Abstract
Influenza A(H5N1) vaccination strategies that improve the speed of the immunological response and cross-clade protection are desired. We compared the immunogenicity of a single 15-μg or 90-μg dose of A/H5N1/Indonesia/05/05 (clade 2) vaccine in adults who were previously primed with A/H5N1/Vietnam/1203/2004 (clade 1) vaccine. High-dose vaccine resulted in significantly higher titers to both clade 1 and 2 antigens. Clade 2 titers were unaffected by the previous dose of clade 1 vaccine. Low-dose priming with a mismatched pandemic influenza A(H5N1) vaccine would improve the rapidity, magnitude, and cross-reactivity of the immunological response following a single high-dose, unadjuvanted, pandemic vaccine.
Keywords: H5N1, influenza, vaccine, prime-boost
In 1997, avian influenza A(H5N1) emerged, rapidly spread through avian populations, and caused small numbers of highly lethal human infections. The virus continues to smolder and has evolved genetically to give rise to a number of virus clades and subclades that are antigenically diverse [1].
Vaccination remains a critical component of pandemic planning and management; however, many avian strains of influenza virus are poorly immunogenic. Even adjuvanted influenza A(H5N1) vaccines require 2 doses to generate seroprotection rates of ≥70% [2]. Yet pandemics occur at unpredicted intervals and evolve quickly, making 2 doses impractical.
A homologous influenza A(H5N1) booster vaccine given months to years after the priming doses allows brisk hemagglutination inhibition (HI) and a rapid neutralizing antibody response with higher titers than those seen after the primary 2-dose series and more cross-clade reactivity to other strains of influenza A(H5N1) [3, 4]. Priming with 2 doses of a clade 3 influenza A(H5N1) vaccine, followed by late administration of a single heterologous high dose of clade 1 vaccine, resulted in higher clade 1 titers of antibody than seen after 2 doses in previously unprimed subjects [5]. In this study, further data on the effects of cross-protection achieved by late administration of a single high-dose or low-dose heterologous booster vaccine are provided.
MATERIALS AND METHODS
Study Design
This was a randomized, double-blinded, multicenter, phase 1/2 study evaluating a single heterologous high or low dose of inactivated A/H5N1/Indonesia/05/05 (clade 2) vaccine given 1.4–3.7 years after receipt of priming clade 1 vaccine (NCT00680069). Eligible subjects were healthy adults aged ≥19 years who had participated previously in influenza A(H5N1) studies wherein they received ≥2 doses of clade 1 virus A/H5N1/Vietnam/1203/2004 vaccine (manufactured by Sanofi Pasteur or Novartis Vaccines) or placebo, including influenza A(H5N1) vaccines with and vaccines without Alhydrogel or MF59 and vaccine doses that ranged from 3.75 to 90 μg of hemagglutinin (HA). The study was approved by the institutional review boards at all 7 institutions.
Subjects provided written informed consent and were randomized to receive a single 0.5-mL intramuscular dose of 15 or 90 μg of inactivated, monovalent, subvirion, reverse genetically derived A/H5N1/Indonesia/05/05 (clade 2) vaccine (Sanofi Pasteur). Influenza A(H5N1)–naive subjects were randomized to receive 2 doses of 15 or 90 μg of clade 2 influenza A(H5N1) vaccine, separated by 28 days. Subjects were stratified by age (19–64 years and ≥65 years) and previous priming dose (dichotomized as low dose [defined as 3.75, 7.5, or 15 μg] and high dose [defined as 30, 45, or 90 μg]) of clade 1 (A/VN/1203/04) vaccine.
Subjects were monitored for local and systemic reactions for 8 days after vaccination, using a memory aid. Unsolicited adverse events (AEs) were captured through day 28 for previously vaccinated subjects and through day 56 for naive subjects. Severe AEs were captured through day 180 (or day 208 for the naive population). AEs were reported using a 4-point scale (absent, mild, moderate, and severe). Serum samples were obtained on days 0, 28, 56 (from the naive population only), and 180.
Laboratory Assays
Serum HI and plaque neutralization antibody titers were measured against the A/Vietnam/1203/2004 (clade 1) and A/Indonesia/05/05 H5N1 (clade 2) viruses, as previously described [6]. Serum samples were tested at a starting dilution of 1:10. Negative samples were assigned a titer of 1:5. Each assay was performed in duplicate for each specified time point by Southern Research (Birmingham, Alabama). The geometric mean titer (GMT) of duplicates was calculated at each time point and used in analysis.
Statistical Analysis
The proportion of subjects with a postvaccination titer of ≥1:40, the proportion of subjects with a ≥4-fold increase from prevaccination to a titer of ≥1:40 (seroconversions), and the GMT were calculated, with 95% confidence intervals (CIs), on day 0, day 28, and 6 months after receipt of vaccine. The relationship between the proportion of responders and covariates, including previous clade 1 dosage, receipt of vaccine with adjuvant, subject age category (19–64 or ≥65 years), clade 2 dosage, peak clade 1 antibody response in previous trial, elapsed time since last dose of priming vaccine, and receipt of 2007/2008 and/or 2008/2009 seasonal influenza vaccines, was examined using logistic or linear regression models. The immunogenicity analysis cohort includes subjects who received at least 1 dose of vaccine and contributed both prevaccination and postvaccination blood samples for testing.
RESULTS
Of 1704 previously vaccinated subjects, 517 were enrolled. Ethnicity and sex did not vary significantly across groups. The mean age for subjects was 54.8 years. The study design and subjects included for analysis are presented in Supplementary Figure 1.
Immunologic Responses After Low-Dose Versus High-Dose Clade 2 Vaccine in Subjects Previously Primed With Clade 1 Vaccine or Naive Subjects
The effects of the clade 2 booster dose for 480 clade 1 vaccine–primed subjects and of the 2 clade 2 vaccine doses for 32 naive subjects were assessed (Table 1). Previously vaccinated recipients of the high-dose clade 2 booster generated a higher clade 2 HI GMT 28 days after vaccination (126.7 [95% CI, 103.3–155.5] vs 29.4 [95% CI, 23.4–36.9]; P < .0001). Similarly, the proportion of individuals who demonstrated HI seroconversion was greater in the high-dose group (78%; 95% CI, 71–82), compared with the low-dose group (41%; 95% CI, 35–48; P < .0001; Supplementary Figure 2).
Table 1.
Hemagglutination Inhibition Assays of Clade 1 or Clade 2 Influenza Virus Antigen for Clade 1 Vaccine–Primed Subjects and Naive Subjects
| Subjects, Clade 2 Vaccine Dose | Day 0, GMT (95% CI) |
Day 28 After Dose 1, GMT (95% CI) |
Day 28 After Dose 2, GMT (95% CI)a |
6 mo After Last Dose, GMT (95% CI) |
||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Subjects, No. | Clade 1 Antigen | Clade 2 Antigen | Subjects, No. | Clade 1 Antigen | Clade 2 Antigen | Subjects, No. | Clade 1 Antigen | Clade 2 Antigen | Subjects, No. | Clade 1 Antigen | Clade 2 Antigen | |
| Overall | ||||||||||||
| Prior vaccinees | ||||||||||||
| 15 μg | 240b | 7.4 (6.6–8.4) | 5.3 (5.1–5.5) | 241 | 29.4 (23.4–36.9) | 28.0 (21.6–36.1) | … | … | … | 238 | 10.9 (9.4–12.6) | 10.0 (8.7–11.5) |
| 90 μg | 239 | 7.3 (6.4–8.2) | 5.2 (5.0–5.4) | 238c | 126.7 (103.3–155.5) | 130.2 (102.4–165.6) | … | … | … | 237 | 15.3 (13.0–18.0) | 13.7 (11.8–15.9) |
| Naive subjects | ||||||||||||
| 15 μg | 14 | 5.0 | 5.0 | 14 | 7.8 (4.0–15.3) | 9.1 (3.8–21.7) | 14 | 7.4 (4.1–13.4) | 14.9 (5.0–44.2) | 14 | 6.2 (4.1–9.6) | 7.2 (4.5–11.8) |
| 90 μg | 18 | 6.8 (4.7–9.8) | 5.2 (4.8–5.6) | 18 | 9.8 (6.0–16.2) | 8.7 (5.4–14.2) | 17 | 8.3 (5.0–13.8) | 20.4 (8.5–48.8) | 17 | 5.8 (4.7–7.1) | 9.6 (5/9–15.6) |
| By subgroup | ||||||||||||
| Prior vaccinees | ||||||||||||
| Low dose, age 19–64 y | ||||||||||||
| 15 μg | 56 | 6.0 (5.0–7.2) | 5.1 (4.9–5.2) | 56 | 36.9 (.36–.64) | 42.8 (23.5–78.1) | … | … | … | 55 | 11.1 (8.0–15.5) | 10.7 (7.8–14.5) |
| 90 μg | 59 | 6.0 (5.1–7.2) | 5.0 | 59 | 141.4 (94.9–210.7) | 195.4 (127.4–299.6) | … | … | … | 59 | 13.7 (9.9–18.9) | 13.0 (9.9–17.2) |
| High dose, age 19–64 y | ||||||||||||
| 15 μg | 80d | 6.2 (5.4–7.3) | 5.0 | 81 | 30.3 (20.4–44.9) | 29.9 (18.9–47.3) | … | … | … | 79 | 9.2 (7.2–11.7) | 9.6 (7.5–12.3) |
| 90 μg | 79 | 7.9 (6.2–10.1) | 5.3 (4.9–5.9) | 79 | 180.9 (128.4–254.9) | 157.2 (101.4–243.7) | … | … | … | 77 | 15.9 (11.7–21.6) | 13.4 (10.1–17.8) |
| Low dose, age ≥65 y | ||||||||||||
| 15 μg | 52 | 8.2 (6.2–10.9) | 5.3 (4.9–5.9) | 52 | 17.3 (11.5–26.0) | 13.3 (8.9–19.8) | … | … | … | 52 | 8.6 (6.6–11.3) | 7.1 (5.8–8.7) |
| 90 μg | 50 | 8.1 (6.2–10.7) | 5.2 (4.8–5.5) | 50e | 70.6 (44.7–111.4) | 72.1 (42.0–123.7) | … | … | … | 50 | 15.8 (11.3–22.1) | 13.6 (10.0–18.4) |
| High dose, age ≥65 y | ||||||||||||
| 15 μg | 52 | 11.2 (7.9–15.8) | 6.0 (5.1–7.1) | 52 | 37.4 (22.7–61.8) | 33.4 (19.0–58.8) | … | … | … | 52 | 17.6 (12.5–24.8) | 14.3 (10.2–20.1) |
| 90 μg | 51 | 7.1 (5.5–9.1) | 5.2 (4.8–5.5) | 50 | 113.9 (71.1–182.5) | 108.6 (63.6–185.5) | … | … | … | 51 | 16.1 (11.4–22.8) | 15.1 (10.8–21.2) |
| Naive subjects | ||||||||||||
| Age 19–64 y | ||||||||||||
| 15 μg | 11 | 5.0 | 5.0 | 11 | 6.2 (3.8–10.2) | 7.5 (3.0–18.8) | 11 | 6.6 (3.5–12.5) | 13.3 (4.0–44.3) | 11 | 5.2 (4.8–5.5) | 6.2 (4.3–9.0) |
| 90 μg | 12 | 5.6 (4.4–7.2) | 5.0 | 12 | 10.0 (5.1–19.6) | 8.9 (4.6–17.1) | 11 | 7.8 (3.9–15.5) | 13.7 (3.9–47.9) | 11 | 5.7 (4.3–7.5) | 8.0 (4.4–14.8) |
| Age ≥65 y | ||||||||||||
| 15 μg | 3 | 5.0 | 5.0 | 3 | 17.8 (.1–4221.3) | 17.8 (.1–4221.3) | 3 | 11.2 (.3–364.1) | 22.4 (0–14 372.4) | 3 | 12.6 (.2–671.9) | 12.6 (.2–671.9) |
| 90 μg | 6 | 10.0 (3.2–31.6) | 5.6 (4.2–7.6) | 6 | 9.4 (3.3–26.7) | 8.4 (3.1–22.8) | 6 | 9.4 (3.3–26.7) | 42.4 (13.0–138.2) | 6 | 5.9 (3.8–9.3) | 13.3 (4.7–37.7) |
Abbreviations: CI, confidence interval; GMT, geometric mean titer.
a Prior vaccinees did not receive a second dose of vaccine.
b Data are for 241 individuals who were tested in the Clade 2 Antigen Assay.
c Data are for 239 individuals who were tested in the Clade 2 Antigen Assay.
d Data are for 81 individuals who were tested in the Clade 2 Antigen Assay.
e Data are for 51 individuals who were tested in the Clade 2 Antigen Assay.
Clade 1 vaccine–primed subjects who received a single 90-μg dose of clade 2 vaccine had a higher clade 2 HI GMT (130.2; 95% CI, 102.4–165.6) than clade 1 vaccine–naive subjects who received two 90-μg doses of clade 2 vaccine (20.4; 95% CI, 8.5–48.8; P = .0001). Similar trends were seen when HI seroconversion rates were analyzed in clade 1 vaccine–primed subjects (77%; 95% CI, 71–82), compared with naive subjects (41%; 95% CI, 18–67; P = .001). For naive subjects, there was no difference between high-dose and low-dose groups (P = .80). Six months following the last dose, clade 2 HI titers were higher in the 90-μg dose group (P = .002), but there was no difference between primed and naive subjects or between age groups.
Immune Response to Heterologous Clade 1 Antigen Following Clade 2 Vaccination
In clade 1 vaccine–primed subjects, the 90-μg clade 2 vaccine booster dose resulted in a higher clade 1 HI GMT, compared with the 15-μg clade 2 vaccine dose (P < .0001). Additionally, a higher proportion of clade 1 vaccine–primed subjects in the 90-μg clade 2 vaccine dose group had a 4-fold rise to a titer of ≥1:40 of clade 1 HI (74%; 95% CI, 68–80), compared with the 15-μg clade 2 vaccine dose group (38%; 95% CI, 32–45; P < .0001). For both naive subject groups, the clade 1 HI titers were low.
Factors Associated With Immune Responses in Clade 1 Vaccine–Primed Subjects
Linear regression models identified positive associations between day 28 clade 2 log HI titer and clade 2 vaccine dose (P < .0001) and the peak clade 1 antibody response in the previous trial (P = .002). Longer intervals between the priming clade 1 vaccine dose and the clade 2 vaccine booster dose were associated with a higher clade 2 response (P = .003). Subjects aged ≥65 years had lower clade 2 titers than younger subjects (P = .05), and subjects who received 2007/2008 trivalent inactivated influence vaccine (TIV) and/or 2008/2009 TIV had lower clade 2 titers than subjects who did not receive TIV in either of the past 2 years (P = .0001; Table 2). There was no association between clade 2 HI titers and previous clade 1 vaccine dose.
Table 2.
Hemagglutination Inhibition Assay for Clade 2 Influenza Virus Antigen, by Receipt of 2007/2008 and/or 2008/2009 Seasonal Influenza Vaccine and Nonreceipt of Either Seasonal Influenza Vaccine
| Subjects, Clade 2 Vaccine Dose | Day 0, GMT (95% CI) |
Day 28 After Dose 1, GMT (95% CI) |
Day 28 After Dose 2, GMT (95% CI)a |
6 mo After Last Dose, GMT (95% CI) |
||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Subjects, No. | No Seasonal Vaccine | Subjects, No. | Seasonal Vaccine | Subjects, No. | No Seasonal Vaccine | Subjects, No. | Seasonal Vaccine | Subjects, No. | No Seasonal Vaccine | Subjects, No. | Seasonal Vaccine | Subjects, No. | No Seasonal Vaccine | Subjects, No. | Seasonal Vaccine | |
| Prior vaccinees | ||||||||||||||||
| 15 μg | 43 | 5.0 | 196 | 5.4 (5.1–5.6) | 43 | 61.3 (29.5–127.3) | 196 | 23.5 (18.0–30.7) | … | … | … | … | 43 | 14.8 (9.9–22.3) | 193 | 9.2 (7.9–10.6) |
| 90 μg | 50 | 5.2 (4.8–5.8) | 188 | 5.2 (5.0–5.4) | 50 | 396.7 (272.0–578.6) | 188 | 95.7 (72.8–125.7) | … | … | … | … | 48 | 19.2 (13.2–27.7) | 188 | 12.6 (10.8–14.8) |
| Naive subjects | ||||||||||||||||
| 15 μg | 3 | 5.0 | 10 | 5.0 | 3 | 22.4 (0–14372.4) | 10 | 7.3 (3.1–17.3) | 3 | 80.0 (.1–53 207.5) | 10 | 7.8 (2.8–21.7) | 3 | 11.2 (1.3–98.0) | 10 | 6.6 (3.5–12.4) |
| 90 μg | 3 | 5.0 | 15 | 5.2 (4.7–5.8) | 3 | 12.6 (.5–328.0) | 15 | 8.1 (4.8–13.6) | 3 | 100.8 (.1–80 850.7) | 14 | 14.5 (6.8–30.8) | 3 | 17.8 (.5–578.0) | 14 | 8.4 (5.2–13.5) |
Abbreviations: CI, confidence interval; GMT, geometric mean titer.
a Prior vaccinees did not receive a second dose of vaccine.
Microneutralization Assays
All analyses discussed above were performed on results of the microneutralization assays. There were no differences in the trends or statistical significances seen between the HI and microneutralization assays (Supplementary Table 1).
Safety and Reactogenicity
Both high-dose and low-dose clade 2 vaccine were equally well tolerated, with 28% of all subjects reporting solicited systemic symptoms (only 1% were severe) and 48% experiencing mild-to-moderate local reactogenicity events, with only a single report of redness >50 mm in diameter. No differences were seen in subjects who were previously primed. The younger population experienced a greater incidence of reactogenic events than the elderly population, although reactions were mild to moderate in severity.
DISCUSSION
Owing to the continued circulation and ongoing antigenic evolution of influenza A(H5N1) strains, there is concern that one of the influenza A(H5N1) strains could develop pandemic potential. Mathematical modeling has suggested that rapid production and distribution of a vaccine containing antigens from a poorly matched strain could slow disease spread and reduce the number of persons who become ill [7, 8]. A number of studies have analyzed the influenza A(H5N1) cross-clade protection of various vaccines. Vaccines with MF59 and ASO3 adjuvants, but not unadjuvanted vaccine, enhanced cross-clade protection [9–11].
In this study, previous priming with a clade 1 vaccine improved the immune response to the heterologous clade 2 vaccine even when compared to 2 doses of clade 2 vaccine in the naive population. Improved immune response following priming required a high dose of vaccine antigen in the boost regimen. There was no association between clade 2 HI titers and previous clade 1 vaccine dose, which suggests that even low-dose priming afforded significant benefit when followed by a late heterologous booster dose of higher antigenic content. The antibody response declined within 6 months, irrespective of prior priming. Although additional drifted influenza A(H5N1) strains were not tested, these results suggest that heterologously primed individuals might achieve a 70% seroconversion rate to new influenza A(H5N1) clade strains following a single high-dose vaccination.
In this study, antibody titers increased when the interval between priming and booster vaccinations was increased to >3 years. This is consistent with findings by Belshe et al, which demonstrated an improved antibody titer if booster doses were given ≥6 months after the last priming dose of vaccine [11]. Receipt of prior seasonal influenza vaccines was a associated with reduced HI titers. This phenomenon has been seen in previous influenza A(H5N1) vaccine trials and with seasonal influenza vaccine responses [12, 13] The exact immunological mechanism underlying the reduced immune response in those having received seasonal influenza vaccines is unknown, although Nolan et al speculated that the immune response in previously vaccinated individuals may be directed to common although less functional B-cell epitopes [12]. The antigenic distance hypothesis suggests that the immune response to the second virus vaccine is negatively influenced by preexisting cross-reactive antibodies produced in response to the first vaccine and/or may stimulate memory clones that target the first vaccine antigen [14]. Given the cross-reactivity of some of the stem epitopes between H1, H3, and H5 influenza virus strains, it is possible that the H5 vaccine administered to individuals recently vaccinated with seasonal influenza vaccine stimulates preexisting small number of H1 and H3 HA stem plasmablasts rather than a new response to the H5 HA head [15].
Analysis of the effect of adjuvant on subsequent response to a late booster dose was limited by the numbers of subjects in some of the adjuvant groups, although the trends suggested that MF59 improved the immunologic response following the clade 2 boost (data not shown). Previous studies have shown that nonadjuvanted priming doses might reduce late heterologous booster doses that incorporate AS03. Further studies are needed to evaluate the interactions between adjuvanted and nonadjuvanted vaccines.
Small numbers of naive subjects were studied in this trial. HI titers were significantly higher following the second vaccination, but there was no difference between high-dose and low-dose groups. In prior influenza A(H5N1) trials, higher vaccine doses have been associated with a much more robust immune response both in HI and microneutralization assays. The small cohort size may explain this discrepancy, and additional studies are necessary to assess the immunogenicity of clade 2 vaccines more fully.
In conclusion, even low-dose priming with influenza A(H5N1) vaccine that is genetically mismatched to a pandemic strain improved the rapidity of onset, the titer, and the cross-reactivity of the immunological response following a single high-dose, unadjuvanted, pandemic vaccine. Antigen sparing is important in a pandemic setting and would most likely be achieved by the addition of adjuvants, but adjuvanted vaccines may not be appropriate for all populations.
Supplementary Data
Supplementary materials are available at The Journal of Infectious Diseases online (http://jid.oxfordjournals.org). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.
Notes
Acknowledgments. We thank all of our research subjects; the staff at each of the clinical sites; and the clinical and laboratory personnel who supported this study, including Diana Noah, PhD (Southern Research Institute), Jack Stapleton, MD, Nancy Wagner, and Geri Dull (University of Iowa), Rowena Dolor, MD, MHS, Christopher Woods, MD, MPH, Lynn Harrington, Beth Patterson, Lori Hendrickson, and Kathlene Chmielewski (Duke University), Vicki Smith, Jesse LePage, and Michelle Dickey (Cincinnati Children's Hospital), Jill Barrett (EMMES), Lisa Chrisley and Mary Lou Mullen (University of Maryland), Shanda Phillips, Belinda Gayle Johnson, Sandra Yoder, and Michael Rock (Vanderbilt University), Annette Nagel (Baylor College of Medicine), and Irene Graham, Edwin Anderson, Janice Tennant, Tammy Blevins, Mahendra Mandava, and Yinyi Yu (Saint Louis University).
Financial support. This work was supported by the Vaccine and Treatment Evaluation Unit Program (contracts HHSN272200800008C [to the University of Iowa], HHSN272200800002 [to Baylor College of Medicine], HHSN272200800006C [to Cincinnati Children's Hospital], HHSN272200800001C [to the University of Maryland], HHSN272200800007C [to Vanderbilt University], and HHSN272200800003C [to St. Louis University]), the National Center for Advancing Translational Sciences (clinical and translational science awards UL1 TR000442 [to the University of Iowa] and UL1TR000445 [to Vanderbilt University]), the General Clinical Research Center (award M01RR 16500 to the University of Maryland), and the National Center for Research Resources (grant K12-RR-023250 to W. H. C.), National Institutes of Health.
Potential conflicts of interest. E. W. has served as a consultant for Merck and a data safety monitoring board member for Novartis and has received research funding as an investigator from bioCSL, Glaxo Smith Kline, Merck, Novartis, and Pfizer. R. B. has served as a consultant to Medimmune and as a speaker for Medimmune, Sanofi, and Merck. All other authors report no potential conflicts.
All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.
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