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Safety and Immunogenicity of Multiple and Higher Doses of an Inactivated Influenza A/H5N1 Vaccine

  1. John H. Beigel1,
  2. Jocelyn Voell1,
  3. Chiung-yu Huang1,
  4. Peter D. Burbelo2 and
  5. H. Clifford Lane1
  1. 1National Institute of Allergy and Infectious Diseases and
  2. 2National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland
  1. Reprints or correspondence: John H. Beigel, MD, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892 (jbeigel{at}niaid.nih.gov)
 
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Abstract

BackgroundH5N1 avian influenza represents an episodic zoonotic disease with the potential to cause a pandemic, and antiviral resistance is of considerable concern. We sought to generate high-titer H5N1 antibodies in healthy volunteers for the purpose of developing hyperimmune intravenous immunoglobulin

MethodsWe conducted a dose-escalating, unblinded clinical trial involving 75 subjects aged 18-59 years. Three cohorts of twenty-five subjects were enrolled sequentially and received 90, 120, or 180 μg of H5N1 A/Vietnam/1203/04 vaccine in 4 doses administered ∼28 days apart

ResultsNo statistically significant dose-related increases in the geometric mean titers (GMTs) of serum hemagglutination inhibition antibody were observed when the 90-μg, 120-μg, and 180-μg cohorts were compared. When the cohorts were analyzed together to determine the effect of additional vaccinations, the GMTs of hemagglutination inhibition antibody after the first, second, third, and fourth vaccinations were 1:15.7, 1:22.2, 1:36.0, and 1:32.0, respectively (first vaccination vs. baseline, P<.001; second vs. first vaccination, P=.02; and third vs. second vaccination, P<.001). The microneutralization GMTs after the first, second, third, and fourth vaccinations were 1:17.5, 1:33.1, 1:55.7, and 1:68.4, respectively (P<.001 for all comparisons)

ConclusionThe results of our study suggest that a third and fourth dose of the H5N1 A/Vietnam/1203/04 vaccine may result in higher hemagglutination inhibition and microneutralization GMTs, compared with the GMTs resulting from fewer doses. There was no benefit to increasing the dose of the vaccine

Trial registrationClinical Trials.gov identifier: NCT00383071

H5N1 avian influenza is currently an episodic zoonotic disease affecting at least 385 people in 14 countries. If this virus acquires the ability for sustained human-to-human transmission, it could trigger the next influenza pandemic. It has previously been noted that there is a limited repertoire of antiviral drugs and increasing levels of antiviral resistance have been noted [1]. Recent studies reported that some H5N1 viruses isolated in Southern China and Southeast Asia were resistant to amantadine [2, 3]. The currently circulating H5N1 viruses are susceptible to oseltamivir and zanamivir, although there have been descriptions of resistance to oseltamivir occurring in isolates recovered from patients who had received this drug [4] and in circulating avian strains [5]

Treatment with anti-influenza antibodies could potentially be of benefit in the treatment of avian influenza. Luke et al assessed the use of passive immunotherapy during the 1918 pandemic, when severely ill patients were sometimes treated with plasma from convalescing survivors. In the 8 published reports, the overall case fatality rate was 16% among 336 patients who received blood products from convalescent patients, compared with a rate of 37% among 1219 untreated patients [6]. Zhou et al reported prompt defervescence and cessation of viral shedding in a patient infected with H5N1 who was treated with plasma from a convalescent patients [7]

A murine monoclonal antibody (MAb) targeting a conserved site on the HA protected mice against lethal H1N1 and H2N1 influenza challenges [8]. MAbs to H1, given either intact or as Fab fragments, prevented lethal H1N1 infection in severe combined immunodeficiency mice [9, 10, 11]. Humanized murine MAbs prevented death when given to mice up to 3 days after an otherwise-lethal H5N1 virus challenge [12]. Similarly, human MAbs developed from survivors that had been infected with H5N1 protected mice when administered up to 72 h after H5N1 virus challenge [13]

Although MAbs may have therapeutic potential, pooled human immunoglobulin from convalescent patients or vaccinees may be more readily available, more expeditiously approved for human use, and more likely to prevent the emergence of escape mutations due to its polyclonal nature. In view of this, we sought to determine a vaccination strategy to generate high titers of anti–influenza H5N1 antibodies in healthy volunteers for the purpose of developing a hyperimmune intravenous immunoglobulin

In previous studies that used the influenza H5N1 vaccine (rgA/Vietnam/1203/04 X A/PR/8/34), 451 healthy adults aged 18–64 years received placebo or 2 vaccine doses of 7.5, 15, 45, or 90 μg by intramuscular injection [14]. A dose-response relationship without a demonstrated plateau was shown between vaccine and immunogenicity. Additional studies that used purified influenza hemagglutinin (HA) vaccine (influenza A/Taiwan/1/86 [H1N1]) with HA doses up to 405 μg showed that increasing doses of HA resulted in increasingly higher levels of serum hemagglutination inhibiting and neutralizing antibody [15]. Therefore, we sought to determine whether additional and/or higher doses of the H5N1 A/Vietnam/1203/04 vaccine led to improved immunogenicity and higher antibody titers. We also sought to evaluate the luciferase immunoprecipitation system (LIPS) as a new platform for the assessment of immune response to the vaccine

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Materials and Methods

Vaccine

The vaccine used in this study is a monovalent inactivated subvirion H5N1 vaccine (rgA/Vietnam/1203/04 X A/PR/8/34) that contains 90 μg/mL A/H5N1 HA, as determined by single radial immunodiffusion (sanofi pasteur)

Study Design and Subjects

We conducted a single-center, dose-escalating unblinded clinical trial. Written informed consent was obtained from prospective subjects prior to screening. Healthy, nonpregnant adults aged 18–59 years who had no known allergy to the vaccine components (including eggs) and met the standard United States Food and Drug Administration criteria for plasma donation were eligible for this study. The study was conducted in accordance with an institutional review board–approved protocol

Study Procedures

Three cohorts of 25 subjects each were enrolled sequentially. The cohorts were assigned to receive 90, 120, or 180 μg of H5N1 vaccine. Each subject was to receive 4 doses of vaccine in the deltoid or gluteal muscle, with each dose separated by ∼28 days. Subjects were allowed to choose the site of vaccination and were observed for 30 min after each vaccination

During the 7 days after each vaccination, subjects recorded the presence and severity of local and systemic symptoms on a diary card. Blood samples for antibody assays were collected before each vaccination and 4 weeks after the final dose. After the vaccination phase of this protocol was completed, subjects with a hemagglutinin inhibition (HAI) titer above a given threshold (the threshold was initially 1:640 but was decreased to 1:160 by the end of the study) were asked to begin plasmapheresis

All reported adverse events and serious adverse events that occurred through day 112 (4 weeks after the fourth vaccination) or while the subject was undergoing plasmapheresis were recorded and reviewed by an independent safety monitoring committee. Stopping rules for safety were evaluated after each cohort was vaccinated at a given study time point, prior to receipt of additional vaccinations, and prior to beginning enrollment of subjects at higher vaccine doses. Adverse events judged not to be related or unlikely to be related to the study interventions were excluded from this analysis

Laboratory Assays

HAI assayHAI assays were performed by Southern Research Institute (Birmingham, AL) using the influenza rgA/Vietnam/1203/04 XA/PR/8/34 influenza (H5N1) vaccine strain. HAI assays were performed in accordance with established procedures by use of horse erythrocytes. Serum samples were tested at an initial dilution of 1:10, and those that were negative for antibody at this dilution were assigned a titer of 1:5. Previous studies of this vaccine reported the initial dilution as 1:20, and those negative for antibody at this dilution were assigned a titer of 1:10 [14]

Serum samples were tested every 2 weeks in batches to determine eligibility for plasmapheresis (results not shown). At the end of the study, samples were tested in duplicate in accordance with good laboratory practice recommendations (the tests were performed by different operators on different days). Because the values for 83 (23.6%) of 352 samples run in duplicate were found to differ from one another by more than 2-fold, a third run was added for all samples. The geometric mean titer (GMT) was calculated for each serum sample. Replicate values not within 2-fold of the other values were excluded from inclusion in the GMT calculation in accordance with procedures established prior to commencement of this study

Microneutralization assayMicroneutralization assays were performed by Southern Research Institute in accordance with established procedures using influenza rgA/Vietnam/1203/2004 x A/PR/8/34 influenza (H5N1). Serum samples were tested at an initial dilution of 1:20, and those that were negative were assigned a titer of 10. Serum samples were tested separately and in triplicate. The GMT was calculated for each serum sample. Replicate values not within 2-fold of the other values were excluded from inclusion in the GMT calculation by procedures established prior to commencement of this study

LIPS and generation of Ruc-antigen fusion constructs pREN2, a mammalian Renilla luciferase (Ruc) expression vector [16] was used to generate HA fusion protein constructs. A plasmid template for the HA of H5N1 Vietnam 1203 (obtained from the Centers for Disease Control and Prevention) was amplified by use of polymerase chain reaction and used to generate 2 nonoverlapping HA DNA fragments. One of the fragments, HA-1, lacked the signal peptide and encoded aa 19–321 of the HA; the HA-2 fragment encoded aa 332–550. Following subcloning into pREN2, the resulting constructs generated C-terminal fusions to Ruc. The plasmid DNA corresponding to each of these different pREN2 expression vectors was prepared using a Qiagen Midi preparation kit. DNA sequencing confirmed the integrity of these 2 DNA constructs

LIPS analysisExtracts containing the Ruc-HA-1 and Ruc-HA-2 fusion proteins were prepared from transfected Cos1 with buffer A containing 50% glycerol, as described elsewhere [17]. A master plate was constructed by diluting patient serum samples 1:10 in assay buffer. For evaluation of antibody titers with LIPS analysis, 40 μL of buffer A, 10 μL of diluted human serum (1 μL equivalent), and 50 μL of Ruc antigen from the Cos1 cell extract diluted in buffer A were added to each well of a second polypropylene plate in which the assay was conducted. By use of these extracts, the immunoprecipitation assay was performed in a 96-well plate format at room temperature. The input for these immunoprecipitation assays for HA-1 and HA-2 proteins was 3.1×106 and 1.63×106, respectively. After the final wash, the plate was blotted, and the luminescence units were measured in a microplate luminometer (Centro LB 960; Berthold) using coelenterazine substrate mix. All luminescence unit data presented were obtained from the mean of 2 independent experiments and corrected for background

Statistical Analysis

Binary variables were compared between different dose cohorts by means of Fisher’s exact test. The 95% exact confidence interval (CI) of a proportion was constructed using the binomial distribution. Geometric mean and geometric standard deviation were calculated for antibody titers, and the comparison of antibody titer between dose groups after a vaccination was performed by applying analysis of variance to log2 titers. To use all available antibody data and account for correlation among multiple antibody titers for the same study subject, the generalized estimating equations (GEE) method [18] was employed to analyze log2 titers. For all GEE analyses, an exchangeable correlation structure was used as the working assumption

All P values are 2-sided, and P values of less than .05 were considered to be statistically significant. Data analyses were performed with Stata (version 10.0; Stata)

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Results

Seventy-five subjects were enrolled between December 2006 and March 2007. Seventy-one subjects received all 4 vaccinations, and 69 of these subjects had serum samples available for all time points, including day 112 (4 weeks after the fourth vaccination). Three subjects withdrew before receiving all vaccinations (2 subjects received 1 vaccination, and 1 subject received 2 vaccinations). Two additional subjects received all vaccinations but did not return for the final follow-up visit. The subjects who did not complete the study included 3 subjects with scheduling conflicts and/or an inability to dedicate the time needed for the study, 1 subject with an ankle fracture, and 1 subject with a small bowel obstruction from a previous appendectomy. The remaining subject was lost to follow-up after receiving 4 vaccinations

The baseline demographic and clinical characteristics of enrolled subjects are shown in table 1. At baseline, no statistically significant differences were noted between the dose cohorts with respect to age, sex, race, or ethnicity

Figure 1
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Figure 1

Aggregate hemagglutination inhibition (HAI) and microneutralization (MN) geometric mean titers among 75 subjects enrolled in a trial of an inactivated influenza A/H5N1 vaccine, according to days since first vaccination. Error bars are 95% confidence intervals

Figure 2
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Figure 2

Proportion of subjects enrolled in a trial of an inactivated influenza A/H5N1 vaccine with a low (<1:40), medium (⩾1:40 and <160), and high (⩾160) antibody titer (hemagglutination inhibition [HAI] and microneutralization [MN]), according to study time point

Figure 3
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Figure 3

Aggregate geometric mean titer of antibody to H5N1 hemagglutinin (HA)–1, as determined by use of the luciferase immunoprecipitation system, according to study time point. Error bars are 95% confidence intervals

Figure 4
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Figure 4

Geometric mean titer of antibody to H5N1 hemagglutinin (HA)–1, as determined by use of the luciferase immunoprecipitation system, according to study time point in 12 subjects for whom hemagglutination inhibition showed no immune response during all planned vaccinations. Error bars are 95% confidence intervals

Figure 5
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Figure 5

Aggregate hemagglutination inhibition (HAI) and microneutralization (MN) geometric mean titers, according to vaccination site and days after first vaccination. Error bars are 95% confidence intervals

Table 1
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Table 1

Demographic and Clinical Characteristics of 75 Subjects Enrolled in a Trial of an Inactivated Influenza A/H5N1 Vaccine

Safety

Two serious adverse events were reported during the study period. One subject was hospitalized after the first vaccination for small bowel obstruction related to a previous appendectomy. This event was judged not to be related to the study vaccine. One subject who did not disclose a previous history of sickle cell anemia was hospitalized after the fourth vaccination with a vaso-occlusive crisis. This event was judged unlikely to be related to the study. No subjects died during the study period

Injection Site Reactogenicity

Pain and tenderness at the injection site were the most commonly reported adverse events. Of 75 subjects, 61 (83%) complained of pain and/or tenderness at the injection site after receiving 1 or more vaccinations. The frequency of pain and/or tenderness decreased with subsequent injections (reports of these events were received from 49 [65%] of 75 subjects, 45 [62%] of 73 subjects, 41 [57%] of 72 subjects, and 33 [48%] of 69 subjects after the first, second, third, and fourth vaccinations, respectively). There were 6 subjects who reported a total of 9 episodes erythema at the injection site. There were 5 subjects who reported a total of 9 episodes of pruritus at the injection site. Mild edema at the injection site was reported by 2 subjects

Systemic Reactogenicity

Excluding events indicative of local reactogenicity, 98 adverse events were reported by 36 subjects. The most commonly reported events were malaise, headache, and myalgia. The frequency of systemic adverse events decreased with subsequent injections (reports of such events were received from 26 [35%] of 75 subjects, 16 [22%] of 73 subjects, 11 [15%] of 72 subjects, and 9 [13%] of 69 subjects after the first, second, third, and fourth vaccinations, respectively). See table 2 for a full list of adverse events. A total of 11 episodes of self-limited lymphadenopathy were observed in 4 subjects, all occurring in the ipsilateral inguinal chain after vaccine injection into the gluteal muscle. No dose related increase in systemic reactogenicity was observed (table 2)

Table 2
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Table 2

Adverse Events Occurring in 75 Subjects Enrolled in a Trial of an Inactivated Influenza A/H5N1 Vaccine, According to Event Grade and Vaccine Dose Received

Immunogenicity

HemagglutinationBaseline HAI titers did not differ significantly between the cohorts (table 1). The primary end point was the proportion of subjects in each cohort who achieved a serum neutralizing antibody titer against influenza A/H5N1 of 1:320. The proportion of subjects who reached titers of 1:40 was also of interest in this study, because a titer of 1:40 is traditionally considered protective [14]. A larger percentage of subjects in the 90-μg cohort reached an HAI titer of 1:40 after 1 vaccination (50% [12 of 24], 12% [3 of 25], and 29% [7 of 25] for the 90-μg, 120-μg, and 180-μg cohorts, respectively; P=.02, by use of Fisher’s exact test). Otherwise, no significant differences were detected between cohorts after the same number of vaccinations with respect to the proportion of subjects who reached HAI titers of 1:40, 1:80, 1:160, and 1:320)

The HAI geometric mean titers (GMTs) for the 90-μg, 120-μg, and 180-μg cohorts for each study time point are shown in table 3. No statistically significant, dose-related increases in the GMTs of serum HAI were observed when the 90-μg, 120-μg, and 180-μg cohorts were compared after each vaccination. Accounting for within-subject correlation, the GEE analysis applied to log2 titers measured after day 0, for which both study day and cohort group were included as categorical variables, showed a statistically significant increase in log2 titer of 0.48 (95% CI 0.12–0.85), 1.22 (95% CI, 0.85–1.59), and 1.12 (95% CI, 0.73–1.50) from first vaccination to second, third, and fourth vaccinations, respectively. However, the GEE analysis suggested that the log2 titer was not significantly different between the dose groups (P=.07 for comparison of the 120-μg and 90-μg cohorts, and P=.37 for comparison of the 180-μg and 90-μg cohorts)

Table 3
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Table 3

Comparison of Hemagglutination Inhibition (HAI) and Microneutralization Geometric Mean Titers Observed in Subjects Enrolled in a Trial of an Inactivated Influenza A/H5N1 Vaccine, According to Dose Received and Study Time Point

Given the limited sample size and the lack of difference in GMTs between the cohorts, we analyzed all cohorts together to determine the effect of additional vaccinations beyond what was previously published. The HAI GMT for the combined cohort after first, second, third and fourth vaccinations was 1:15.7 (95% CI, 11.0–22.5), 1:22.2 (95% CI, 15.6–31.7), 1:36.0 (95% CI, 25.7–50.5), and 1:32.0 (95% CI, 22.7–45.3), respectively (figure 1). The paired t test showed that the mean HAI log2 titer increased significantly after each additional vaccination through the third vaccination (first vaccination vs. baseline, P<.001; second vs. first vaccination, P=.02; third vs second vaccination, P<.001). The difference between the third and the fourth vaccination was not statistically significant (P=.88). The proportion of subjects with low (<1:40), medium (⩾1:40 and <160), or high (⩾160) antibody responses (HAI and microneutralization) at each study time point are shown in figure 2

MicroneutralizationThe microneutralization GMTs for the 90-μg, 120-μg, and 180-μg cohorts for each study time point are shown in table 3. No statistically significant, dose-related increases in the serum microneutralization GMTs were observed when the 90-μg, 120-μg, and 180-μg cohorts were compared after the first 3 vaccinations. After the last vaccination, the GMT for the 180-μg group was significantly higher than that for the 90-μg group (P=.02), but it was not significantly different from that of the 120-μg group (P=.35). All cohorts were again combined together to determine the effect of additional vaccinations. The microneutralization GMT for the combined cohort after first, second, third, and fourth vaccinations was 1:17.5 (95% CI, 14.7–20.8), 1:33.1 (95% CI, 26.5–41.3), 1:55.7 (95% CI, 45.9–67.6), and 1:68.4 (95% CI, 56.5–82.9), respectively. The paired t test showed that the mean microneutralization log2 titer increased significantly after each additional vaccination (all P<.001)

LIPSTo test whether we could use a surrogate for the HAI assay, we studied the cohorts for changes in antibodies against HA as detected by use of the LIPS. Two different HA fragments from the Vietnam 1203 strain were tested. One of the constructs, designated HA-1, corresponded to the N-terminal 300 amino acids of the HA, whereas a C-terminal protein HA-2 corresponded to the C-terminal 218 amino acids. Both the HA-1 and HA-2 constructs were highly expressed in Cos1 cells and used in a high throughput screening method to measure antibodies in the vaccinated individuals. Analysis of anti–HA-2 antibodies showed that many individuals had a high level of HA-2 antibodies on day 0, which increased slightly over the duration of the study. Because HA-2 is more conserved across influenza subtypes, this result likely reflects preexisting antibodies generated during seasonal influenza vaccination or infections

For the combined cohort, the GMT for antibodies to H5N1 HA-1 as measured by use of LIPS at baseline and after the first, second, third, and fourth vaccinations was 98.5 (95% CI, 47.8–203.0), 345.0 (95% CI, 147.4–807.6), 4421.2 (95% CI, 2870.8–6809.1), 15,038.6 (95% CI, 11,894.0–19,014.6), and 26,447.5 (95% CI, 21972.2–31834.4), respectively (figure 3). Measurements of anti–HA-1 seem to be positively correlated with HAI; GMTs for subjects with 1:5, 1:5–1:20, and >1:20 HAI titers were 370.7 (95% CI, 218.0–630.3), 3192.7 (95% CI, 1238.5–8223.7), and 11,520.5 (95% CI, 8574.9–15,477.9), respectively. However, the difference in anti-HA-1 among subjects with an HAI titer >1:20 was not statistically significant. Interestingly, 12 subjects who had no immune response to the vaccine as measured by HAI (i.e., their HAI titer was 1:5 through all vaccinations and during follow-up) showed a demonstrable and statistically significant increase in anti–HA-1 antibodies after each vaccination (all P<.003) (figure 4). For this subset, the microneutralization GMT was 1:10.4 on day 0 (95% CI, 9.8–11.0) and increased after each vaccination to reach a value of 1:63.5 on day 112 (95% CI, 39.7–101.43)

Immunogenicity by vaccine siteThe vaccine is formulated at 90 μg/mL, and standard practice would limit intramuscular injection volumes to 1 mL in the deltoid and 2 mL in the gluteus. Therefore, the 90-μg cohort received 1 injection in the deltoid or gluteus, whereas the 120-μg and 180-μg cohorts received 2 injections into the deltoid (1 shot into each), or 1 shot in the gluteus. Unexpectedly, but likely in an effort by subjects to minimize anticipated discomfort, the majority of subjects in the 90-μg cohort elected to receive 1 shot into the deltoid (74 [81%] of 91 vaccinations), whereas the majority of subjects in the 120-μg and 180-μg cohorts elected to receive 1 shot in the gluteus (74 [76%] of 97 vaccinations and 64 [74%] of 87 vaccinations, respectively). The GEE analysis showed that, adjusted for study time point, the receipt of vaccination in the deltoid was associated with a 0.88 (95% CI 0.18–1.58; P=.01) increase in mean log2 HAI titer and a 0.24 (95% CI −0.16–0.63; P=.25) increase in microneutralization titer, compared with receipt of vaccination in the gluteus (figure 5)

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Discussion

The present study was conducted to determine the optimal vaccination schedule for the generation of high-titer anti–influenza A/Vietnam/1203/04 intravenous immunoglobulin that could be used as a potential therapeutic agent for the treatment of avian influenza. Our results extend previous observations related to the immunogenicity of the H5N1 influenza virus subvirion vaccines. Our study demonstrated GMTs after receipt of 2 doses of 90 μg/mL vaccine that are similar to those observed previous studies involving this vaccine, after differences in the methodology used to determine HAI titer (accounting for a 1-dilution correction) were incorporated (1:26.8 vs. 1:56.3) [14]

The results of our study suggest that some of the barriers to developing adequate antibody titers (which are generally accepted to be 1:40) may be overcome by an additional dose of vaccine, which increased both the GMT and the percentage of the cohort that achieved a titer >1:40. One previous study evaluated the efficacy of a third vaccination dose given 6 months after the primary series [19]. In that study, the HAI GMT titer for the 90-μg cohort was 1:53.3 (95% CI, 38.1–74.7) 28 days after the primary series of 2 vaccinations but decreased to 1:25.6 (95% CI, 18.9–34.6) after 6 months. After a third vaccination at the 6-month time point, the HAI titer increased to 1:69.8 (95% CI, 49.2–99.1). However, this increase was not maintained, and the titer had decreased to 1:18.5 (95% CI, 14.5–23.6) by 6 months after the third vaccination

The HAI assay did not perform well in our study. The HAI assay underestimated the microneutralization titers and did not reflect the overall immunogenicity of the vaccine (as demonstrated by the antibodies measured by use of LIPS). Whether the antibodies generated in the absence of an HAI response would be protective is unclear, and this question deserves further study

One surprising finding was that an additional antigen dose did not appear to generate a higher antibody titer. Interpretation of this data is confounded by the apparent 0.88-fold decrease in HAI antibody for those subjects who received the vaccine in the gluteus. Given the local reactogenicity and the injectate volume required, the use of alternative vaccination locations such as the gluteus may curtail local adverse effects but may also diminish immunogenicity

No published studies have evaluated the effect of vaccination site on antibody response to the influenza vaccine. A randomized trial that used the hepatitis B vaccine noted significantly lower antibody titers in those subjects who were vaccinated in the arm, compared with those who were vaccinated in the buttock (GMT values of 1454 vs. 85 mIU/mL) [20]. Some of this difference was attributable to needle length, and the disparity between the groups was partially improved when a 5.08-cm (2-inch) needle was used for gluteus injections instead of a 2.54-cm (1-inch) needle (GMT, 387 vs 85 mIU/mL). Our study used a standard 3.81-cm (1.5-inch) needle for all injections. The source of these results in our study is unclear and may represent difference in antigen processing at different vaccination sites or simply be related to the volume of injectate when vaccinations were given in the gluteus. The influence of injection site on influenza vaccine immunogenicity deserves further study

In the process of this study, we have collected 7.5 L of plasma from 3 subjects with a plasma HAI GMT of 1:256. This plasma will be processed into an intravenous immunoglobulin product for further studies. Although we did not achieve the objective of obtaining a large amount of anti-H5N1 influenza plasma for intravenous immunoglobulin therapeutic studies, these findings did help elucidate vaccination hyperimmunization strategies for further studies

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Acknowledgments

We are grateful to the volunteers for their generous contributions to this study, our clinic for their hard work executing this study, and the Safety Monitoring Committee (John Treanor, Jim Campbell, and Ben Schwartz) for their time and oversight of the study

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Footnotes

  • ↵Potential conflicts of interest: none reported

    Financial support: Intramural Research Programs of the National Institute of Allergy and Infectious Diseases (to J.B. and H.C.L.); National Institute of Dental and Craniofacial Research (to P.B.); National Institutes of Health

    The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the United States government

  • Received December 2, 2008.
  • Accepted February 26, 2009.
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  1. J Infect Dis. (2009) 200 (4): 501-508. doi: 10.1086/599992
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