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Journal of Virology, July 1999, p. 5903-5911, Vol. 73, No. 7
Influenza Branch1 and
Infectious Disease Pathology Activity,2
Division of Viral and Rickettsial Diseases, National Center for
Infectious Diseases, Centers for Disease Control and Prevention,
Atlanta, Georgia 30333
Received 5 January 1999/Accepted 24 March 1999
During 1997 in Hong Kong, 18 human cases of respiratory illness,
including 6 fatalities, were caused by highly pathogenic avian
influenza A (H5N1) viruses. Since H5 viruses had previously been
isolated only from avian species, the outbreak raised questions about
the ability of these viruses to cause severe disease and death in
humans. To better understand the pathogenesis and immunity to these
viruses, we have used the BALB/c mouse model. Four H5N1 viruses
replicated equally well in the lungs of mice without prior adaptation
but differed in lethality for mice. H5N1 viruses that were highly
lethal for mice were detected in multiple organs, including the brain.
This is the first demonstration of an influenza A virus that replicates
systemically in a mammalian species and is neurotropic without prior
adaptation. The mouse model was also used to evaluate a strategy of
vaccination against the highly pathogenic avian H5N1 viruses, using an
inactivated vaccine prepared from nonpathogenic
A/Duck/Singapore-Q/F119-3/97 (H5N3) virus that was antigenically
related to the human H5N1 viruses. Mice administered vaccine
intramuscularly, with or without alum, were completely protected from
lethal challenge with H5N1 virus. Protection from infection was also
observed in 70% of animals administered vaccine alone and 100% of
mice administered vaccine with alum. The protective effect of
vaccination correlated with the level of virus-specific serum antibody.
These results suggests a strategy of vaccine preparedness for rapid
intervention in future influenza pandemics that uses antigenically
related nonpathogenic viruses as vaccine candidates.
During May through December 1997, an
outbreak of avian influenza A (H5N1) virus in Hong Kong caused 18 human
cases (6 fatal) of respiratory illness (7, 11, 30). The H5N1
infections in humans were preceded by the circulation of highly
pathogenic H5N1 viruses in birds, first in poultry farms and later in
wholesale and retail poultry markets in Hong Kong (5, 8,
28). The H5N1 viruses isolated from both chickens and humans
possessed hemagglutinin (HA) molecules with multiple basic amino acids
adjacent to the cleavage site between HA1 and HA2 and were lethal for
experimentally infected chickens, features characteristic of highly
pathogenic avian influenza A viruses (8, 26, 29, 30). The
fact that the H5N1 viruses resulted in severe or fatal respiratory
disease in the majority of infected persons aged 13 to 60 years was of particular concern since this age group is not normally considered to
be at increased risk for death and complications from influenza (6, 31).
The antigenic and genetic analysis of 16 H5N1 viruses isolated from
humans identified two closely related but distinguishable groups,
represented by A/Hong Kong/156/97 (HK/156) virus (group A) and A/Hong
Kong/483/97 (HK/483) virus (group B). Group B viruses share a
substitution at residue 156 of HA1, which creates a potential glycosylation site absent in group A viruses (3). An
asymmetric cross-reactivity between the groups was demonstrated by
postinfection ferret serum in hemagglutination inhibition (HI) assays.
Antiserum raised to group B viruses exhibited a greater degree of
cross-reactivity with group A viruses compared with the reactivity of
group A virus antiserum for group B viruses (3). All
internal genes, like those encoding the surface glycoproteins, were of
avian origin (8, 27, 29), indicating that the H5N1 viruses
that infected humans in Hong Kong had crossed the species barrier
without genetic reassortment with a human influenza virus. Neither the
HA nor the NA (neuraminidase) genes of the human H5N1 viruses isolated from the outbreak showed evidence of adaptive changes (3). Furthermore, Matrosovich et al. (22) have reported that H5N1 virus isolated from the index case (HK/156) possessed an HA with receptor specificity typical of avian viruses.
Previous studies had demonstrated that humans were not susceptible to
infection with a highly pathogenic H5 virus that caused high mortality
in poultry in Pennsylvania in 1983 (2). The ability of the
H5N1 viruses from Hong Kong to cause severe respiratory illness,
multiorgan dysfunction, and a high rate of mortality in humans raised
questions concerning the mechanism(s) of pathogenicity and the
development of prevention and control measures in preparation for an
actual pandemic caused by highly pathogenic avian viruses. In this
situation, traditional methods for developing inactivated vaccines for
humans would be greatly compromised by the need to prepare and perform
safety testing of reassorted vaccine candidates under biosafety level
3-plus (BSL-3+) containment conditions (1).
We investigated the mouse as a mammalian model for the study of H5N1
influenza virus pathogenesis and immunity. We report here on four human
H5N1 viruses that replicated efficiently in the lungs, without any
adaptation, and have different levels of lethality for BALB/c mice. In
addition, we demonstrate the utility of this animal model for
evaluating protective immunity to human H5N1 influenza viruses and a
strategy for effective vaccination against the pathogenic H5N1
influenza viruses that uses the antigenically related, but
nonpathogenic, avian A/duck/Singapore/Q/F119-3/97 (dk/Sing) (H5N3) virus.
Viruses.
The influenza viruses used in this study were the
H5N1 group A viruses HK/156 and A/Hong Kong/486/97 (HK/486); the H5N1
group B viruses HK/483 and A/Hong Kong/485/97 (HK/485); the H5N3
dk/Sing virus; and the reassortment human influenza A virus X-31, which possesses the surface glycoprotein genes of A/Aichi/2/68 (H3N2) and the
internal protein genes of A/Puerto Rico/8/34 virus. A U.S. Department
of Agriculture permit was obtained before work with avian influenza
viruses was begun. Virus stocks were propagated in the allantoic
cavities of 10-day-old embryonated hen eggs under conditions that were
found to be optimal for virus replication for H5N1 viruses (37°C,
24 h) or dk/Sing and X-31 viruses (34°C, 48 h). Virus
stocks were aliquoted and stored at
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
A Mouse Model for the Evaluation of Pathogenesis
and Immunity to Influenza A (H5N1) Viruses Isolated from
Humans
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
70°C until use. The passage
histories of the viruses are detailed in Table
1. Fifty percent tissue culture
infectious dose (TCID50) and 50% egg infectious dose
(EID50) titers were determined by serial titration of
viruses in Madin-Darby canine kidney (MDCK) cells and eggs, respectively. Titers were calculated by the method of Reed and Muench
(24).
TABLE 1.
Characteristics of influenza A H5N1 viruses
Laboratory facility. Because of the potential risk to humans and poultry, all experiments using infectious pathogenic avian H5N1 viruses, including work with animals, were conducted using BSL-3+ containment procedures (25). Investigators were required to wear appropriate respirator equipment (RACAL Health and Safety Inc., Frederick, Md.). Work performed with the nonpathogenic virus dk/Sing was conducted under BSL-2 conditions.
Infection of mice and challenge experiments.
Female BALB/c
mice, 6 to 8 weeks old (Charles River Laboratories, Wilmington, Mass.),
were used in all experiments. Mice were lightly anesthetized with
CO2, and 50 µl of infectious virus diluted in
phosphate-buffered saline (PBS) was inoculated intranasally (i.n.).
Fifty percent mouse infectious dose (MID50) and 50% lethal dose (LD50) titers were determined by inoculating groups of
seven mice i.n. with serial 10-fold dilutions of virus. Four days
later, three mice from each group were euthanized, and lungs were
collected and homogenized in 1 ml of cold PBS. The homogenates were
frozen at 70°C and later thawed for ease of handing. Solid debris
was pelleted by centrifugation, and tissues were titrated for virus infectivity in eggs. The four remaining mice in each group were checked
daily for disease signs and death for 14 days postinfection (p.i.).
MID50 and LD50 titers were calculated by the
method of Reed and Muench (24) and were expressed as the
EID50 value corresponding to 1 MID50 or
LD50. Lung virus titers were used for the determination of
MID50 because we had previously determined that the H5N1
viruses used in this study replicated to higher titers (
100-fold) in lung tissue than in upper respiratory tract tissues.
Immunohistochemistry. Tissues from infected mice were fixed in formalin and were processed for immunohistochemistry by using a two-step biotin-streptavidin method essentially as previously described (32). The primary polyclonal antibody used was a goat anti-Tern/South Africa/61 (H5N3) (National Institute of Allergy and Infectious Diseases, Bethesda, Md.) antibody which recognizes both surface glycoproteins and internal proteins of the virus.
Antibody assays.
Immune sera from blood samples collected
from the orbital plexus were treated with receptor-destroying enzyme
from Vibrio cholerae (Denka-Seiken, Tokyo, Japan) as
previously described (19) before testing for the presence of
HI antibody by standard methods (20). Individual mice were
considered to have responded to vaccination if serum HI titers were
40. Titers of neutralizing antibody were determined essentially as
previously described (16) except that a lower cell
concentration (1.5 × 105 MDCK cells/ml) was used.
Neutralization titers are expressed as the reciprocal of the highest
dilution of serum that gave 50% neutralization of 100 TCID50 of virus.
Vaccine and adjuvant preparations and immunizations. dk/Sing virus was concentrated from allantoic fluid and purified on a linear sucrose gradient as described previously (10). The HA content of purified dk/Sing virus was estimated to be 30% of the total viral proteins by densitometric analysis of viral protein bands separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Inactivated whole-virus vaccine was prepared by treating purified virus at a concentration of 1 mg/ml with 0.025% formalin at 4°C for >3 days. This treatment resulted in the complete loss of infectivity of the virus. Groups of mice were lightly anesthetized with CO2 and injected intramuscularly (i.m.) with either 10 µg of dk/Sing vaccine (=3 µg of HA) alone, 10 µg of vaccine mixed with alum, or PBS mixed with alum in a volume of 0.1 ml. A 2% alum (Alhydrogel; Superfos Biosectors, Kvistgaard, Denmark) suspension was mixed with an equal volume of vaccine in PBS or PBS only for 6 h at 4°C before immunization. Mice received two inoculations at an interval of 3 weeks.
Statistical analysis. Statistical significance of the data was determined by using Fisher's exact or Student's t tests.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In vitro and in vivo growth characteristics of human H5N1 influenza viruses. Two group A and two group B H5N1 viruses isolated from patients representing the spectrum of disease severity observed during the outbreak were selected for use in this study. Their origin and growth characteristics in MDCK cells, eggs, and mice are shown in Table 1. For comparison, the characteristics of a human H3N2 reassortant virus X-31 are also shown. HK/156 and HK/486 are group A viruses, and HK/483 and HK/485 are group B viruses. All human H5N1 viruses and X-31 virus had high infectivity titers in MDCK cells and eggs. With the exception of HK/156 virus, the H5N1 viruses had similar MID50 titers determined by the detection of virus in the lungs of mice 4 days p.i. Ten to 100 times more HK/156 virus than the other H5N1 viruses was required to infect mice. The lethality of the four H5N1 viruses for mice varied considerably. HK/483 and HK/485 viruses were the most lethal for mice, whereas HK/156 virus was considerably less lethal. The lethality of HK/486 was substantially lower than that of the other H5N1 viruses. The nonpathogenic avian H5 virus, dk/Sing, could also infect mice (MID50 = 104.2) but was not lethal, even at high doses (106 EID50 [data not shown]).
Lethality of H5N1 viruses in mice. A further experiment compared the time courses of lethal infection for the H5N1 viruses. Mice (10 per group) infected i.n. with 104, 103, or 102 MID50 of virus were observed for 14 days. The percentages of mice surviving the infections are shown in Fig. 1. Mice infected with any dose of HK/483 virus rapidly succumbed 4 to 8 days later. Likewise, 100% of mice infected with any dose of HK/485 virus died 6 to 10 days p.i. Mice infected with 104 MID50 of HK/156 showed significant (90%) mortality by day 9. However, the lethality of HK/156 virus was reduced to 40 and 10% when 103 and 102 MID50 of virus, respectively, were used to infect mice. In contrast to the lethal outcomes with the three H5N1 viruses, none of the mice infected with 103 or 102 MID50 of HK/486 virus died and only 1 of 10 mice infected with 104 MID50 of virus died. These results demonstrated that HK/483 and HK/485 viruses were substantially more lethal for BALB/c mice than were HK/156 and HK/486 viruses.
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), HK/485 (
), HK/156 (
), or
HK/486 (
) virus and examined daily for 14 days.
Detection of H5N1 viruses in mouse tissues. To investigate further the differences in pathogenesis of the human H5N1 viruses in mice, we examined the degree of replication in different tissues. Groups of mice were infected with 100 MID50 of one of three H5N1 viruses or X-31 virus. The mean virus titers (expressed as log10 EID50 per milliliter ± SD) were determined on day 4 p.i., when virus titers in the lungs were maximal, and on day 6 p.i., a time point that immediately preceded the death of mice infected with the highly lethal HK/483 virus (Fig. 2). Infection of mice with each of the H5N1 viruses resulted in high titers of virus in the lungs on days 4 and 6 p.i. Viruses were recovered from the brains of mice infected with HK/483 and HK/156 viruses with titers increasing 15- to 30-fold from days 4 to 6 p.i. The brains of mice infected with HK/483 virus had 50-fold higher titers of virus on day 6 p.i. than those of mice infected with HK/156 virus (P < 0.05). In contrast, mice infected with HK/486 virus had undetectable virus in brains on either day p.i. Virus was also present in the livers, spleens, and blood of mice infected with HK/483 virus on day 4 and 6 p.i., but the titers of virus on day 6 were lower than those on day 4. The kidneys of mice infected with HK/483 virus also yielded substantial titers of virus. A level of virus similar to that of HK/483 was observed in organs and blood of mice infected with HK/485 virus (data not shown). In contrast, mice infected with HK/156 virus had low levels of virus in the livers and kidneys on day 4 and undetectable levels of virus in spleens and blood at either time point. Mice infected with HK/486 or X-31 virus had no detectable virus in these organs on either day p.i.
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) and 6 (
) p.i., and titrated in
eggs. The mean virus titers from three mice per group are shown. The
limit of virus detection was 101.2 EID50/ml for
lungs and 100.8 EID50/ml for blood and other
tissues. * and 
, P < 0.01 and P <
0.05, respectively, versus groups infected with HK/156 or HK/486
virus.
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Immunogenicity of an inactivated whole virus dk/Sing (H5N3) vaccine. One strategy for developing vaccines against pathogenic avian influenza viruses is the use of a closely related avian virus that is nonpathogenic for poultry and presumably for humans. Such a virus could be handled safely in the laboratory and, on a larger scale, in a vaccine-manufacturing plant. One such virus is dk/Sing virus, which is antigenically related to the human H5N1 viruses and shares 92 to 93% amino acid sequence homology in the HA1 portion of the HA molecule (4).
In preliminary experiments, we had observed that protection from lethal challenge with HK/483 virus in mice previously infected with HK/156, HK/486, or dk/Sing virus correlated with the presence of serum HI antibody against homologous viruses. All mice (n = 37) with serum HI antibody titers of80 were completely protected from
death after challenge with HK/483 virus. Ninety-three percent of mice
(n = 30) with an HI titer of 40 were protected from
death. In contrast, all mice (n = 27) with HI antibody
titers of 
20 died within 7 days of challenge. These results indicated
that serum HI antibody titers of 40 may be sufficient to protect mice from a lethal challenge with H5N1 virus.
To evaluate the potential of the dk/Sing vaccine candidate and the
utility of the mouse model for vaccine efficacy studies, a group of
mice (n = 22) was immunized with 10 µg of purified dk/Sing vaccine. A second dose was administered 3 weeks after the
first. Because avian viruses have been reported to elicit minimal, if
any, serum HI antibody responses in mammalian species (17),
another group of mice (n = 20) was immunized with two doses of 10 µg of vaccine mixed with alum, an adjuvant licensed for
use in humans. A negative control group of mice (n = 17) received alum alone.
The serum HI antibody response to HK/156 virus measured in individual
serum samples collected 3 weeks after the first and second i.m.
vaccination is shown in Fig. 4. One dose
of vaccine alone failed to elicit a response (HI titer 40) in
mice. A second dose of vaccine was required to induce a substantial HI
antibody response in 65% of mice (geometric mean titer [GMT] = 51).
Although the GMT of mice receiving one dose of vaccine with alum
(GMT = 26) was significantly greater than those of mice receiving
one dose of vaccine alone (P < 0.001), only 40% of
animals responded. The administration of a second dose of vaccine with
alum resulted in 100% of mice responding and greatly elevated HI
antibody titers (GMT = 710) compared with mice receiving a second
dose of vaccine alone (P < 0.001). These results
indicated that two doses of the vaccine, with or without alum, were
required to elicit responses in most mice. The addition of alum to the
vaccine greatly augmented the HI antibody response after a second dose.
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Protective efficacy of dk/Sing vaccine. We next investigated the protective efficacy of the dk/Sing vaccine against a lethal challenge with the group B virus HK/483. This virus was chosen for two reasons. First, HK/483 virus was the most pathogenic of the four H5N1 viruses examined in this study (Table 1 and Fig. 1). Second, the group B viruses have been characterized with reference ferret antiserum to have decreased reactivity with antibody raised to group A H5N1 or dk/Sing viruses. We considered challenge with HK/483 virus to be a more stringent test of protective efficacy.
Four months after vaccination, mice were infected i.n. with 50 LD50 of HK/483 virus. Each vaccine group was then divided into mice (n = 10 to 12) that were monitored for signs of disease and death for 14 days or mice (n = 7 to 10) that were euthanized on day 6 p.i. to determine the level of virus replication in the lung and brain. Day 6 was chosen to evaluate protection from infection because unvaccinated mice were shown previously to have substantial titers of virus in both lungs and brains at this time point. As shown in Table 3, control mice receiving alum alone died 6 to 7 days p.i. with high titers of virus in the lungs and brains. In contrast, 100% of mice vaccinated with dk/Sing virus, with or without alum, survived lethal challenge. Thirty percent of mice that received vaccine alone had low levels of virus in the lungs on day 6 p.i. Virus titers in these mice ranged from 103.0 to 104.0 EID50/ml. No virus was detected in the brains of mice from either vaccine group. When considered as a group, protection from infection in mice receiving vaccine alone was highly significant compared with the control group (P < 0.001). Protection from infection was augmented by the addition of alum to the dk/Sing vaccine. All mice receiving dk/Sing vaccine with alum were completely protected from infection in both lungs and brains. These results demonstrated that vaccination of mice with two doses of dk/Sing (H5N3) vaccine induced a high degree of protection from infection and death following challenge with a highly lethal human H5N1 virus.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The introduction of influenza A (H5N1) viruses into humans served as an important reminder that another influenza pandemic is highly likely, if not inevitable. The H5N1 outbreak highlighted the potential of pandemic strains to arise directly from avian species, the natural reservoir of all 15 known HA subtypes of influenza A viruses. The outbreak also demonstrated that an avian virus could cross the species barrier to replicate in humans and cause severe disease. The H5N1 viruses resulted in a high rate of mortality in an age group not normally considered to be at increased risk for death and complications from influenza. For these reasons, the H5N1 viruses warrant further study in an effort to understand their pathogenesis in humans and to establish prevention and control measures in preparation for an actual pandemic.
Although the ferret model has been widely used for the study of other influenza A viruses, it has not yet been shown to be a suitable animal model for the study of human H5N1 viruses in vivo. In contrast, the BALB/c mouse was found to be a useful model system for investigation of human H5N1 virus pathogenesis and immunity. The H5N1 viruses replicated in mouse lungs to high titers and caused lethal disease without the prior adaptation that is generally required for human influenza A viruses to possess these properties (18). Furthermore, the highly lethal HK/483 and HK/485 viruses which caused severe or fatal disease in humans were also highly lethal in mice. These viruses replicated systemically and were neurotropic in mice, two properties which have not previously been demonstrated for an influenza A virus without prior adaptation to the host. Although the four H5N1 viruses examined in this study replicated to similarly high titers in the lungs of mice, differences in the lethality of the viruses were observed. HK/483 and HK/485 viruses were highly lethal for BALB/c mice; as little as 1 MID50 of HK/483 virus was sufficient to kill mice. HK/156 virus required a substantially higher MID50 to elicit a lethal infection in mice, and the lethality of HK/486 virus was substantially less than that of the HK/483 and HK/485 viruses. Shortridge et al. (28) have also recently reported that high dose (106 EID50) of HK/156 virus was highly lethal for BALB/c mice. However, in contrast to the results presented here, Gubareva et al. (15) reported that HK/156 virus was lethal for BALB/c mice even at low infectious doses. One reason for the differences in the lethality between the HK/156 virus preparations may be that the HK/156 virus used in the Gubareva study had been passaged twice in mouse lungs prior to characterization for lethality. In the present study, the viruses were only passaged in vitro prior to characterization in vivo. The differences in pathogenicity observed among H5N1 viruses did not correlate with passage history. We have tested the lethality of HK/486 and HK/483 virus stocks which had fewer passages in eggs or which had been passaged only in MDCK cells (HK/486). In each case, the viruses exhibited lethality for mice similar to that reported here (data not shown). It is noteworthy that HK/486 virus, which was substantially less lethal for mice, also caused mild disease in the child from whom it was isolated.
A feature of the highly pathogenic HK/483 viruses was the presence of elevated levels of virus in the spleen, liver, and blood at day 4 p.i. Disease progression was associated with the presence of viral antigens in subepithelial tissues in the lungs. More striking still were substantial titers of virus and viral antigens in the brains of mice infected with HK/483 virus, that increased during the infection and peaked on day 6 just before death. Mice infected with the less pathogenic HK/156 virus had significantly less virus in the brain on day 6 p.i. than did mice infected with HK/483 virus (P < 0.05). Mice infected with HK/486 had no detectable virus in the brain at days 4 and 6. Taken together these results suggest that the dissemination of virus to organs other than the lungs may depend on the extent of viremia that occurs early in the infection. In addition, the more pathogenic viruses may replicate more efficiently in other organs, including the brain. In this study, H5N1 group B viruses were highly pathogenic, whereas group A viruses were of intermediate to low pathogenicity. It is not clear from these results whether the additional glycosylation site in HA1, present in group B viruses but not group A viruses, contributes to the pathogenicity of these viruses. Sequence analysis of the HA gene of viruses used in this study confirmed the presence of multiple basic amino acids adjacent to the cleavage site in all viruses, including HK/486 which was substantially less lethal for mice (data not shown). Therefore, further studies are required to determine the molecular basis for the differences in lethality of the H5N1 viruses.
The mouse model also provided a means to evaluate a strategy for vaccination against H5 viruses. Currently, vaccine strains with an optimal antigenic match are achieved by producing high-growth reassortants from a circulating virus that has the desired antigenic characteristics. Preparation of vaccine candidate strains from the highly pathogenic H5N1 viruses by reassortant or by genetic manipulation and gene rescue techniques must be conducted under BSL-3+ conditions. Extensive BSL-3+ safety testing of those candidate strains would be needed and may delay vaccine production in a pandemic situation. An alternative strategy for the rapid and safe development of a vaccine against a highly pathogenic avian virus is to use an antigenically related nonpathogenic virus that can be handled under laboratory (BSL-2) conditions. This study demonstrated that a vaccine prepared from the nonpathogenic dk/Sing (H5N3) virus effectively protected mice from lethal challenge with the antigenically related but highly pathogenic human HK/483 (H5N1) virus. High titers of serum HI or neutralizing antibody and complete protection from infection and death were achieved in mice administered vaccine in the presence of alum. Mice administered vaccine alone had substantially lower serum HI and neutralizing antibody titers. One hundred percent of these animals were protected from death, but only 70% were protected from infection. Vaccine-induced serum HI titers of 40 or greater were sufficient to protect mice from lethal infection with H5N1 virus, whereas higher titers of antibody were required to protect mice completely from infection. Garcia et al. (13) reported that an inactivated vaccine prepared from low pathogenic A/chicken/Hidalgo/232/94 (H5N2) virus effectively protected chickens from lethal infection with a highly pathogenic influenza H5N2 virus. On the other hand, Kodihalli et al. (21) recently reported that a DNA vaccine encoding HK/156 virus HA, but not A/Turkey/Ireland/1/83 (H5N8) HA, protected BALB/c mice from infection with a lethal dose of HK/156 virus. This lack of protection with a heterologous H5 HA may reflect the greater amino acid sequence difference between the viruses used by Kodihalli et al. (12% difference in HA1) compared with the dk/Sing and HK/483 viruses used in the present study (8% difference in HA1). The present study demonstrated that increased levels of protection from infection correlated with increased serum antibody titers. Therefore, differences between the results of the two studies may, alternatively, reflect differences in the ability of the DNA-based vaccine compared with the protein-based vaccine used in the present study to elicit adequate titers of neutralizing antibody.
The protection afforded by the dk/Sing (H5N3) vaccine against lethal challenge with an H5N1 virus suggested that an antigenically related NA was not necessary for the protective effect observed in mice in this study. However, other investigators have demonstrated that vaccines composed of purified NA protected mice from lethal challenge (12), and the presence of NA-specific antibody has been associated with reduction in the magnitude of disease in humans (9). Therefore, it remains desirable to have both major surface glycoproteins of a candidate vaccine strain antigenically matched with the virus causing disease in humans.
Our studies also demonstrated that alum adjuvant substantially augmented the antibody response to H5 vaccine when mice received two doses of vaccine with adjuvant. However, studies in humans demonstrated that alum was ineffective for enhancing HI antibody to a recombinant H3 HA vaccine (23). These studies were performed with adults who would have previously been infected with related H3N2 viruses and therefore would have cellular responses primed to H3 viruses (14). Because a vaccine against a novel HA subtype may not have this advantage, the effectiveness of adjuvants for vaccination against pandemic influenza should be explored further.
The efficacy of the dk/Sing vaccine supports previous proposals to develop a library of vaccine strains representing different influenza A subtypes that could be evaluated in humans in preparation for future pandemic. Improved surveillance in avian and animal reservoirs may distinguish influenza A subtypes that pose a pandemic threat. These candidate vaccines would be available for a rapid intervention strategy following the emergence and spread of a virus with pandemic potential.
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ACKNOWLEDGMENTS |
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We thank Dennis Alexander, Central Veterinary Laboratory, Surrey, United Kingdom, for providing the dk/Sing virus originally isolated by Yueh Lee-Lin, Veterinary Laboratory Branch, Animal Health and Inspection Division, Singapore; Jing Huang for the confirmatory sequencing of H5N1 virus stocks; Thomas Rowe for preparation of figures; and Kanta Subbarao for critical review of the manuscript.
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ADDENDUM IN PROOF |
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A recent study by Gao et al. (P. Gao, S. Watanabe, T. Ito, H. Goto, K. Wells, M. McGregor, A. J. Cooley, and Y. Kawaoka, J. Virol. 73:3184-3189, 1999) has also demonstrated heterogeneity in the pathogenicity for BALB/c mice of influenza A (H5N1) viruses isolated from humans.
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FOOTNOTES |
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* Corresponding author. Mailing address: Influenza Branch, Mailstop G-16, DVRD, NCID, Centers for Disease Control and Prevention, 1600 Clifton Road, N.E., Atlanta, GA 30333. Phone: (404) 639-3591. Fax: (404) 639-2334. E-mail: jmk9{at}cdc.gov.
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REFERENCES |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Journal of Virology, July 1999, p. 5903-5911, Vol. 73, No. 7
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Leneva, I. A., Goloubeva, O., Fenton, R. J., Tisdale, M., Webster, R. G.
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Perkins, L. E. L., Swayne, D. E.
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O’Neill, E., Krauss, S. L., Riberdy, J. M., Webster, R. G., Woodland, D. L.
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Nishimura, H., Itamura, S., Iwasaki, T., Kurata, T., Tashiro, M.
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Hoffmann, E., Stech, J., Leneva, I., Krauss, S., Scholtissek, C., Chin, P. S., Peiris, M., Shortridge, K. F., Webster, R. G.
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Cauthen, A. N., Swayne, D. E., Schultz-Cherry, S., Perdue, M. L., Suarez, D. L.
(2000). Continued Circulation in China of Highly Pathogenic Avian Influenza Viruses Encoding the Hemagglutinin Gene Associated with the 1997 H5N1 Outbreak in Poultry and Humans. J. Virol.
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Tumpey, T. M., Lu, X., Morken, T., Zaki, S. R., Katz, J. M.
(2000). Depletion of Lymphocytes and Diminished Cytokine Production in Mice Infected with a Highly Virulent Influenza A (H5N1) Virus Isolated from Humans. J. Virol.
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Hiromoto, Y., Yamazaki, Y., Fukushima, T., Saito, T., Lindstrom, S. E., Omoe, K., Nerome, R., Lim, W., Sugita, S., Nerome, K.
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Dybing, J. K., Schultz-Cherry, S., Swayne, D. E., Suarez, D. L., Perdue, M. L.
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Talon, J., Salvatore, M., O'Neill, R. E., Nakaya, Y., Zheng, H., Muster, T., Garcia-Sastre, A., Palese, P.
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