Immune Responses of Healthy Subjects to a Single Dose of Intramuscular Inactivated Influenza A/Vietnam/1203/2004 (H5N1) Vaccine after Priming with an Antigenic Variant
- Nega Ali Goji1,
- Carrie Nolan1,
- Heather Hill2,
- Mark Wolff2,
- Diana L. Noah3,
- Tracy B. Williams3,
- Thomas Rowe3 and
- John J. Treanor1
- 1University of Rochester Medical Center, Rochester, New York
- 2EMMES Corporation, Rockville, Maryland
- 3Southern Research Institute, Birmingham, Alabama
- Reprints or correspondence: Dr. John Treanor, Prof. of Medicine, Infectious Diseases Unit, Room 3-6308, University of Rochester Medical Center, 601 Elmwood Ave., Rochester, NY 14642 (john_treanor{at}urmc.rochester.edu).
-
Presented in part: 44th annual meeting of the Infectious Diseases Society of America, Toronto, 12–15 October 2006 (abstract LB-2090).
Abstract
Background. We administered a single dose of influenza A/Vietnam/1203/2004 (H5N1, clade 1) vaccine to subjects who had received 2 doses of influenza A/Hong Kong/156/1997 (H5N1, clade 0) vaccine in 1998.
Methods. Thirty-seven subjects previously vaccinated with a baculovirus-expressed recombinant hemagglutinin A/Hong Kong/156/1997 vaccine in 1998 received a single intramuscular dose of 90 µg of inactivated subvirion A/Vietnam/1203/2004 vaccine in 2006. Serum antibody was measured before vaccination and 28 and 56 days after vaccination. Antibody responses were compared with those measured after one or two 90-µg doses in H5-naive subjects.
Results. On day 28 after a single dose, the geometric mean titer (GMT) of hemagglutination-inhibition antibody in primed subjects was 64.0 (95% confidence interval [CI], 37.8–108.5), with 68% responding (4-fold increase in antibody level to a titer of ≥1:40). In contrast, H5-naive subjects who received two 90-µg doses had a day 56 (28 days after the second dose) GMT of 27.7 (95% CI, 20.3–38.0), with only 43% responding.
Conclusions. This study suggests that priming can result in immune responses to a single dose of an antigenically variant strain of H5N1 influenza virus and could be a useful strategy for pandemic control.
Trial registration. ClinicalTrials.gov identifier: NCT00240903.
Severe disease in humans due to avian influenza viruses of the H5N1 subtype has raised considerable concern regarding the potential emergence of these viruses in pandemic form [1, 2]. A cornerstone of planning for control of such pandemics is the development of effective vaccines for H5N1. However, the observation that 2 doses of inactivated vaccine are required to generate serum antibody responses [3–5] poses a potential logistical obstacle to the use of vaccines for pandemic control.
In one previous study, serum antibody responses to a 2-dose schedule of an H5 vaccine generated from the avian influenza A/duck/Singapore/1997 virus (antigenically related to the A/Hong Kong/1997 virus but not highly pathogenic in birds) and administered with the oil-in-water adjuvant MF59 were significantly enhanced when the subjects were revaccinated 16 months later with a single dose of the same vaccine [6]. In addition, there were no adverse events of concern on revaccination.
These results suggested that one strategy for pandemic control could involve prevaccination of some segments of the population before the emergence of a pandemic so that effective protection could be achieved with a single-dose schedule if and when a pandemic does emerge. However, this strategy is complicated by the observation of continued antigenic drift among H5 viruses in birds, with the evolution of at least 3 genetically distinct lineages, or clades [7]. This means that a pandemic H5 virus might be an antigenic variant of the virus used for a prevaccination strategy. If this were the case, it would be important to document that a priming effect can occur when individuals are revaccinated with variants in a different clade.
The availability of individuals who received a baculovirus-derived recombinant A/Hong Kong/156/1997 (clade 0) H5 hemagglutinin vaccine in 1998 [4] provided an opportunity to evaluate this question by revaccinating these individuals with the more recent A/Vietman/1203/2004 (clade 1) vaccine. We expected that if the previous clade 0 exposure resulted in significant priming, then revaccination with a clade 1 vaccine should result in more vigorous responses than those observed when the same vaccine was administered to H5-naive subjects [5]. Therefore, we administered a single 90-µg dose of subvirion A/Vietnam/1203/2004 vaccine to subjects who had previously received A/Hong Kong/156/1997 vaccine and compared their results to the those we observed in H5-naive subjects who had received 2-dose 90-µg vaccination with A/Vietnam/1203/2004 vaccine in a previous study [5].
Methods
Study design. The study was conducted as an open-label phase 1 evaluation of a single dose of inactivated subvirion H5N1 vaccine. The subjects for the study were individuals who had participated in a previous study evaluating baculovirus-expressed recombinant H5 (rH5) hemagglutinin of the A/Hong Kong/156/1997 (H5, clade 0) vaccine [4]. In that study, conducted in late 1997 and early 1998, subjects received 2 doses of rH5 vaccine (Protein Sciences Corporation; lot 2769P) at either 25, 45, or 90 µg or 90 µg followed by 10 µg, at intervals of either 21, 28, or 42 days. Vaccine was administered in a total volume of 1 mL by intramuscular injection in the deltoid muscle of the nondominant arm by means of a 1-inch 25-gauge needle. There were a total of 117 recipients of rH5 vaccine in 12 groups in this study who were eligible for the present study. Subjects who had received the rH5 vaccine at any dose or schedule were recruited using an existing database of previous subjects. To participate, subjects had to be in good health, not pregnant, and not receiving immunosuppressive medications.
All subjects received a single 90-µg dose of inactivated, egg-derived, subvirion, reverse genetically derived (rg) A/Vietnam/1203/2004 vaccine (Sanofi Pasteur; lot V109:5C) from the same lot of vaccine used in our previous trial [5] administered to the deltoid of the nondominant arm by means of a 1-inch 25-gauge needle, in open-label fashion. Serum samples for assessment of hemagglutination-inhibition (HI) and microneutralization (MN) responses were obtained before vaccination and on days 28 and 56 after vaccination. Subjects were monitored for local and systemic reactions to the vaccine for 7 days after vaccination by means of the same assessment tools used in our previous evaluation of the subvirion A/Vietnam/1203/2004 vaccine [5] and for unsolicited adverse events for 56 days after vaccination. Adverse events were reported using a 4-point scale, as follows: 0 indicates absence; 1 indicates mild, or just barely noticeable; 2 indicates moderate, or resulting in some impairment in normal activities; and 3 indicates severe, or that the adverse event prevented normal daily activities.
The study was approved by the Institutional Review Board of the University of Rochester, and all subjects gave written informed consent before participation. Subject in trials used to generate control serum had given consent for testing their samples against related viruses.
Vaccines and viruses. The reference virus for the production of the H5N1 vaccine was a rg A/Vietnam/1203/2004 × PR8 reassortant virus generated from the human isolate influenza A/Vietnam/1203/2004 (H5N1) virus by means of a plasmid rescue system [8]. The hemagglutinin was modified to replace the cleavage site with that of a low pathogenicity avian virus, and the vaccine was produced in eggs as described elsewhere [5]. The vaccine was produced as a partially purified subvirion preparation and formulated at a concentration of 90 µg/mL, as determined by single radial immunodiffusion using sheep antisera to bromelain-cleaved native H5 hemagglutinin in the agar.
Laboratory assays. Serum HI assays were performed using the homologous rg A/Vietnam/1203/2004 × PR8 virus by standard microtiter techniques [9], with horse red blood cells as the indicator cells [10]. Serum samples were tested at a starting dilution of 1:10, and negative samples were assigned a value of 1:5 for calculation purposes.
Serum neutralizing antibody to the rg A/Vietnam/1203/2004 × PR8 virus was assessed under enhanced biosafety level 2 conditions by microtiter neutralization in MDCK cells [11] at the Southern Research Institute. Serum samples were tested at a starting dilution of 1:10, and negative samples were assigned a value of 1:5 for calculation purposes.
To characterize the effect of prior vaccination on the breadth of the antibody response, we also evaluated titers of HI antibody to H5N1 antigenic variants in postvaccination serum. These assays were performed as described above but used the wild-type A/Vietnam/1203/2004 (clade 1), A/Indonesia/05/2005 (clade 2), and A/Hong Kong/156/1997 (clade 0) viruses under enhanced biosafety level 3 conditions.
Statistical analysis. The following end points were determined for the immunologic assays: the rate of achievement of a postvaccination serum MN titer of 1:40 or greater; the response rate, defined as a 4-fold or greater increase in titer between prevaccination and day 28 to a titer of at least 1:40; and the geometric mean titer (GMT) of postvaccination antibody. All rates and mean titers were calculated with exact 95% confidence intervals.
For evaluation of the hypothesis that priming with the A/Hong Kong/156/1997 vaccine would result in booster responses to the A/Vietnam/1203/2004 vaccine, we compared these end points with the comparable end points after one or two 90-µg doses of subvirion A/Vietnam/1203/2004 vaccine in H5-naive subjects from the previous study of this vaccine [5]. This comparison was done using existing data—that is, serum samples from the 90-µg group of the previous study were not reassayed for comparison with this study. However, titers from the previous study were recalculated by defining the same starting dilution as 1:10 rather than as 1:20; this resulted in each result being 1 dilution lower than previously reported and means that results from the studies were compared using the same starting-dilution definition. Antibody assays in the present study were conducted in the same laboratory as the previous study and by the same methodology, and quality-assurance/quality-control analysis of the performance of the MN and HI assays over the time period covered by the 2 determinations showed minimal change in the titers of the positive and negative control serum.
The breadth of antibody response was evaluated on the basis of titers of HI antibody to H5N1 antigenic variants in postvaccination serum. A subset of control serum samples were selected from among those obtained on day 28 after the administration of 2 doses of unadjuvanted vaccine in age-matched H5-naive subjects and were matched for similar titers of HI antibody to the rg A/Vietnam/1203/2004 × PR8 virus. These control serum samples and the serum samples from the present study were then retested in HI assays against the wild-type A/Vietnam/1203/2004 (clade 1), A/Indonesia/05/2005 (clade 2), and A/Hong Kong/156/1997 (clade 0) viruses.
Rates of local and systemic solicited adverse events over 7 days and of unsolicited adverse events over 56 days were tabulated by level of severity.
Results
Review of records from the study done in 1998 revealed that 117 subjects were in study groups that were potentially eligible for this study. Of these individuals, 70 were no longer at their previous address and were not listed in the local telephone directory. Of the remaining 47 subjects who could be contacted, 5 had developed exclusionary conditions, and 5 were unable to participate because scheduling conflicts. Thus, a total of 37 subjects from the 1998 study who had received the Hong Kong vaccine and who met all current entry criteria were enrolled in the present study. All subjects received a single 90-µg dose of A/Vietnam/1203/2004 vaccine between March and April of 2006 and made all follow-up visits; there were no dropouts, and no subjects were lost to follow-up. Table 1 compares the demographics of the subjects in this study with those who received 90 µg of H5 vaccine in the previous evaluation of A/Vietnam/1203/2004 vaccine in H5-naive subjects. In general, the 2 populations were comparable, although there was slightly more ethnic diversity in the H5-naive population participating in the more recent study of the A/Vietnam/1203/2004 vaccine. The male-to-female ratio was similar in both subject populations, and a similar proportion of subjects recalled having received conventional trivalent inactivated influenza vaccine within the previous year. As expected, the participants in the revaccination study were slightly older than those in the comparison group
Figure 1
Rates and severity of local and systemic adverse events during the first 7 days after the first dose (D1) or the second dose (D2) of 90 µg of subvirion H5N1 vaccine in naive subjects in the previous study [5] and after 1 dose in H5 vaccine-primed (revaccinated [RV]) subjects in the present study. Symptoms are graded on the following scale: mild indicates any noticeable symptom, moderate indicates some limitation in activity, and severe indicates that the adverse event prevented normal daily activities.
Table 1
Demographics of the enrolled population (H5 primed) and comparison group (H5 naive).
The immune response to vaccination is shown in table 2. The primed subjects did not have detectable antibody to the A/Vietnam/1203/2004 virus or to the A/Hong Kong/156/1997 virus (data not shown) at the beginning of the study. A single 90-µg dose resulted in serum HI and MN titers that were significantly higher than those seen after 1 dose in vaccine-naive subjects and were actually higher than those seen after 2 doses in these subjects. Among primed subjects who received a single dose of vaccine, a decrease in antibody titer was noted by day 56 after vaccination, but this drop was not statistically significant for either HI or MN antibody. Primed subjects had more frequent responses to a single dose of subvirion vaccine than did naive subjects and were more likely to achieve HI and MN titers of 1:40 or greater on day 28 than were naive subjects after either 1 or 2 doses of vaccine.
Table 2
Comparison of serum hemagglutination-inhibition (HI) and microneutralization (MN) titers against the A/Vietnam/1203/2004 virus (H5N1, clade 1) after receipt of one or two 90-µg doses of subvirion A/Vietnam/1203/2004 vaccine in subjects previously vaccinated with A/Hong Kong/156/1997 (H5, clade 0) vaccine (H5 primed) or in subjects with no previous exposure to H5 viruses (H5 naive) in a separate study.
The response to a single dose of the A/Vietnam/1203/2004 vaccine was somewhat dependent on whether the subject had responded to the A/Hong Kong/156/1997 virus after 2 doses of rHA A/Hong Kong/156/1997 HA vaccine in 1997 (table 3). Although the numbers of subjects are small, the frequency of both HI and MN responses as well as the GMT achieved on day 28 were higher in those who had responded to the vaccine in 1998 than in those who did not. However, there was not a clear relationship between the dose of vaccine received in 1998 and the ability to respond in 2006 or between the interval between doses in 1998 (21, 28, or 42 days) and the response in 2006 (data not shown).
Table 3
Relationship between (1) the dose of A/Hong Kong/156/1997 (H5, clade 0) vaccine received and the neutralizing antibody response to A/Hong Kong/156/1997 in 1998 and (2) hemagglutination-inhibition (HI) and microneutralization (MN) response on day 28 to A/Vietnam/1203/2004 (H5N1, clade 1) vaccine in 2006.
To characterize the effect of prior vaccination on the breadth of the antibody response, serum from the primed subjects as well as titer- and age-matched H5-naive subjects were retested in HI assays against the wild-type A/Vietnam/1203/2004 (clade 1), A/Indonesia/05/2005 (clade 2), and A/Hong Kong/156/1997 (clade 0) viruses (table 4). Because of the matching process, titers of antibodies to the homologous virus were similar between the 2 groups. However, subjects who had been primed with A/Hong Kong/156/1997 and received a single dose of A/Vietnam/1203/2004 had slightly higher titers and were more likely to have a titer of >1:40 against both A/Indonesia/05/2005 and A/Hong Kong/156/1997 viruses, compared with their naive counterparts.
Table 4
Effect of previous vaccination on serum titers of hemagglutination-inhibition (HI) antibody to antigenically drifted viruses, including A/Vietnam/1203/2004 (VN/04; clade 1), A/Indonesia/05/2005 (IN/05; clade 2), and A/Hong Kong/156/1997 (HK/97; clade 0).
Vaccination of primed subjects was not associated with increased local or systemic reactogenicity, compared with naive subjects receiving one or two 90-µg doses of subvirion vaccine. Figure 1 shows the results of 7-day reactogenicity assessments after a single dose in primed subjects or after 1 or 2 doses of the same vaccine in H5-naive subjects. There were no differences in the rates or severity of local or systemic adverse events after either dose of vaccine in naive subjects or on revaccination. No subjects reported fever, and there were no vaccine-attributable serious adverse events in either group.
Discussion
The serum HI and MN responses to a single dose of vaccine in this study relative to the responses seen to a single dose in immunologically naive subjects support the hypothesis that previous vaccination with a clade 0 vaccine can prime for responses to a clade 1 vaccine. The results of this study are consistent with previous reports that a third dose of H5 vaccine can generate substantial increases in antibody level (both for the A/duck/Singapore/1997 (H5N3) vaccine [6] and H5N1 vaccines [12]) but extend these findings to the situation where priming and revaccination use antigenically variant viruses, which may be more representative of the likely scenario for pandemic control. Somewhat similar observations were made in 1977, when persons primed by infection with H1N1 viruses before 1957 were found to respond to a single dose of inactivated H1N1 vaccine 20 years later, in 1977 [13].
Seasonal influenza vaccination of young, immunologically naive children with inactivated influenza vaccine presents some of the same challenges that will be faced by those organizing pandemic vaccination campaigns, with a requirement for 2 doses to be administered within a relatively short period of time. Thus, there has also been interest in determining the effects of priming on responses to subsequent doses in this population. These studies have shown that, in the absence of a change in vaccine formulation, similar responses are seen in young children who receive 2 doses separated by 6 months (a single dose in the spring followed by a single dose in the fall) or 2 doses separated by 28 days (two doses in the fall) [14]. If the 2 antigens were minor antigenic variants (e.g., 1 dose of A/Panama/1999 [H3N2] followed by 1 dose of A/Wyoming/2003 [H3N2]), the response was very similar to the response to 2 doses of A/Wyoming/2003, with clear evidence of priming for A/Wyoming responses by previous A/Panama vaccination. However, it appeared that a single dose of B/Hong Kong/1999 (Victoria lineage) did not prime for a response to a single dose of A/Jiangsu/1999 (Yamagata lineage) [15]. Although not strictly comparable because our study involved a 3-dose rather than a 2-dose schedule, these studies suggest that there may be limits to the extent of antigenic variation that would still permit effective cross-priming.
Influenza A H5N1 viruses are continuing to undergo antigenic evolution in birds, as demonstrated by the emergence of viruses belonging to clade 2, which are represented by the A/Indonesia/05/2005 virus and multiple subclades [7]. If this situation continues, then it is likely that a pandemic virus will not be an exact match for any stockpiled vaccine. Therefore, there is intense interest in strategies that might result in a more broadened immune response capable of recognizing antigenic variants. To assess the effect of multiple doses on the cross-reactivity of the antibody response, we compared the recognition of A/Hong Kong/156/1997 and A/Indonesia/05/2005 viruses by HI tests in serum samples from this study and in a set of samples from recipients of 2 doses of A/Vietnam/1203/2004 vaccine who were matched for a similar titer of antibody to the homologous A/Vietnam/1203/2004 virus. Although the differences were small, subjects who had received H5 vaccines derived from 2 different clades had higher titers of antibody to Hong Kong and Indonesia viruses and were more likely to have titers of 1:40 or greater, relative to those who had received 2 doses of vaccine from a single clade (Vietnam). In other studies evaluating multiple doses with a single clade, it has been necessary to use oil-in-water adjuvants to broaden the range of reactivity against antigenically distinct strains [16, 17].
In some studies, it has been observed that, when an individual is primed with 1 influenza variant within a subtype and subsequently vaccinated with a second influenza variant of the same subtype, the second vaccination may result in an antibody response to the first variant that is as vigorous or more vigorous than the response to the second variant, a phenomenon referred to as “original antigenic sin” [18]. All other things being equal (which they might not be, for example, with regard to assay sensitivity), one might expect under these circumstances that the previously primed subjects would manifest HI responses to the A/Hong Kong/1997 virus that were at least of the same frequency and magnitude as the responses to A/Vietnam/2004. Clearly, that was not seen in the present study, in which responses to A/Vietnam appear to be much more frequent and of higher titer than the responses to A/Hong Kong.
One potential explanation for the phenomenon of original antigenic sin is related to the effects of prevaccination antibody on the stimulation of B cells. Given that naive B cells have a lower affinity for antigen, preexisting antibody might compete for binding of antigen to the naive cells, resulting in preferential stimulation of the memory B cells and thus in an immune response consistent with original antigenic sin [19]. Because the primed subjects in our study did not have detectable antibody to the A/Hong Kong/1997 virus at the beginning of the study, this could be an explanation for why original antigenic sin-like manifestations were not observed.
It will be useful to confirm these findings in subjects receiving other combinations of H5 vaccines. If the finding that priming can result in enhanced responses to single-dose vaccination schedules is confirmed, then prepandemic vaccination programs could be considered, especially in populations of first responders, health care workers, and the military. Such populations might then be able to be effectively and rapidly vaccinated with a single dose of a vaccine specific for an emerging pandemic if it were to occur.
Footnotes
-
Potential conflicts of interest: J.J.T. has received grant support and speaker's fees from Sanofi Pasteur and has received grant support from Protein Sciences Corporation. All other authors report no potential conflicts.
-
Financial support: National Institutes of Health (contract N01 AI 25460); US General Clinical Research Center (grant M01 RR00044).
- Received January 16, 2008.
- Accepted April 28, 2008.
- © 2008 by the Infectious Diseases Society of America
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