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Background Widespread infections of avian species with avian influenza H5N1 virus and its limited spread to humans suggest that the virus has the potential to cause a human influenza pandemic. An urgent need exists for an H5N1 vaccine that is effective against divergent strains of H5N1 virus.
Methods In a randomized, dose-escalation, phase 1 and 2 study involving six subgroups, we investigated the safety of an H5N1 whole-virus vaccine produced on Vero cell cultures and determined its ability to induce antibodies capable of neutralizing various H5N1 strains. In two visits 21 days apart, 275 volunteers between the ages of 18 and 45 years received two doses of vaccine that each contained 3.75 µg, 7.5 µg, 15 µg, or 30 µg of hemagglutinin antigen with alum adjuvant or 7.5 µg or 15 µg of hemagglutinin antigen without adjuvant. Serologic analysis was performed at baseline and on days 21 and 42.
Results The vaccine induced a neutralizing immune response not only against the clade 1 (A/Vietnam/1203/2004) virus strain but also against the clade 2 and 3 strains. The use of adjuvants did not improve the antibody response. Maximum responses to the vaccine strain were obtained with formulations containing 7.5 µg and 15 µg of hemagglutinin antigen without adjuvant. Mild pain at the injection site (in 9 to 27% of subjects) and headache (in 6 to 31% of subjects) were the most common adverse events identified for all vaccine formulations.
Conclusions A two-dose vaccine regimen of either 7.5 µg or 15 µg of hemagglutinin antigen without adjuvant induced neutralizing antibodies against diverse H5N1 virus strains in a high percentage of subjects, suggesting that this may be a useful H5N1 vaccine. (ClinicalTrials.gov number, NCT00349141
[ClinicalTrials.gov]
.)
All these studies were carried out with vaccines manufactured by conventional methods (i.e., with the use of embryonated chicken eggs and modified, attenuated reassortant viruses produced by reverse genetics).7 We have devised a strategy for the development of an H5N1 vaccine that involves the use of a wild-type virus (i.e., the strain circulating in nature) grown in a Vero cell culture. This strategy has the advantage that the lead time for pandemic vaccine production can be reduced, since the generation of attenuated reassortants is not required, although the requirement for the use of enhanced biosafety level 3 (BSL-3) facilities for such a strategy is a relative drawback. In addition, cell culture provides a robust manufacturing platform that eliminates dependence on embryonated chicken eggs, which would be an advantage in the event of limited availability of such eggs during a pandemic caused by a highly pathogenic avian virus. This technique was used to develop a whole-virus vaccine that was highly immunogenic in animal models.8 We report on the safety and immunogenicity of this vaccine, using formulations with and without alum adjuvant.
Methods
Study Design and Objective
From June 2006 through September 2006, we enrolled a total of 284 men and women between the ages of 18 and 45 years in a randomized, partially blinded (between groups) clinical trial at three sites: one in Austria and two in Singapore. The study was designed by its sponsor, Baxter. Data were collected by the investigators and were held and analyzed by Baxter. The manuscript was written by a subgroup of industry and academic authors; all authors contributed to the content, had full access to the data, and vouch for the completeness and accuracy of the data and data analysis.
The appropriate local review boards and ethics committees approved the protocol for the study, which was conducted in compliance with Good Clinical Practice guidelines and the provisions of the Declaration of Helsinki. The study investigators were unaware of assignments to study groups. (For details of the study design, see the Supplementary Appendix, available with the full text of this article at www.nejm.org.)
The objective was to identify the immunogenicity and safety of various doses of inactivated H5N1 whole-virus vaccine in formulations with and without adjuvant. The primary immunogenicity outcome was the number of subjects with hemagglutination-inhibition and neutralizing antibodies to the vaccine strain (A/Vietnam/1203/2004) 21 days after the first and second doses of vaccine. The primary safety outcome was any systemic reaction after the first and second doses.
Vaccine
The monovalent avian influenza H5N1 whole-virus vaccine (Baxter) was produced with the wild-type strain A/Vietnam/1203/2004, which was obtained from the Centers for Disease Control and Prevention and was inactivated with formalin and ultraviolet light. The vaccine was manufactured in Vero cell culture in an enhanced BSL-3 facility (as required for wild-type H5N1 virus), as described previously.9
Randomization and Follow-up
Subjects were eligible to participate if they were clinically healthy, understood the study procedures, provided written informed consent, and agreed to keep a daily record of symptoms. Women were required to have a negative pregnancy test at screening and before each vaccination.
Subjects were recruited in three study cohorts in a dose-escalating manner and were randomly assigned to receive two 0.5-ml injections into the deltoid muscle at an interval of 21 days (range, 19 to 23) with an H5N1 whole-virus formulation containing 3.75 µg, 7.5 µg, 15 µg, or 30 µg of hemagglutinin antigen with a 0.2% alum adjuvant or 7.5 µg or 15 µg of hemagglutinin antigen without adjuvant. There was no placebo group. Subjects and investigators were unaware of the dose of vaccine administered within the subgroups (Figure 1, and the Supplementary Appendix). Blood samples were taken for serologic testing before the first dose of vaccine and on day 21 after the first and second doses.
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Assays
We evaluated all immunogenicity outcomes against the influenza-virus strain used in the vaccine (A/Vietnam/1203/2004) according to hemagglutination-inhibition and virus-neutralization assays. To assess cross-reactivity of antibodies, all assays were also conducted with known related influenza strains — for example, an original prototype clade 3 strain (A/Hong Kong/156/1997) and a clade 2 strain (A/Indonesia/05/2005).
Using a hemagglutination-inhibition or virus-neutralization assay, we investigated secondary immunogenicity outcomes by analyzing the antibody response 21 days after the first and second doses of vaccine; the increase in the antibody response 21 days after the first and second doses, as compared with baseline; and the number of subjects with seroconversion (which we defined as a minimum increase by a factor of 4 in the titer) 21 days after the first and second doses, as compared with baseline.
The hemagglutination-inhibition assay is the standard test for detection of antibodies against influenza after infection or vaccination. However, this assay may be insensitive for the detection of anti-H5 antibodies.10,11 For this reason, immunogenicity analyses focused on a determination of functional neutralizing-antibody responses. Since most licensing authorities typically request data regarding hemagglutination-inhibition assays or single radial hemolysis, these determinations are also reported but only for the vaccine virus strain A/Vietnam/1203/2004. (For details on hemagglutination-inhibition and virus-neutralization assays and single radial hemolysis,12,13,14 see the Supplementary Appendix.)
Statistical Analysis
The protocol called for the recruitment of 45 subjects per study group. With this number of subjects, the 95% confidence interval for the percentage of subjects with an antibody response that was associated with protection did not extend more than 15% from the observed rate, assuming a seroprotection rate of approximately 80%.
We used the likelihood-ratio chi-square test to compare the number of subjects with local or systemic reactions within 7 days after vaccination among the various vaccine formulations. For binary variables (i.e., seroprotection and seroconversion), response rates and 95% confidence intervals were computed for each strain and time point. The confidence intervals were interpreted in a descriptive manner, and no adjustment for multiplicity was made.15
In addition, for the log-transformed values of virus-neutralization titers and single radial hemolysis, a longitudinal analysis was performed within a repeated mixed-model framework of analysis of covariance. Changes from baseline were analyzed, accounting for the fixed effects of vaccine formulation, day, sex, age, baseline titer, interaction between the vaccine formulation and day, and random effects for subjects. Vaccine formulations without adjuvant were compared with formulations with adjuvant within this model. Comparisons were also made between groups receiving 7.5 µg and 15 µg of hemagglutinin antigen without adjuvant. We calculated the proportion of subjects with a virus-neutralization titer of 1:20 or more and that of subjects with results of 25 mm2 or more on single radial hemolysis, using a generalized linear model with repeated measurements and the general-estimating-equations method (see the Supplementary Appendix).
Results
Study Population
A total of 275 subjects between the ages of 18 and 45 years received the first dose of vaccine, and 257 received the second dose. All vaccinated subjects were included in the safety analysis. Two subjects who initially gave their consent withdrew from the study because of nonserious adverse events, including four events in one subject (chills, fatigue, malaise, and insomnia) and one event in the second subject (papular rash); the majority of these symptoms abated within 24 hours. Immunogenicity data were available for 258 subjects for the first dose of vaccine and for 249 subjects for the second dose of vaccine.
Safety
The rates of occurrence of injection-site and systemic reactions during the first 7 days after each dose of vaccine are presented in Table 1. No serious, vaccine-related adverse events were recorded. There were two serious adverse events recorded in two subjects: hospitalization due to a contusion of the left foot and hospitalization for an elective abortion.
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There were no significant differences between the vaccine formulations with respect to local reactions after the first dose and the second dose of vaccine (P=0.32 and P=0.97, respectively, for all comparisons). With respect to systemic reactions, a slight difference was observed between the vaccine formulations after the first dose of vaccine (P=0.01), a finding that was largely due to an unexpectedly low rate of headache observed in the group receiving the 30-µg formulation with adjuvant. No difference was shown regarding systemic reactions after the second dose of vaccine (P=0.15).
Immune Response
At 21 days after the first and second doses, functional neutralizing antibodies against strain A/Vietnam/1203/2004 were detected in patients receiving any of the six formulations. Table 2 shows the rates of response in subjects with a virus-neutralization titer of 1:20 or more, and Table 3 shows the geometric mean increase (GMI) of the titer from baseline and the percentage of seroconversion. Numerically, the formulations without adjuvant induced the highest rates of a virus-neutralization titer of 1:20 or more after the first dose (40.5% and 39.5% for 7.5 µg and 15 µg without adjuvant, respectively) and the second dose (76.2% and 70.7% for 7.5 µg and 15 µg without adjuvant, respectively) (Table 2). Similar results were obtained with respect to GMI (Table 3), since the highest GMIs were obtained for the formulations without adjuvant (5.3 and 5.7 for 7.5 µg and 15 µg without adjuvant, respectively) (Table 3). Among subjects with seroconversion (an increase in the titer by a factor of at least 4 after immunization), the highest rates of response were again seen in subjects who received a 7.5-µg or 15-µg formulation without adjuvant (69.0% and 68.3%, respectively) (Table 3).
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40) and single radial hemolysis (i.e., the proportion of subjects with an area of 25 m2 on single radial hemolysis). Numerically, the formulations without adjuvant again were more immunogenic than those with adjuvant. On single radial hemolysis, the percentage of seroprotection 21 days after the second dose of vaccine without adjuvant was 78.6% for the 7.5-µg dose and 61.0% for the 15-µg dose. Single radial hemolysis for H5N1 antibodies appeared to be more sensitive than hemagglutination-inhibition assay, since the equivalent values for hemagglutination-inhibition assay were 47.6% and 26.8%, respectively.
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Cross-Neutralization
The 7.5-µg and 15-µg formulations without adjuvant showed high levels of cross-reactivity against the A/Hong Kong strain (76.2% and 78.0%, respectively, with a neutralizing titer of 1:20) (Table 2). The responses against the clade 2 strain were somewhat lower (with rates of a virus-neutralization titer of 1:20 of 45.2% and 36.6% for the 7.5-µg and 15-µg formulations without adjuvant, respectively) (Table 2).
We also analyzed the virus-neutralization response to the heterologous strains using the mixed model. Results were similar to those for the homologous strain. Formulations without adjuvant elicited significantly higher immune responses than those with adjuvant. Antibody titers increased significantly from baseline, independently of the vaccine dose (Table 4, and Tables 3 and 4 in the Supplementary Appendix).
The reverse cumulative distribution curves for antibody titers after the first and second doses of vaccine against all three strains support the finding of higher immunogenicity from the formulations without adjuvant (Figure 2). Analysis of rates of seroprotection with homologous and heterologous immune responses showed results that were consistent with those obtained by direct analysis of values of virus-neutralization titers and single radial hemolysis (Tables 6 and 7 in the Supplementary Appendix).
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It has been reported that whole-virus trivalent influenza vaccines are more immunogenic than subvirion vaccines but are also more prone to cause adverse reactions.5 In our study, a monovalent whole-virus H5N1 vaccine had a side-effect profile similar to that of subvirion H5N1 formulations described previously.2,3,16 Most important, the low rate of fever among subjects in our study (2 to 7%) compares favorably with that reported both for subvirion H5N1 vaccines and for an egg-derived whole-virus H5N1 vaccine with adjuvant.2,3,6,16 However, it should be noted that reporting systems and characteristics of the subjects differ among the various studies.
With respect to immunogenicity, the highest neutralizing-antibody response after the second dose of vaccine (76.2%) was obtained with the 7.5-µg formulation without adjuvant, which was equivalent to a rate of seroconversion of 69.0% and represented an increase by a factor of 4 or more in the neutralization titer after two doses of vaccine (Table 2 and Table 3). These data are also similar to the levels of immunogenicity reported in a study of an egg-derived whole-virus H5N1 vaccine, in which 96% of subjects who received two doses of 5-µg or 10-µg formulations had a neutralization titer of 1:20 or more,6 although differences in assay systems must be taken into account in making such direct comparisons.
Lower rates of seroprotection and seroconversion (as defined in the guidelines of the Committee for Proprietary Medicinal Products17) were obtained with the hemagglutination-inhibition assay than with the virus-neutralization assay, which supports the finding that the hemagglutination-inhibition assay is less sensitive for detection of anti-H5 antibodies, as reported previously.10,11 In our study, single radial hemolysis, which is considered to have a sensitivity equivalent to that of the hemagglutination-inhibition assay for seasonal influenza strains,18 was shown to be more sensitive than the hemagglutination-inhibition assay for H5N1.
The lack of enhancement of vaccine immunogenicity by the use of alum adjuvant at the doses studied here was consistent with data from a previous study, which showed that no effect of alum adjuvant was seen with a 15-µg dose of subvirion vaccine, and a 7.5-µg formulation without alum was more immunogenic than the formulation with adjuvant.3 In the previous study, an enhanced immune response with the use of alum was seen only with the 30-µg formulation. We did not investigate this dose without alum in our study.
However, other studies have described substantial positive effects of other adjuvants on H5N1 immunogenicity. The use of an oil-in-water–based emulsion in a 3.8-µg dose of split-virus vaccine resulted in 82% seroconversion, as compared with 4% seroconversion without adjuvant.16 The addition of another oil-in-water–based adjuvant (MF-59) to an H5N3 vaccine was also associated with a substantial increase in antibody response.19
Our data also showed that the whole-virus clade 1–based vaccine can induce a substantial cross-neutralizing response against clade 2 and clade 3 strains. The results described in Table 2 are encouraging: after two doses of 7.5-µg of the formulation without adjuvant, the proportions of subjects with neutralizing titers of 1:20 or more were 45% of those immunized against the clade 2 Indonesia strain and 76% of those immunized against the clade 3 Hong Kong strain. However, there is no available evidence to indicate which neutralizing titer is sufficient to confer protection. Most studies of H5N1 split-virus and whole-virus vaccines have not described attempts to determine the cross-reactivity of antibodies to other H5N1 virus strains. However, a recent study of a novel split-virus vaccine with adjuvant also showed high levels of cross-neutralization against a clade 2 strain.16 In addition, in a study involving 15 subjects, two doses of an H5N3 vaccine with MF-59 as adjuvant induced intermediate levels of cross-reactivity to antigenically distinct H5N1 strains, and three doses induced high levels of cross-reactivity.20
The apparent absence of a dose–response relationship in our study may be surprising. However, it is in agreement with a number of studies of vaccine for pandemic influenza. Leroux-Roels et al. reported no relationship between the dose of antigen and the neutralizing-antibody response for H5N1 formulations with adjuvant,16 and there appeared to be an inverse dose–response relationship with respect to responses to the clade 2 strain. A number of other studies involving other pandemic-strain vaccines — H9N2,21 H5N3,19 and H2N222 — have shown no dose–response relationship or even a reduced response at higher doses. The reasons for these findings are unclear, but at least with respect to vaccines with adjuvant, it has been speculated that the ratio of adjuvant to antigen may be critical in determining the immune-enhancing effect rather than the antigen concentration alone.19 For other viral vaccines, particularly those with soluble proteins, it has been reported that there are distinct dose–response relationships for induction of various cytokines. In many studies, responses similar to those mediated by type 2 helper T cells have been elicited at low doses of vaccine, and responses similar to those mediated by type 1 helper T cells have been elicited at higher doses.23 Further studies focusing on T-cell responses will be required to investigate this phenomenon. In addition, these studies will be extended by the use of antigen doses lower than 3.75 µg to confirm and extend the results obtained in our study.
Our study provides initial safety and immunogenicity data for a whole-virus H5N1 vaccine produced on Vero cell culture. It also shows that a broadly reactive immune response to clade 2 and clade 3 of H5N1 virus can be obtained with the use of a low-dose clade 1 vaccine without adjuvant. Since we observed no significant dose–response relationship, the 7.5-µg formulation without adjuvant has been chosen for further development.
Supported by Baxter.
Drs. Ehrlich, Berezuk, Fritsch, Löw-Baselli, Vartian, Bobrovsky, Pavlova, Pöllabauer, Kistner, and Barrett report being employed by Baxter and having an equity interest in the company; Drs. Kistner and Barrett, holding patents on influenza vaccines derived from Vero cell cultures; Dr. Müller, receiving consulting and lecture fees and grant support (to the Medical University of Vienna) from Baxter; Dr. Tambyah, serving as a member of the Asia–Pacific Advisory Committee on Influenza and receiving consulting fees from Baxter, Merlion Pharmaceuticals, and Janssen-Cilag, lecture fees from Pfizer, Wyeth and IBC Asia, and grant support from Baxter and Interimmune; and Dr. Montomoli, receiving lecture fees and grant support (to the University of Siena) from Baxter. No other potential conflict of interest relevant to this article was reported.
This study is dedicated to the memory of Dr. Michel Canavaggio, head of bioscience research and development at Baxter and a great supporter of this project, who died in July 2006, about 6 weeks after the initiation of the study.
We thank the following members of the Baxter research and development team for their critical role in this study: L. Grillberger, K. Howard, W. Mundt, M. Reiter, H. Savidis-Dacho, C. Tauer, and W. Wodal; N. Cox and S. Klimov of the Centers for Disease Control and Prevention for providing the H5N1 viruses; and J. Wood of the National Institute for Biological Standards and Control for providing the reference standards.
Source Information
From the Department of Global Research and Development, Baxter BioScience (H.J.E., G.B., S.F., A.L.-B., N.V., R.B., B.G.P., E.M.P., O.K., P.N.B.), and the Department of Clinical Pharmacology, Medical University of Vienna, Vienna General Hospital (M.M., C.J.) — both in Vienna; Changi General Hospital (H.M.L.O.) and the National University of Singapore and National University Hospital (P.A.T., D.F.) — all in Singapore; and the University of Siena, Siena, Italy (E.M.).
Drs. Ehrlich and Müller contributed equally to this article.
Address reprint requests to Dr. Müller at the Department of Clinical Pharmacology, Medical University of Vienna, Vienna General Hospital (AKH), Währinger Gürtel 18-20, 1090 Vienna, Austria, or at markus.mueller{at}meduniwien.ac.at.
References
In addition to the authors, the following investigators contributed to the trial: Data Monitoring and Safety Board: E. Marth, R. Konior, F. Sonnenburg; Baxter Clinical Study Team: K. Birthistle, T. Dvorak, S. Geyer, M. Kraft, M.C. Leitgeb, F. Maritsch, L. Phillipson, E. Robotka. Austria: Medical University of Vienna, Vienna General Hospital (Study Center Management), Vienna: A. Abrahim, M. Bauer, M. Brunner, A. Cornea, C. Drucker, Z. Erdogan, J. Griss, B. Heinisch, F. Kovar, E. Lackner, C. Lambers, O. Langer, I. Leitner, C. Marsik, W. Poeppl, M. Popovic, R. Sauermann, R. Schaberl, G. Sodeck, C. Thallinger, F. Traunmueller, C. Wagner, M. Zeitlinger; Singapore: Changi General Hospital (Study Center Management): S.K. Chua, S. Chuin, R. Fong, A.S. Foo, A.G. Koh, P.K. Lim, S.Y. Yap, L.H. Yew; National University Hospital (Study Center Management): J.W.L. Goh, L.Y. Hsu, C.W.P. Loke, J.Y.C. Ng, E.L. Toh, P. Weatherill, Y.P. Zhou.
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