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Safety and Immunogenicity of Nonadjuvanted and MF59-Adjuvanted Influenza A/H9N2 Vaccine Preparations

  1. Robert L. Atmar1,2,
  2. Wendy A. Keitel1,2,
  3. Shital M. Patel1,2,
  4. Jacqueline M. Katz3,
  5. Dewei She4,
  6. Hana El Sahly1,2,
  7. Justine Pompey3,
  8. Thomas R. Cate1,2, and
  9. Robert B. Couch1,2
  1. 1Departments of Medicine and Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas
  2. 2Departments of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas
  3. 3Departments of Influenza Branch, Centers for Disease Control and Prevention, Atlanta, Georgia
  4. 4Departments of EMMES Corporation, Rockville, Maryland
  1. Reprints or correspondence: Dr. Robert L. Atmar, 1 Baylor Plaza, MS BCM280, Houston, TX 77030 (ratmar{at}bcm.edu).
 
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Abstract

Background. Influenza A/H9N2 viruses can infect humans and are considered to be a pandemic threat. Effective vaccines are needed for these and other avian influenza viruses.

Methods. We performed a phase I, randomized, double-blind trial to evaluate the safety and immunogenicity of a 2-dose schedule (administered on days 0 and 28) of 4 dose levels (3.75, 7.5, 15, and 30 µg of hemagglutinin) of inactivated influenza A/chicken/Hong Kong/G9/97 (H9N2) vaccine with and without MF59 adjuvant. Vaccine safety was assessed with a diary and selected blood tests. Immunogenicity was measured using serum hemagglutination inhibition (HAI) and microneutralization (MNt) antibody assays.

Results. Ninety-six healthy adults (age, 18–34 years) were enrolled in the study. Arm discomfort was more common in groups that received adjuvant, but adverse effects of the vaccination were generally mild. Geometric mean serum HAI and MNt antibody titers to the influenza A/chicken/Hong Kong/G9/97 (H9N2) virus strain for all vaccine groups were similar on day 0 but were significantly higher (P < .001) on both days 28 and 56 for the MF59-adjuvanted vaccine groups than for groups given nonadjuvanted vaccine. Other measures of immunogenicity were also higher in the adjuvanted vaccine groups. HAI and MNt geometric mean titers measured after the administration of a single dose of MF59-adjuvanted vaccine were similar to those measured after 2 doses of nonadjuvanted vaccine.

Conclusions. The combination of MF59 adjuvant with a subunit vaccine was associated with improved immune responses to an influenza A/H9N2 virus. The adjuvanted vaccine was immunogenic even after a single dose, raising the possibility that a 1-dose vaccination strategy may be attainable with the use of adjuvanted vaccine.

The recent emergence of novel avian influenza virus strains in human populations (influenza A/H5N1, A/H7N7, and A/H9N2 viruses) has bolstered ongoing efforts to develop pandemic preparedness plans [1–4]. One of the cornerstones of pandemic preparedness is the development and production of safe and immunogenic vaccines [5]. The threat of pandemic influenza in 1976 (swine influenza) and again in 1977 (Russian influenza) led to inactivated influenza virus vaccine development programs that provided important insights into variables influencing the immune responses to immunization [6, 7]. Factors affecting the immunogenicity of inactivated influenza virus vaccines include the dose of hemagglutinin (HA) included in the vaccine, the number of doses administered (1 or 2), the use of an adjuvant, vaccinee age, prior antigenic experience and underlying disease of the vaccinee, and the type of product used (whole virus, split virus, or purified surface antigen).

Influenza A/H9N2 viruses are panzootic in poultry in Eurasia and constituted 75% of the influenza viruses isolated from birds in the live poultry markets of Hong Kong during 2001–2003 [8]. Three distinct genetic viral lineages exist in poultry in Asia, and 2 of these—represented by Quail/Hong Kong/G1/1997 (G1-like) and Duck/Hong Kong/Y280/1997 (Y280-like)—have caused infection in humans [1, 9]. Influenza A/H9N2 viruses belonging to these 2 sublineages have been shown to bind to sialic acid residues found on human (terminal α2,6Gal linkage) as well as avian (terminal α2,3Gal linkage) cells and, thus, may have a greater potential to infect humans than other avian influenza subtypes [10]. Therefore, these viruses have been identified as posing a potential pandemic threat for which vaccine development is warranted [4].

As part of this vaccine development effort, we conducted a dose-ranging study to evaluate the safety and immunogenicity of a 2-dose regimen of nonadjuvanted or MF59-adjuvanted subunit influenza A/H9N2 vaccine in young healthy adults. In this article we present results that indicate that the administration of 2 doses and the use of an adjuvant enhance the immune response to the study vaccine.

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Methods

Subjects. Study participants were healthy, nonpregnant adults (age, 18–34 years). The upper age limit was selected to exclude persons born before 1970, on the basis of a previous report suggesting that persons born before 1968 are primed to the H9 antigen [11]. Exclusion criteria at enrollment included the following characteristics: a known allergy to eggs or other components of vaccine; prior receipt of an influenza A/H9N2 vaccine; abnormal laboratory values for blood tests; pregnancy, a desire to become pregnant in the next 3 months, or lactation; an inability or unwillingness to practice contraception during the course of the trial; immunosuppression as a result of underlying illness or treatment; acute or chronic illness; use of any medication other than for contraception; receipt of experimental vaccines or medications in the month prior to study entry; receipt of immunoglobulin or other blood product within 3 months prior to enrollment; receipt of licensed inactivated or live vaccines within the preceding 2 weeks or 4 weeks, respectively; and a history of a severe reaction following vaccination with a contemporary influenza vaccine. Eligible participants were screened and enrolled during the period March–April 2005 after providing written, informed consent in accordance with protocols approved by the Baylor College of Medicine Institutional Review Board.

Vaccines. Chiron Vaccines (Siena, Italy) prepared the study product, which consisted of influenza A/H9N2 purified surface HA and neuraminidase (NA) antigens in a suspension for injection in prefilled syringes. The virus used to produce the vaccine was prepared at the Centers for Disease Control (CDC; Atlanta, GA) and is a high-growth reassortant containing HA and NA antigens from the Y280-like influenza A/chicken/Hong Kong/G9/97 virus strain and internal genes from an H1N1 high growth reassortant vaccine strain influenza A/Johannesburg/82/96 X A/Puerto Rico/8/34 [12]. Single-dose syringes contained 3.75, 7.5, 15, or 30 µg of the vaccine strain HA in a volume of 0.5 mL. Adjuvanted vaccine preparations contained the same HA concentration adsorbed to MF59C.1 adjuvant (9.75 mg squalene, 1.175 mg polysorbate 80, 1.175 mg sorbitan trioleate, 0.66 mg sodium citrate, 0.04 mg citric acid). The manufacturer completely prepared all vaccine formulations. Nonadjuvanted vaccine doses were confirmed to be the intended vaccine doses through repeat testing. Fisher Bioservices (Rockville, MD) encoded the study vaccine and provided it to the study site.

Study procedures. Subjects were screened for good health by obtaining a health history, performing a targeted physical examination, and obtaining blood tests (complete blood cell count; determination of serum glucose, serum transaminases, serum creatinine, and creatine kinase levels; and serum pregnancy tests). Exclusion criteria were reviewed at screening and on the day of each vaccination. Urine pregnancy tests were performed on the day of each vaccination for all female subjects in the study. Within 4 weeks of screening, eligible subjects were enrolled and randomized to 1 of 8 vaccine groups (3.75, 7.5, 15, or 30 µg of HA, with or without MF59 adjuvant) using the IDES data management system (EMMES Corporation). After randomization (day 0), all subjects received the initial dose of 0.5 mL of study vaccine by intramuscular injection into the deltoid muscle. Four weeks (on day 28) after the initial dose, subjects received a second dose of the same vaccine preparation into the opposite arm. Subjects remained in the clinic for at least 20 min after each injection and were seen in the clinic 2 and 7 days later for safety evaluations. The subjects maintained a daily memory aid (diary) for 1 week after each vaccination to record their oral temperature, the presence of any local or systemic symptoms, and the use of any medication. Screening blood tests were repeated 1 week after each vaccination, and serum samples for antibody studies were collected prior to each vaccination (days 0 and 28) and 4 weeks after the second vaccination (day 56).

Solicited and unsolicited adverse events were graded on a scale from 0 to 3, where 0 indicated absence of the symptom, 1 indicated mild symptoms (easily tolerated), 2 indicated moderate symptoms (interfere with activity), and 3 indicated severe symptoms (incapacitating). Solicited adverse events included injection site symptoms (pain, tenderness, erythema, or induration) and systemic symptoms (feverishness, headache, malaise, nausea, or myalgia). Fever was considered to be the presence of an oral temperature of ⩾37.8°C (⩾100°F). The diameter of measurements of injection site erythema and induration were graded as follows: 0, less than 0.5 cm; 1, small (0.5–4.9 cm); 2, medium (5–10 cm); or large (>10 cm). Serious adverse events were defined as life-threatening adverse events, significant or persistent disability, hospitalization, or death. Vaccine allocation was concealed from each study subject and from all investigative staff except for the vaccine administrator, who had no other contact with the subjects.

Serologic testing. Tests for serum hemagglutination-inhibiting antibody (HAI) were performed as described [13], except reagent concentrations were altered to permit a starting dilution of 1:4 and turkey RBCs were used instead of RBCs from chickens. The test strains, influenza A/chicken/Hong Kong/G9/97 and influenza A/Hong Kong/1073/99, were grown in embryonated chicken eggs and were inactivated with β-propiolactone by Dr. Elena Govorkova (St. Judes Children's Research Hospital; Memphis, TN).

Microneutralization assays were performed at the CDC using previously described methods [14]. Pre- and postvaccination serum samples were tested together in duplicate, beginning with a dilution of 1:20. Serial 2-fold dilutions were made to a final dilution of 1:2560, and end point titers represented the highest serum dilution that resulted in 50% neutralization of 100 TCID50 (tissue culture infectious dose, 50%) of virus. Test strains were the same as for HAI and were grown in embryonated chicken eggs; allantoic fluid was harvested and stored at -80°C until use.

Statistical procedures. Reactogenicity end points were compared with Fisher's exact test between groups. Proportions and 95% CIs were used to describe the following immunogenicity end points: the proportion of subjects in each vaccine dose group who achieved a ⩾4-fold increase in titer between pre- and postimmunization serum samples, as measured by HAI or mNT (seroconversion rate), and in the proportion of patients who achieved a serum HAI titer of ⩾32 following each vaccination. Proportions were compared using a χ2 test. If none of the 12 subjects that were enrolled in each dose level/adjuvant group experienced a given adverse event, the upper bound of a 1-sided 95% CI for the probability of that event was ∼0.22. Other measures of immunogenicity included the determination of geometric mean titers and mean fold increase (seroconversion factor), with corresponding 95% CIs for serum HAI and MNt antibody levels, following each vaccination. The effect of adjuvant on the immunogenicity outcome was analyzed using a general linear model with the consideration of repeated measurement under the assumption that titer, as a logarithmic function, has a normal distribution. All analyses were performed using SAS software, version 8.2 (SAS Institute), except Spearman correlation coefficients, which were obtained using R 2.1.1 software (R Foundation).

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Results

Study subjects. Ninety-six subjects (12 per vaccine group) were enrolled during the period from 31 March 2005 through 7 April 2005. Demographic data are presented in table 1. All but 1 subject completed the study; a single person was not revaccinated after she was found to be pregnant at her day 28 visit. She subsequently terminated the pregnancy.

Figure 1
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Figure 1

Serum hemagglutination inhibition antibody levels to the influenza A/chicken/Hong Kong/G9/1997 (H9N2) virus strain from samples obtained before (on day 0) and 4 weeks after each vaccination (on days 28 and 56). Vaccine groups are indicated by hemagglutinin content. Bars, upper limits of 95% CIs; +, presence of MF59 adjuvant.

Figure 2
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Figure 2

Correlation of day 56 serum hemagglutination inhibition and microneutralization antibody titers to the influenza A/chicken/Hong Kong/G9/1997 (H9N2) virus strain among all vaccinees. Numbers in parentheses represent the number of specimens represented by the point in the graph.

Table 1
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Table 1

Demographic characteristics of study population, by vaccine type and dose.

Reactogenicity. Solicited adverse signs and symptoms observed in the week following vaccination are shown in table 2. Mild injection site symptoms (pain or tenderness) were the most commonly observed adverse reactions and were more frequent in the groups receiving adjuvanted vaccines. Local symptoms peaked on the evening of vaccination and resolved by the second day in all but 4 subjects (1 each from the groups that received doses of 7.5 µg without adjuvant, 7.5 µg with adjuvant, 15 µg with adjuvant, and 30 µg without adjuvant). Systemic symptoms occurred throughout the 7-day follow-up period, and frequencies of these adverse events were similar in adjuvanted and nonadjuvanted vaccine groups (table 2). No differences in local and systemic adverse event rates were observed by vaccine dose and/or by adjuvant status except for pain and tenderness at the injection sites. Subjects who received 15 µg doses (with or without adjuvant) tended to experience less pain than subjects in any other dose group after the first vaccination (P < .037). Subjects who received vaccine with adjuvant experienced more pain than those who received vaccine without adjuvant in each dose group for both the first and second vaccinations (P < .037), except for subjects who received 15-µg doses (first or second vaccination) and 7.5-µg doses (second vaccination). Adjuvant greatly increased the experience of tenderness for both the first and second vaccinations in each dose group (P < .01), except the groups that received a dose of 15 µg as a second vaccination.

Table 2
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Table 2

Maximum intensity of local and systemic reactions to vaccinations, by vaccination type and dose.

One subject reported a severe upper respiratory tract infection (associated with severe malaise and nausea) that started 1 day after receiving the initial dose of the 15-µg, nonadujvanted vaccine. Parainfluenza virus type 3 was isolated from a combined nasal wash/throat swab sample during the illness. Three men (1 each having received the 7.5-µg dose without adjuvant, the 15-µg dose without adjuvant, and the 15-µg dose with adjuvant) had grade 3 (severe; ⩾5 times the upper limit of normal) elevations in serum creatine kinase levels that were attributed to excessive physical activity during the follow-up period, and 1 woman (who received a dose of 30 µg without adjuvant) had an unexplained grade 3 elevation in serum creatine kinase at day 56. No serious adverse events occurred during the study.

Immunogenicity. One-third of the subjects had a baseline serum HAI antibody titer of ⩾16 to the influenza A/chicken/Hong Kong/G9/97 virus strain, whereas only 5% of subjects had a neutralizing antibody titer of ⩾20. Geometric mean serum HAI and MNt antibody titers among the vaccine groups to the influenza A/chicken/Hong Kong/G9/97 virus strain were similar at baseline (day 0), but they were higher in the MF59-adjuvanted groups than in the nonadjuvanted groups at both days 28 and 56 (P < .001, by general linear model; table 3 and figure 1). There was no evidence of a dose effect in a general linear model as measured by serum HAI (P = .92) or MNt (P = .54) antibody titers when controlling for adjuvant status, and there was no evidence of an interaction effect between adjuvant status and dose group in the analyses of HAI (P = .61) or MNt (P = .38) assay results. HAI and MNt geometric mean titers were similar after a single dose of MF59-adjuvanted vaccine to those achieved after 2 doses of nonadjuvanted vaccine (table 3). A strong correlation between serum HAI and MNt antibody titers was noted at days 28 (r = .85) and 56 (r = .93; figure 2).

Table 3
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Table 3

Serum immune responses to the influenza A/Chicken/Hong Kong/G9/97 virus strain homologous vaccine antigen, by vaccine type and dose.

All but 1 person who received adjuvanted vaccine achieved a day 56 serum HAI antibody titer of ⩾32, compared with only 63% of persons who received nonadjuvanted vaccine (P < .001, by χ2 test). Similarly, all but 2 subjects (with baseline serum HAI titers of 32 and 128, respectively) who received adjuvanted vaccine had a ⩾4-fold increase in serum HAI antibody titer, compared with 58% of persons who received nonadjuvanted vaccine (P < .001, by χ2 analysis). Similar results were observed in the MNt assay. The magnitude of change in serum antibody level (as measured by seroconversion factor) and the seroconversion rate were also consistently greater in the adjuvant group (table 3).

Antibody responses to the heterologous but related influenza A/Hong Kong/1073/99 (H9N2) virus strain were also measured. No subject had a preexisting serum HAI antibody level >8, and none had serum MNt antibody activity of ⩾20. In contrast with results obtained using homologous antigen for testing, responses to this heterologous antigen occurred in a minority of subjects by all measures of immunogenicity, and no subject achieved a serum HAI antibody level of 32. Seroconversion rates for each vaccine group are shown in table 4. Neither dose nor presence of adjuvant had significant effects on immune responses to this heterologous antigen.

Table 4
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Table 4

Seroconversion rates (95% CI) to the heterologous H9N2 strain, influenza A/Hong Kong/1073/99, by vaccine type and dose.

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Discussion

There are a number of strategies for developing immunogenic vaccines against avian influenza strains that are potential pandemic threats. These include the use of standard egg-grown vaccines containing a higher-than-usual (>15-µg) dose of viral HA, the addition of adjuvants, the production of vaccine antigen in cell-based substrates, and the use of DNA vaccines [5, 15, 16]. We evaluated an egg-grown influenza A/H9N2 virus vaccine containing doses of 3.75–30 µg of HA, with and without MF59 adjuvant. The MF59-adjuvanted vaccine was well tolerated, and vaccines containing as little as 3.75 µg of viral HA were highly immunogenic. In addition, use of the adjuvant elicited immune responses after a single dose of vaccine that was comparable with responses observed after 2 doses of nonadjuvanted vaccine. There was increased local reactogenicity associated with the MF59 adjuvant that was similar to that observed in other studies of MF59-adjuvanted influenza virus vaccines [17, 18].

Several recent studies have assessed the safety and immunogenicity of different doses of inactivated H5 and H9 influenza virus vaccines. Thus far, the H5 vaccines have been less immunogenic and may require higher doses and/or adjuvant approaches. Only ∼50% of subjects developed neutralizing antibody responses after receiving two 90-µg doses of a recombinant baculovirus-expressed H5 HA vaccine [19] or two 90-µg doses of an inactivated subvirion H5 virus vaccine [20]. Immune responses were less common in cohorts receiving lower vaccine doses. Addition of MF59 adjuvant to a surface-antigen influenza A/H5N3 vaccine significantly improved immune responses following 2 doses of vaccine, compared with nonadjuvanted preparations [17].

Two studies have evaluated responses to influenza A/Hong Kong/1073/99 (H9N2) virus strain vaccines. Stephenson et al. [11] evaluated the safety and immunogenicity of 2 doses, administered 21 days apart, of 7.5, 15, or 30 µg of HA of whole-virus or subunit vaccines without adjuvant in healthy adults. The vaccines were well tolerated. Seroconversion rates were similar among recipients of whole-virus and subunit virus vaccines, but persons born after 1968 were less likely to respond to either vaccine. Hehme et al. [21] also observed that 1 or 2 doses of 15 µg of a whole-virus influenza A/Hong Kong/1073/99 (H9N2) strain vaccine, as well as lower doses (1.9–7.5 µg), when combined with alum adjuvant, were immunogenic in persons 18–60 years of age.

The findings in our study were similar to those of Hehme et al. [21], in that immune responses were observed among recipients of nonadjuvanted vaccine. All subjects in our study were born in 1970 or later, so we were not able to evaluate whether the subjects' year of birth influenced response rates (as was suggested by Stephenson et al. [11]). The use of adjuvant was associated with improved immune responses, even when using vaccine with one-fourth the standard HA content. The lack of a dose response is also similar to the observations of Hehme et al. [21], who failed to observe a clear dose response following administration of an alum-adjuvanted influenza A/H9N2 vaccine. It is possible that even lower (<3.75-µg) vaccine doses could lead to acceptable immune responses. The higher microneutralization geometric mean titer in the 30-µg dose group after 2 doses suggests a possible dose response among the nonadjuvanted vaccine groups, but more subjects per study group—and possibly even higher antigen doses—would be needed to clearly demonstrate this effect.

Stephenson et al. [22] noted cross-reactive serum antibody responses to H5 variants following administration of 2 or 3 doses of MF59-adjuvanted H5N3 vaccine. For this reason, we examined the serologic responses to an influenza A/H9N2 virus strain from the other major circulating lineage that infects humans [9]. Immune responses to the heterologous influenza A/Hong Kong/1073/99 (H9N2) virus strain were infrequent in the current study. Of note, however, is that the administration of an inactivated, alum-adjuvanted influenza A/chicken/Hong Kong/G9/1997 virus strain vaccine to BALB/c mice also failed to elicit detectable cross-reactive neutralizing antibodies to the influenza A/Hong Kong/1073/99 (H9N2) virus strain, but partial protection against challenge with the influenza A/Hong Kong/1073/99 (H9N2) virus strain was observed [23].

The limitations of this study are that it was not designed to be able to detect modest differences in dose-related immune responses and that the lack of an apparent dose response may be due to insufficient sample size. In addition, only young, healthy adults were included in the study, so the effects of age or health status on immune responses were not evaluated.

In summary, the combination of MF59 adjuvant with a purified surface antigen vaccine was associated with improved immune responses to an avian influenza A/H9N2 virus. The adjuvanted vaccine was immunogenic even after a single dose, raising the possibility that a 1-dose vaccination strategy may be attainable with the use of adjuvanted vaccine. Expanded studies are planned to more precisely define the immune responses to these vaccines in this population and in older adults.

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Acknowledgments

We thank Dr. Elena Govorkova (St. Jude Children's Research Hospital; Memphis, TN) for provision of inactivated H9N2 antigen for use in HAI assays; Drs. Patricia Winokur, Rebecca Brady, and Richard Hamill for serving on the Safety Monitoring Committee; Annette Nagel (study coordinator) and other clinical staff of the VRPRU; Diane Nino and technical staff for performance of HAI serologic assays; Heather Hill, Robin Cessna, and Ken Wilkins at EMMES Corporation for contributions to data management and analysis; and the National Institute of Allergy and Infectious Diseases Influenza Team (Sonnie Kim, Jean Hu-Primmer, Lydia Falk, and Linda Lambert).

Financial support. National Institutes of Health (contract N01-AI-30039).

Potential conflicts of interest. All authors: no conflicts.

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Footnotes

  • Received April 28, 2006.
  • Accepted July 31, 2006.
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  1. Clin Infect Dis. (2006) 43 (9): 1135-1142. doi: 10.1086/508174
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