonpregnant women aged 18–49 years. Subjects were excluded if they had received an influenza A/H5N1 vaccine previously; had a contraindication to receiving an influenza vaccine; had an acute illness and/or a temperature of >38.0°C within 1 week of vaccination; were immunosuppressed; were actively infected with human immunodeficiency virus, hepatitis B virus, or hepatitis C virus; or had a chronic medical condition that might interfere with the evaluation of immune responses (including diseases such as chronic liver disease, significant renal disease, and diabetes mellitus). Additional exclusion criteria also included a history of Guillain-Barré syndrome, alcohol or drug abuse in the prior 5 years, or a major psychiatric disorder. Subjects were also not allowed to receive another investigational product or to participate in another interventional trial while enrolled in this study.
Ethical Approval of the Study Protocol
The study was conducted in accordance with the Declaration of Helsinki, The Belmont Report ,and Good Clinical Practice regulations. The study was approved by the Baylor College of Medicine Institutional Review Board in Houston, Texas, prior to any subjects being consented. The study is registered on ClinicalTrials.gov (identifier NCT00439335).
The study product was an inactivated monovalent subvirion influenza A/H5N1 vaccine (rgA.Vietnam/1203/2004; batch 04-067; lot UD08854 [sanofi pasteur, Swiftwater, PA]) that was produced using methods similar to those used to manufacture Fluzone, a licensed seasonal trivalent influenza vaccine. The vaccine used in this study was provided in multidose vials containing 387 μg/mL of influenza A/H5N1 HA (H5/HA), as determined by single radial immunodiffusion. The placebo used was sodium chloride 0.9% injection, USP (lot 33-248-DK).
The intradermal injection was performed using the Mantoux technique (needle and syringe) by an experienced vaccinator, and bleb formation was confirmed. The intramuscular injection was performed using standard techniques. All injections were given in the deltoid region.
After signing the informed consent document, subjects were screened for eligibility by review of inclusion/exclusion criteria, medical history, medication list, a targeted physical examination if indicated by medical history, and pregnancy testing (for females of childbearing potential). Inclusion/exclusion criteria were reviewed prior to randomization. Urine pregnancy tests were performed on the day of each vaccination for female subjects, as indicated.
Enrolled subjects were randomized at a ratio of 1:1 to one of two groups, using the Internet Data Entry System (AdvantageEDC, EMMES). Subjects in group 1 received 0.1 mL H5 HA by the intradermal route in one arm and 0.1 mL of saline placebo by the intramuscular route in the opposite arm. In group 2, subjects received 0.1 mL H5 HA by the intramuscular route in one arm and 0.1 mL of saline placebo by the intradermal route in the opposite arm.
After randomization (on day 0), all subjects received the first dose of study vaccine and placebo, as indicated. The subject and study staff assessing safety parameters and adverse events were unaware of the vaccine group assignments. The study vaccine and placebo were administered by unblinded vaccinators. To prevent unblinding of the subject, the syringes were labeled “left” or “right” and “intramuscular” or “intradermal” to designate the arm and route, respectively. In addition, the subject was asked to look in the opposite direction when the product was administered in each arm. Four weeks after the initial vaccination (on day 28), eligibility was reassessed, and eligible subjects received a second dose of study vaccine and placebo by the same route as the initial injection. Subjects remained in the clinic for at least 20 minutes after each injection and were seen in the clinic 2 and 7 days later for safety evaluations. All subjects recorded their oral temperature, the presence of any injection site or systemic symptoms, and the use of any medication(s) on a memory aid daily for 1 week after each vaccination.
Solicited and unsolicited adverse events were graded on a scale of 0–3, where 0 indicated absence of symptoms, 1 indicated mild symptoms that did not interfere with daily activities, 2 indicated moderate symptoms that had some interference with daily activities, and 3 indicated severe symptoms that were incapacitating. Solicited adverse events included injection site symptoms (pain, tenderness, induration, itching, erythema, and pigmentation) and systemic symptoms (feverishness, headache, malaise, nausea, or myalgia). Fever was an oral temperature of ≥37.8°C (≥100°F). The diameter of injection site erythema, induration, and pigmentation were graded as follows: grade 1, mild (diameter, <20 mm); grade 2, moderate (20–50 mm); and grade 3, severe (>50 mm). Unsolicited adverse events that occurred during days 0–56 were recorded. Serious adverse events (SAEs) were recorded throughout the study period (from days 0 to 208, or 6 months after the second dose of vaccine) and were defined as any event that was considered life threatening and resulted in significant or persistent disability, hospitalization, or death.
Blood samples for hemagglutination inhibition (HAI) and microneutralization (Neut) antibody assays were collected prior to each vaccination (on days 0 and 28) and 4 weeks and 6 months after the second vaccination (on days 56 and 208, respectively). An HAI or Neut antibody titer of ≥40 was considered a putative protective titer. Samples that were negative (titer, <10) were assigned a titer of 5. Seroresponse was defined as increases relative to baseline in H5-specific HAI and/or Neut antibody titer of ≥4-fold to at least 40 after vaccination. HAI and Neut assays were performed at Southern Research Institute, Birmingham, Alabama, as described previously [25
This study was designed to explore the immunogenicity of a monovalent inactivated influenza A/H5N1 vaccine administered in a similar dose by the intradermal or intramuscular routes. Our hypothesis was that the influenza A/H5N1 vaccine given by intradermal injection has a slightly greater but acceptable injection site reactogenicity, less systemic reactogenicity, and superior immunogenicity than the same dosage given by intramuscular injection to healthy subjects aged 18–49 years. The sample size of 113 per group (intradermal and intramuscular) was selected to detect a 20% absolute increase in seroresponse frequency in the intradermal vaccine group, assuming a 30% seroresponse frequency in the intramuscular vaccine group.
Frequencies of injection site and systemic reactions after each vaccination were based on the most severe response reported. Comparisons between vaccine groups were performed using the Fisher exact test, in which reactogenicity was dichotomized as either none to mild or moderate to severe. Logistic regression models that controlled for age and sex were used to evaluate differences between vaccine groups for injection site/systemic reactogenicity.
Immune responses were summarized in terms of H5-specific HAI and Neut antibody titers transformed to a logarithmic scale for analyses. Analyses included the distribution of titers (emphasizing the proportion of subjects achieving titers that were ≥40 and a 4-fold rise over baseline) at 28 days after each vaccination. The Fisher exact test and analysis of variance were used to test the difference between groups for dichotomous (titer ≥40, 4-fold rise) and continuous (geometric mean titer [GMT]) measures, respectively. Logistic regression and linear regression models that controlled for age and sex were developed for 4-fold responses and GMT, respectively, to examine the effect of route of administration.