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

  1. David I. Bernstein1,
  2. Kathryn M. Edwards2,
  3. Cornelia L. Dekker3,
  4. Robert Belshe4,
  5. Helen K. B. Talbot5,
  6. Irene L. Graham4,
  7. Diana L. Noah6,
  8. Fenhua He7 and
  9. Heather Hill7
  1. 1Division of Infectious Diseases, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio
  2. 2Pediatric Infectious Diseases, Vanderbilt University Medical Center, Nashville, Tennessee
  3. 3Pediatric Infectious Diseases, Stanford University School of Medicine, Stanford, California
  4. 4Infectious Diseases and Immunology, St. Louis University, St. Louis, Missouri
  5. 5Medicine, Vanderbilt University Medical Center, Nashville, Tennessee
  6. 6Southern Research Institute, Birmingham, Alabama
  7. 7EMMES, Rockville, Maryland
  1. Reprints or correspondence: Dr. David Bernstein, 3333 Burnet Ave, ML 6014, Cincinnati, OH 45229 (david.bernstein{at}cchmc.org).
 
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Abstract

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 µg per dose; vaccine at 15 or 7.5 µg per dose with MF59; or vaccine at 30, 15, or 7.5 µg 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 µg of vaccine per dose with MF59, in which 63% of subjects achieved the predetermined endpoint (hemagglutination-inhibition titer ⩾40) 28 days after the second dose, compared with 29% in the group that received the highest dose (45 µg per dose) of vaccine alone. Conclusions. A 2-dose regimen of subvirion influenza A (H5N1) vaccine was well tolerated. The antibody responses to 15 µg of A/H5 vaccine with MF59 were higher than the responses to 45 µg of vaccine alone.

Trial registration. ClincalTrials.gov identifier: http://www.clinicaltrials.gov/ct2/show/NCT00280033?term=NCT00280033&rank=1NCT00280033.

H5N1 avian influenza viruses continue to cause disease in humans. Although cases have been reported in 12 countries, most of the fatalities have been reported from Vietnam, Indonesia, Egypt, Thailand, China, and Turkey [1]. Outbreaks in birds have been reported in Asia, and an increasing number of cases have been noted in Europe and Africa, elevating concern that the virus will continue to spread to human populations, acquire the ability to spread from person to person, and cause a pandemic. Concern regarding this spread has been heightened by the high mortality rates associated with infection in humans [2, 3]. Vaccination remains the most attractive strategy for preventing or limiting the spread of avian influenza in human populations.

Early trials with H5N1 vaccine and other avian influenza vaccines suggested that at least 2 doses of vaccine were needed to induce acceptable antibody responses and that the antigen content would need to be increased beyond the amount routinely used in seasonal influenza vaccines, to increase the magnitude and frequency of the responses [4–6]. In the most recently published study, Treanor et. al. [5] administered 2 doses of a subvirion H5N1 vaccine at 7.5, 15, 45, or 90 µg per dose to healthy adults. All doses were well tolerated but doses of less than 90 µg induced serum hemagglutination-inhibition (HAI) titers or neutralization titers of ⩾40 in less than 50% of subjects. Among those who received the highest dose of vaccine, an HAI titer of ⩾40 was achieved in 58% of subjects, whereas a neutralization titer of ⩾40 was observed in 54%.

Other reports have indicated that adjuvants enhance the immune response to subvirion H5N1 vaccines [7, 4, 8]. Aluminum-containing adjuvants, the only adjuvants approved for human use in the United States, have been used routinely in a number of vaccines and have been investigated in conjunction with inactivated influenza vaccines. For seasonal influenza vaccines, aluminum hydroxide has not been shown to clearly increase immunogenicity. In one study involving an inactivated H5N1 influenza vaccine, the addition of aluminum hydroxide modestly increased the HAI and neutralization titers (not a statistically significant increase) at the highest vaccine dose tested (30 µg), but not at the lower doses tested (7.5 and 15 µg) [4].

Other adjuvants, including MF59, significantly increase the antibody titers to A/H5N3 [8], and A/H9N2 [7]. MF59 is an oil-in-water emulsion adjuvant that has been approved for use with seasonal influenza vaccines in several countries in Europe since 1997. MF59 has been shown to improve antibody responses to influenza, herpes simplex, cytomegalovirus, and HIV vaccines [9]. In the most recent avian influenza vaccine study involving MF59 [7], HAI and neutralization titers to A/H9N2 were higher after the first and second doses in the groups that received vaccine with MF59, compared with the group that received vaccine alone, following doses of 3.75, 7.5, 15, or 30 µg. In this study, there was no evidence of a dose effect.

In the current study, we evaluated a subvirion influenza A/H5N1 vaccine at doses from 7.5 µg to 45 µg with and without aluminum hydroxide or MF59. Our results with respect to the safety and immunogenicity of these vaccines are reported here.

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Methods

Subjects. Healthy, nonpregnant adults (age 18–64 years) were eligible for participation. Subjects were excluded if they had a known allergy to eggs or other vaccine components; had previously received any influenza A/H5 vaccine; were immunosuppressed; were known to have HIV, hepatitis B, or hepatitis C infection; had a history of Guillain-Barré syndrome; had received blood products in the past 3 months; had received killed vaccines in the past 2 weeks or live vaccines in the past 4 weeks; had an acute or chronic illness that would interfere with evaluations or increase risk; or had a history of severe reactions after influenza vaccination. Subjects were also excluded if they had laboratory values outside of the normal range on their screening blood tests, including hemoglobin level, white blood cell count, platelet count, alanine aminotransferase level, and creatinine level. Eligible subjects were enrolled between March 6 and March 27, 2006. The protocol and consent forms were approved by the institutional review board of each participating center.

Vaccine and adjuvants. Chiron Vaccines (now part of Novartis) prepared the monovalent inactivated subvirion vaccine. The virus used to prepare the working seed and the vaccine was produced by reverse genetics using the modified hemagglutinin and unaltered neuraminidase encoding genes from the influenza A/Vietnam/1203/2004(H5N1) strain and all other genes from A/PuertoRico/8/34 [10]. The vaccine virus was then grown in embryonated eggs by use of a process similar to that used for seasonal vaccine preparation. The vaccine was formulated to contain a hemagglutinin concentration of 60 µg/mL.

Administration of the vaccine at different doses was accomplished by varying the volume of the vaccine and adjuvant mixtures. Vaccine and adjuvant were prepared daily in the investigational pharmacy by withdrawing 0.7 or 0.8 mL of antigen from 3-mL vials containing 60µg/mL of antigen and adding this to vials with equal volumes of adjuvant (0.7 or 0.8 mL) or using antigen alone. After mixing, the product was administered within 8 h, and it was administered within 10 min of drawing the mixture into the syringe. Vaccine and adjuvant were administered at the dose and volume shown in table 1. It should be noted that decreases in the volume administered for doses with reduced antigen content led to decreasing amounts of MF59 and aluminum hydroxide being administered with decreasing doses of antigen, as described above. The MF59 content at the higher dose was 9.75 mg squalene, 1.68 mg polysorbate 80, 1.68 mg sorbitan trioleate, 0.96 mg sodium citrate, and 0.06 mg citric acid; the lower dose contained half these amounts. The aluminum hydroxide content ranged from 0.35 mg to 0.88 mg (table 1).

Study design. This multicenter randomized, double-blind, placebo-controlled trial was conducted at 4 sites in the United States: Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio; Vanderbilt University Medical Center, Nashville, Tennessee; Stanford University School of Medicine, Stanford, California; and St. Louis University, St. Louis, Missouri. After providing informed consent, eligible subjects were randomly assigned to 1 of 9 groups: saline placebo (n = 30); influenza vaccine alone at 45, 30, or 15 µg per dose (n = 90, 60, and 30, respectively); influenza vaccine at 15 or 7.5 µg per dose, combined with MF59 (n = 30, for both groups); or influenza vaccine at 30, 15, or 7.5 µg per dose, combined with aluminum hydroxide (n = 60, 30, and 30, respectively) (table 1). Each subject received 2 intramuscular injections of an identical formulation in the deltoid area, administered approximately 28 days apart.

The choice of doses and the combinations of vaccine with adjuvant preparations used in the study were largely driven by the vaccine, which was available for the trial as a single formulation of 60 µg/mL, and the expected tolerable volumes and concentrations of MF59 and aluminum hydroxide. The sample size was weighted to favor doses that were deemed to be the most likely to be implemented in the future, to obtain more precise estimates of the responses in those dose groups. The study was not designed to test a specific hypothesis. However, sample sizes of 90 and 60 subjects in the groups of greatest interest provided 80% power to detect a difference in the proportion of responders of 50% and 51%, respectively, when compared with a group of 30, assuming a 20% response rate for the smaller group. Additionally, sample sizes of 90 and 60 provided at least 99% and 95% probabilities, respectively, of observing a vaccine-related event, assuming a true occurrence rate of at least 5.0%. In addition, these sample sizes provided the full widths of, at most, 21.5% and 26.4% respectively, based on a 95% Blyth-Still confidence interval of an observed event rate.

After randomization, an unblinded member of the study team who did not otherwise participate in the trial administered the injection. Subjects were observed for 15 min; they then completed a memory aid to record local and systemic adverse events, as well as their daily temperatures for 7 days. Subjects were called 1–3 days and 14–21 days after each vaccination to elicit information about any adverse events. Subjects also returned to the clinic 7–9 days after each vaccination for assessment and to repeat the same laboratory tests outlined earlier. Approximately 28 days after each vaccination, and again at 6 months after the second vaccination, serum samples were obtained for antibody assays. All 7-day safety data available after the subject had received the first dose of vaccine were reviewed prior to administering the second dose of vaccine.

Antibody assays. Microneutralization (MN) and hemagglutination-inhibition (HAI) assays were performed at Southern Research Institute using genetically modified reassortant, rgA/Vietnam/ 1203/2004x A/PR/8/34, as described elsewhere [11, 12]. MN and HAI assays with horse erythrocytes were performed as described elsewhere [5], except that the same starting dilution was defined as 1:10 rather than 1:20, and samples that were negative were assigned a titer of 5. Development of a titer of ⩾40 in both the MNand HAI assays was predefined as indicative of a “responder.” Detectable responses were defined as a titer ⩾10. For both assays, serum samples were tested in duplicate in independent assays, and samples that gave more than 2-fold different results in replicate samples were retested.

Statistical analysis. Data collection for this study was conducted using the Statistical and Data Coordinating Center's (SDCC) Internet-based Electronic Data Capture system. The SDCC was also responsible for the randomization. Because of the limited supply of study vaccine, randomization tables were established at the SDCC each night for the following day, on the basis of only the cumulative enrollment for all sites and the expected number of new enrollees at each site. At the time of randomization, the SDCC had no information about the individuals being randomized, only the total number that investigators planned to randomize. The site staff who evaluated vaccine reactogenicity were fully blinded to the dose and type of vaccine given.

The proportions of solicited adverse events (mild, moderate, or severe) after either the first or second dose of vaccine were based on the most severe response reported. The proportion of individuals with an elevated oral temperature was compared across 9 treatment groups by use of the Fisher exact test, and the proportions of the remaining local and systemic reactions were compared by use of the χ2 test. Pain at the injection site was also analyzed by assigning a numerical scale (0, 1, 2, or 3) to the level of response (none, mild, moderate, or severe). The relationship of pain to dose levels and adjuvant effects was explored with the general linear model, and the result was also verified by use of only an ordinal scale and testing with cumulative logistic regression.

Analytical results for geometric mean titers (GMTs) were determined by converting to a logarithmic scale and assuming normality or using rank order methods. Exact confidence intervals were reported for all proportional endpoints. For subjects with and subjects without preexisting antibodies to the H5N1 virus, geometric mean titers were compared using the Wilcoxon rank test, and the proportions of 4-fold increases from the baseline were compared using the Fisher exact test. All analyses were conducted using SAS (version 8.2; SAS Institute).

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Results

A total of 394 subjects received at least 1 dose of vaccine and 382 received 2 doses (figure 1). The reasons that subjects were withdrawn from the study and their group assignment is also shown in figure 1. The demographic characteristics of the population were comparable among the vaccine groups, except for a significant difference noted in the sex distribution (P < .01 by χ2 [table 2]).

Figure 1
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Figure 1

Trial profile and reasons for subject withdrawal

Figure 2
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Figure 2

Antibody responses over time. The geometric mean antibody titer (GMT) for hemagglutination-inhibiting (HAI) antibody (A) and microneutralization (MN) antibody (B) is shown for the groups that received the largest vaccine dose without adjuvant, the largest dose with aluminum hydroxide adjuvant, and the largest dose with MF59 adjuvant. Antibody evaluations were performed prior to vaccination (day 0), 28 days after the first vaccination (day 28), 28 days after the second vaccination (day 56), and 6 months after the second vaccination (day 208).

Table 1
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Table 1

Dose and volume of vaccine and adjuvant per study design.

Table 2
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Table 2

Demographic characteristics of study subjects, according to vaccine group.

Safety. There were no serious adverse events associated with the vaccine. As shown in table 3, local reactions, especially pain and tenderness were common, whereas systemic adverse events occurred in as few as 0%–3% for fever and as many as 13%–44% for headache in the 7 days following vaccination. No significant differences in vaccine reactogenicity were seen among the groups, except for local pain and tenderness. The incidence and severity of pain was related to both the vaccine dose and the adjuvant. The incidence and severity of pain increased as dose levels increased in each adjuvant group (P = .01, Type III F-test). The incidence and severity of pain were also increased in the groups that received aluminum hydroxide, compared with the groups that received no adjuvant, and the incidence and severity of pain increased further in the groups that received MF59. Therefore, pain was most common after a dose of 15 µg of vaccine with MF59. The pain was, however, mild to moderate and lasted for 1–2 days for the majority of subjects. The incidence of tenderness was also similarly related to vaccine dose level and adjuvant. Neither local or systemic adverse events increased after the second dose, compared with the first. No subject withdrew from the study because of the intensity of local reactions.

Table 3
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Table 3

Incidence of adverse events after the first or second dose of vaccine, according to vaccine group.

Immunogenicity. Antibody responses were measured by HAI and MN at baseline, at 28 days after each dose, and at 6 months after the second dose. After the first dose of vaccine, 7%–29% of subjects in each group had detectable HAI antibody (i.e., titer ⩾10), and 18%–66% had detectable MN antibody (data not shown). However, antibody at the predefined endpoint titer of ⩾40 was observed in only 0%-22% of subjects, as measured by HAI, and in 3%–21% of subjects, as measured by MN (data not shown). The highest percentage of responders was seen in the group receiving 15 µg of vaccine with MF59. The geometric mean titer after the first dose was slightly higher in the group that received 45 µg of vaccine alone (titer 10.8), compared with the group that received 15 µg of vaccine with MF59 (titer 9.5).

After the second dose of vaccine, antibody levels increased in all groups. HAI antibody was detected (titer ⩾10) in 10%–72% of subjects and MN antibody was detected in 45%–97% of subjects (data not shown). As shown in table 4, the highest antibody response was seen in the group that received 15 µg of vaccine with MF59, in which 63% of subjects achieved an HAI titer ⩾40 (GMT 32.6). The vaccine alone induced HAI antibody at a titer ⩾40 in a range from 24% at the lowest dose (15 µg) to 29% at the highest dose (45 µg). The percentages of subjects achieving MN titer ⩾40 and the MN GMTs were somewhat higher, compared with the HAI responses. Thus, the percentage of subjects with MN titer ⩾40 in the group receiving 15 µg of vaccine with MF59 was 81%, with a GMT of 63.0. The addition of aluminum hydroxide did not increase the antibody as measured by HAI or MN to the level observed after vaccine alone at any dose.

Table 4
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Table 4

Antibody response in study subjects 28 days after receipt of the second dose of vaccine.

At 6 months after the second dose, antibody titers had decreased in all groups. The HAI antibody levels over time are shown for groups that received the largest vaccine dose without adjuvant (45 µg), vaccine with aluminum hydroxide (30 µg), and vaccine with MF59 (15 µg) (figure 2A). Similar decreases in MNantibody levels were also seen (figure 2B). At 6 months after the second dose, HAI responses of ⩾40 were observed in 18% of those who received 45 µg of vaccine alone, 5% of those who received 30 µg of vaccine with aluminum hydroxide, and 20% of those who received 15 µg of vaccine with MF59.

At baseline, 0%–7% of subjects had detectable HAI antibody (titer ⩾10), and 3%–18% had detectable MN antibody . To gain insight into whether these low levels of antibody reflected antibody to H5 or were nonspecific background, we compared the antibody response in these subjects after 1 dose of vaccine, reasoning that those with true H5 antibody should be primed and have a higher response to vaccine. The day 28 GMT after dose 1 in subjects shown to have preexisting antibody to the H5N1 virus by HAI assay was significantly higher than that for subjects without detectable antibody (56.3 vs. 19.3; P < .001, Wilcoxon rank test) (table 5), but the percentage with a 4-fold increase in titer did not differ significantly (18% vs. 11%; P < .34, Fisher exact test) (table 6). When the day 28 MN titers were compared, the subjects with preexisting neutralizing antibody had a higher GMT than subjects without preexisting antibody (95.0 vs. 16.8; P < .001, Wilcoxon rank test), and they were more likely to develop a 4-fold increase in antibody level after 1 dose (30% vs. 8%; P = .001, Fisher exact test) (table 6).

Table 5
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Table 5

Comparison of geometric mean titer (GMT) between those with baseline titer <10 and those with baseline titer ⩾10.

Table 6
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Table 6

Comparison percentage of subjects with a 4-fold increase after 1 dose of vaccine, for subjects with baseline titer <10 and subjects with baseline titer ⩾10.

The possibility that these differences were the result of an unequal distribution within vaccine groups was investigated. There were no significant differences in the distribution of subjects with preexisting antibody across the 9 groups for either HAI (P = .37) or MN (P = .37). There were also no significant differences in the baseline GMTs across groups for HAI (P = .28) and MN (P = .71).

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Discussion

All the H5N1 vaccine and adjuvant combinations were generally well tolerated, although local symptoms, especially pain and tenderness, were common. The percentage of subjects who developed pain and/or tenderness increased as the antigen content of the vaccine increased, and the percentage was increased at equivalent doses when combined with an adjuvant. The addition of aluminum hydroxide increased the frequency of pain, compared with vaccine alone, and MF59-adjuvanted vaccines further increased the percentage of subjects who experienced pain. It is, however, important to note that the pain was most often assessed as mild to moderate and did not lead to the withdrawal of any subject before the second dose was administered. An increase in local reactions to vaccine with MF59 has been noted previously when this adjuvant was used with other avian influenza vaccines [7, 8], as well as with seasonal influenza vaccines [13, 14].

The antibody response to the vaccines appeared to be related to both the quantity of vaccine and the inclusion of MF59, but not to the inclusion of aluminum hydroxide. The antibody response to vaccine tended to increase as quantities of vaccine increased. Without an adjuvant, the percentage of subjects who developed neutralizing antibody at a titer of ⩾40 after 2 doses of vaccine increased from 28% in the group that received 15 µg of vaccine to 48% in the group that received 45 µg. The addition of aluminum hydroxide did not increase the antibody response in any of the 3 groups in which it was evaluated (i.e., the groups that received 7.5, 15, or 30 µg of vaccine). In a previous report of an H5N1 vaccine, aluminum hydroxide improved the antibody response to a 30-µg dose of vaccine but did not increase the antibody response to lower doses of vaccine (7.5 or 15 µg) [4]. In fact, although the HAI GMT to the 30-µg dose was increased by adjuvant, the effect was not described as statistically significant, and in the group that received a 7.5-µg dose, the HAI GMT was similarly decreased by the addition of aluminum hydroxide. Thus, it does not appear that the addition of aluminum hydroxide will improve the immunogenicity of subunit avian influenza vaccines.

The addition of MF59, however, increased both the GMT and the percentage of subjects who developed an antibody titer ⩾40. The group receiving the highest dose of vaccine (15 µg) tested with MF59 was the only group in which >50% of the subjects were shown to have developed an antibody titer of ⩾40, by either HAI or neutralization assay. The neutralizing antibody GMT in this group was 63.0, compared with a GMT of 15.8 in the group that received 15 µg of vaccine alone. The antibody response in the group receiving 15 µg of vaccine with MF59 was higher than that in the group receiving 7.5 µg of vaccine with MF59, but it is important to note that the latter group received only half the dose of MF59 that the former group received. The increased antibody response observed here is consistent with previous reports that demonstrate increased antibody titers following vaccination with other A/H5N3 and A/H9N2 avian influenza vaccines [7, 8] as well as seasonal influenza vaccines [13, 14]. Further evaluations that use lower doses of vaccine and consistent doses of MF59 are needed to define the optimum combination for vaccination. Extended studies involving the elderly individuals and young people are also needed to determine whether the addition of MF59 would improve the immunogencity of A/H5 vaccines in these groups.

HAI, and more often MN, antibodies to H5N1 virus were occasionally detected prior to vaccination. Previous studies have also reported a low level of preexisting antibody [5, 4], without apparent exposure to H5N1 viruses. It is unclear whether this represents a true response or was a nonspecific reaction. Neutralizing antibody may be present due to neuraminidase specific antibody, as N1 viruses (H1N1) have circulated and have been part of routine influenza vaccines for some time. It is also possible that this antibody represents heterosubtypic immunity from cross-reacting epitopes from H1, H2, or H3 viruses. It is, therefore, interesting to note that there was some evidence that subjects with preexisting antibody responded better to the first dose of vaccine than subjects without preexisting antibody, which implies that the initial antibody measured may have been H5 specific and that the improved responses were due to a booster response, as opposed to a primary response.

In summary, 2 doses of A/H5N1 vaccine at antigen concentrations ranging from 7.5 to 45 µg per dose were well tolerated. The addition of aluminum hydroxide did not improve antibody response for any antigen dose tested. However, when vaccine antigen was combined with MF59, both HAI and MN antibody responses were substantially increased, so that titers were higher for the group that received 15 µg of vaccine with MF59, compared with titers for the group that received the highest dose tested, 45 µg of vaccine alone. The target HAI antibody titer of ⩾40 was achieved in 63% of the subjects, and the target MN response was detected in 81% of subjects who received 15 µg of vaccine with MF59, the highest dose tested with MF59. Antibody responses were thus as high or higher than those reported previously in studies that used 45–90 µg of A/H5 antigen [5].

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Footnotes

  • Potential conflicts of interest: D. I. B. receives funding for clinical studies from Medimmune, GlaxoSmith-Kline, and Sanofi. K.M.E. receives research funding from Sanofi and Medimmune, and is a consultant for PATH. C.L.D. worked for Chiron Vaccines until 1997, but currently has no relationship or holdings with them. R.B. is a consultant for Medimmune and Chiron; a speaker for Merck, Medimmune and Sanofi; and receives funding from Medimmune and Merck. Other authors report no relevant conflicts.

  • Financial support: National Institutes of Health (N01 AI 25459, to D.I.B.; AI 25462, to K.M.E.; and AI 25464, to R.B.).

  • Received August 1, 2007.
  • Accepted September 27, 2007.
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  1. J Infect Dis. (2008) 197 (5): 667-675. doi: 10.1086/527489
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