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Journal of Virology, May 2001, p. 4896-4901, Vol. 75, No. 10
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.10.4896-4901.2001
Immunity to Influenza A H9N2 Viruses Induced by
Infection and Vaccination
Xiuhua
Lu,
Mary
Renshaw,
Terrence M.
Tumpey,
Gloria D.
Kelly,
Jean
Hu-Primmer, and
Jacqueline M.
Katz*
Influenza Branch, Division of Viral and
Rickettsial Diseases, National Center for Infectious Diseases,
Centers for Disease Control and Prevention, Atlanta, Georgia 30333
Received 8 November 2000/Accepted 20 February 2001
 |
ABSTRACT |
Avian influenza A H9N2 viruses are widespread among domestic
poultry and were recently isolated from humans with respiratory illness
in China. Two antigenically and genetically distinct groups of H9N2
viruses (G1 and G9) are prevalent in China. To evaluate a strategy for
vaccination, we compared G1 and G9 viruses for their relative
immunogenicity and cross-protective efficacy. Infection of BALB/c mice
with representative viruses of either group protected against
subsequent challenge with the homologous or heterologous H9N2 virus in
the absence of detectable cross-reactive serum hemagglutination inhibition antibody. Mice injected intramuscularly with inactivated G1
whole virus vaccine were completely protected from challenge with
either H9N2 virus. In contrast, mice administered inactivated G9
vaccine were only partially protected against heterologous challenge
with the G1 virus. These results have implications for the development
of human vaccines against H9N2 viruses, a priority for pandemic preparedness.
| |
TEXT |
Avian influenza A H9N2 viruses are
circulating in domestic poultry worldwide (1, 13, 14, 15, 24,
32). Although this avian subtype is generally not highly
pathogenic for avian species, these viruses have recently been
transmitted to mammalian species, including humans (2, 15, 22,
27, 28). In Hong Kong, H9N2 viruses were isolated from domestic
pigs in 1998 and 1 year later were isolated from two children with
uncomplicated febrile respiratory illnesses (2, 22, 27,
28). An additional five human cases of H9N2 influenza infection
in southern China have been reported (15). Three
genetically and antigenically distinct (
4-fold differences in titers
in serologic assays) Eurasian H9 sublineages, represented by
A/Quail/Hong Kong/G1/97 (Qa/G1; G1 group), A/Chicken/Hong Kong/G9/97
(Ck/G9; G9 group), and A/Duck/Hong Kong/Y439/97 (Korean group) viruses
have been identified in Asia (12). A surveillance study
conducted in Hong Kong poultry markets in 1999 resulted in the
isolation of G1 group viruses from 16% of quail (n = 101), while the majority of viruses isolated from 4.7% of
chickens (n = 1,180) were antigenically similar to G9 viruses (13). Qa/G1 and Ck/G9 viruses differ by 8% in
their hemagglutinin (HA) amino acid sequences (12). The
H9N2 viruses isolated from humans in Hong Kong are G1 group viruses,
sharing >99% nucleotide homology with the prototype Qa/G1 virus
(22). In contrast, the viruses isolated from swine belong
to the G9 group (22). Furthermore, seroprevalence studies
suggest that G9 viruses may also have been transmitted to humans
exposed to infected poultry in Hong Kong (Jacqueline M. Katz,
unpublished data). All six internal genes of the G1 group viruses and
the PB1 and PB2 genes of the Ck/G9 virus share a high degree of
nucleotide homology with those of the highly pathogenic H5N1 viruses
isolated from humans in 1997 (12, 14, 22), suggesting that
these viruses may share molecular determinants that facilitate their replication in mammalian species.
Both Eurasian H5N1 and H9N2 viruses have proven their ability to
directly infect humans (4, 6, 15, 20, 22, 28, 34, 35).
However, unlike the H5N1 viruses, H9N2 viruses are currently widespread
in domestic poultry in southern China (13, 15, 32) and
thus remain a potential source of further human infections and,
possibly, a new pandemic strain. A virus with the ability to be
efficiently transmitted among humans may arise by mutation of the avian
H9N2 virus genome and/or by reassortment between this avian and a human
influenza A virus. Humans, as well as swine, must now be considered a
likely "mixing vessel" for a reassortment event with pandemic
consequences. Therefore, the development of a human influenza vaccine
for H9N2 viruses is considered a high priority for pandemic
preparedness. To provide a rational basis for vaccine development, we
compare here the relative immunogenicity of G1 and G9 viruses in mice
and evaluate each as a candidate strain for the development of an
inactivated vaccine against H9N2 viruses.
Replication, immunogenicity, and cross-protective efficacy of live
H9N2 viruses.
Qa/G1 and Ck/G9 viruses have been shown to replicate
in BALB/c mice without prior adaptation (14). In this
study, we have used an H9N2 virus isolated in a human, A/Hong
Kong/1073/99 (HK/1073), as a prototype G1 virus together with Ck/G9,
the prototype G9 virus. To compare the infectivity of these viruses, 6- to 8-week-old female BALB/c mice (Charles River Laboratories,
Wilmington, Mass.) were infected intranasally (i.n.) under light
CO2 anesthesia with 106 50% egg infectious
doses (EID50) of HK/1073 or Ck/G9 virus. At various times
postinfection (p.i.), mice were euthanized and lungs were collected and
titrated for virus as previously described (18). As shown
in Fig. 1A, the lung virus titers of mice
infected with the HK/1073 virus were consistently 15-fold higher than those of mice infected with the Ck/G9 virus at each time point tested
(P < 0.05). Neither H9N2 virus replicated in
extrapulmonary organs, caused significant weight loss, or caused fatal
disease in mice following i.n. inoculation. The 50% lethal doses for
HK/1073 and Ck/G9 viruses were >108.0 EID50.
Similar results were obtained with other G1 group (Qa/G1 and another
human isolate, A/Hong Kong/1074/99) and G9 group (A/Swine/Hong Kong/10/98) viruses (data not shown). Thus, all of the H9N2 viruses studied failed to replicate outside the respiratory tract and caused no
morbidity or mortality in mice. In contrast, Guo et al.
(14) reported that Qa/G1 virus caused 37% mortality and replicated in mouse brains. Sequence analysis of the HA and
neuraminidase (NA) genes of the viruses used in the present study
failed to identify any consistent molecular basis for this difference
between studies.
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FIG. 1.
Replication and serum HI antibody production in mice
infected with H9N2 viruses. (A) Kinetics of replication of H9N2 viruses
in mouse lungs. Mice were infected i.n. with 106
EID50 of each virus. Lungs from three mice per group were
harvested at the indicated times and titrated in embryonated eggs. The
limit of virus detection was 101.2 EID50/ml.
(B) Serum HI antibody responses of mice infected with H9N2 viruses. HI
titers are the reciprocal of the highest dilution of serum that
completely inhibited agglutination of 0.5% turkey red blood cells by 4 hemagglutinating units of virus. Antibody titers are expressed as the
mean log2 titer of five mice per group.
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To evaluate the relative immunogenicity of G1 and G9 group viruses,
serum antibodies were measured in mice that had been infected
i.n. 1 month previously with 10
7 to 10
2
EID
50 of either HK/1073 or Ck/G9 virus (Fig.
1B). Sera were
collected
from five mice per group, treated with receptor-destroying
enzyme,
and tested by the hemagglutination inhibition (HI) assay as
previously
described (
21). Homologous HI antibody titers
induced by Ck/G9
virus were
4-fold higher than those induced by the
HK/1073 virus.
Similar differences in serum HI and neutralizing
antibody titers
were obtained when the other G1 and G9 group viruses
listed above
were compared (data not shown). These results indicated
that G9
group viruses were more immunogenic than G1 group viruses,
despite
replicating to lower titers in mouse
lungs.
To better understand the extent of cross-protective immunity between G1
and G9 group viruses, mice infected with either H9N2
virus 1 month
previously were tested for the presence of cross-reactive
serum HI and
neutralizing antibody (
31) and were then challenged
i.n.
with 10
6 EID
50 of either HK/1073 or Ck/G9
virus. As shown in Table
1,
mice infected
with the HK/1073 virus had low HI and neutralizing
antibodies to
HK/1073 virus but no detectable antibody to Ck/G9
virus. Similarly,
sera from mice infected with Ck/G9 virus had
substantial HI and
neutralizing antibody titers to Ck/G9 virus
but failed to react with
heterologous HK/1073 virus. Despite the
lack of cross-reactive
antibody, mice infected with HK/1073 virus
were completely protected
from lung infection with either HK/1073
or Ck/G9 virus on day 4 p.i. Likewise, mice previously infected
with a Ck/G9 virus were
essentially protected from challenge with
virus of either group. Only
one of five mice infected with Ck/G9
virus and subsequently challenged
with HK/1073 virus had a detectable
lung virus titer (10
3.9
EID
50/ml). Similar cross-protection was achieved between
other
G1 and G9 group viruses (data not shown). These results indicated
that infection of mice with live G1 or G9 group virus resulted
in
protection from challenge with the heterologous H9N2 virus
in the
absence of detectable cross-reactive serum HI and neutralizing
antibody
responses.
To further investigate the anti-H9 HA antibody response following
infection, sera and mucosal washes were collected and tested
for
anti-HA-specific immunoglobulin G (IgG), IgA, and IgM antibodies
by
enzyme-linked immunosorbent assay (ELISA) as previously described
(
18,
19) but using a purified baculovirus-expressed
recombinant
HK/1073 HA protein (Protein Sciences Corporation, Meriden,
Conn.;
1 µg/ml). Purified Ck/G9 HA was not available for this study.
No significant differences in serum and mucosal wash antibodies
were
observed among mice infected i.n. with either HK/1073 or
Ck/G9 virus,
despite the fact that antibody was detected with
HK/1073 HA (Fig.
2). These results demonstrated that serum
IgG,
IgA, and IgM antibodies and mucosal wash IgG and IgA antibodies
that cross-react with the HK/1073 G1 group HA are induced in mice
infected with Ck/G9 virus.
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FIG. 2.
Anti-HK/1073 HA antibody responses after infection with
H9N2 viruses. Mice were infected i.n. with 107
EID50 of HK/1073 or Ck/G9 virus. Two months later, antibody
samples were collected from 10 animals per group and were tested by
ELISA for the presence of IgG, IgM, and IgA antibody using a purified
HK/1073 virus HA recombinant protein. Antibody responses are expressed
as reciprocal endpoint titers + standard deviations (SD).
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Immunogenicity and cross-protective efficacy of inactivated H9N2
virus vaccines.
We next compared inactivated whole virus vaccines
prepared from HK/1073 (G1 group) or Ck/G9 (G9 group) virus for their
relative immunogenicity and cross-protective efficacy in mice. The
viruses were concentrated from allantoic fluid and purified on a linear sucrose gradient as described previously (5) and were
inactivated by treatment with 0.025% formalin at 4°C for 3 days. The
HA content of the purified viruses was estimated to be 35 to 39% of
the total viral proteins by densitometric analysis of viral protein
bands separated by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (26). Mice were immunized once
intramuscularly (i.m.) with 10 µg of purified inactivated vaccine, in
the presence or absence of 1% alum (Alhydrogel; Superfos Biosectors,
Kvistgaard, Denmark), as previously described (23). As
shown in Table 2, the administration of
vaccine alone resulted in 100% of mice achieving an HI titer of 40
to the homologous virus. The addition of alum resulted in homologous HI
titers that were 6- to 12-fold higher than titers achieved without
adjuvant. HK/1073 vaccine also induced modest titers of antibody to the
heterologous Ck/G9 virus, which were slightly enhanced by alum. In
contrast, the Ck/G9 vaccine induced only G9 strain-specific HI
antibody, even following coadministration of Ck/G9 vaccine with alum. A
microneutralization assay also failed to detect any cross-reactive
antibody in sera from animals vaccinated with Ck/G9 virus, in either
the presence or absence of alum (data not shown). Next, the protective
efficacy of the H9N2 vaccines was investigated 3 days p.i. (Table 2).
The level of viral replication in the lower and upper respiratory tract
of mice was determined. Lung and anterior nasal (nose) tissue were
collected, homogenized, and titrated for virus infectivity in eggs as
previously described (19). Mice administered HK/1073
vaccine were completely protected from infection of the lung with
homologous HK/1073 virus and only one of seven mice had a low titer of
virus in the nose. The HK/1073 vaccine also substantially protected
mice from challenge with heterologous Ck/G9 virus, and when formulated
with alum, the vaccine was 100% cross-protective. Mean virus titers
were reduced by >104-fold in the lung and by 6-fold in
the nose. While mice immunized with Ck/G9 vaccine were essentially
protected from homologous virus challenge in both the lungs and noses,
only partial protection from heterologous challenge with HK/1073 virus
was demonstrated. Following HK/1073 virus challenge, virus was detected
in the lungs and noses of six of seven mice that received the Ck/G9
vaccine alone. Nevertheless, the mean lung virus titers were
significantly lower than for mice that received alum alone but were
significantly higher than for those that received the HK/1073 vaccine
(P < 0.01). Although the addition of alum to the Ck/G9
vaccine resulted in a further reduction of virus titers, protection
from HK/1073 virus infection was again incomplete. These results
suggest that inactivated vaccines made from HK/1073 viruses offered
more complete protection from heterologous H9 virus challenge than did
the Ck/G9 vaccine. Nevertheless, the Ck/G9 vaccine provided partial
protection from HK/1073 virus challenge in the absence of a
cross-reactive serum HI and neutralizing antibody responses (data not
shown), suggesting a role for cellular immunity or antibody that is not
detected by HI and microneutralization assays.
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TABLE 2.
Protective efficacy of influenza H9N2 inactivated
vaccines against infection with HK/1073 and Ck/G9 viruses
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To determine whether vaccination with Ck/G9 vaccine induced any serum
antibody response to HK/1073 virus HA, IgG, IgM, and
IgA serum
antibodies were measured by ELISA using the HK/1073
recombinant HA
protein. Mucosal antibody responses were not measured
because systemic
vaccination with inactivated influenza vaccine
typically does not
induce mucosal antibodies in mice (
18). As
shown in Fig.
3, vaccination with either of the H9N2
vaccines
without alum induced substantial serum anti-HK/1073 HA IgG and
lower levels of IgM and IgA antibody responses. In particular,
mice
immunized with Ck/G9 vaccine mounted a substantial anti-HK/1073
HA IgG
antibody response but the response was significantly lower
than that of
mice receiving HK/1073 vaccine alone (
P < 0.01).
Coadministration of alum with the vaccines enhanced IgG responses
four-
to eightfold (
P < 0.01) and enhanced IgM responses
two-
to fourfold but did not augment IgA responses. Formulation of
the
Ck/G9 vaccine with alum enhanced the IgG response to HK/1073
HA
eightfold. These results suggest that the partial protection
against
HK/1073 virus challenge observed in mice vaccinated with
Ck/G9 virus
may, in part, be associated with the induction of
non-HI antibody that
is cross-reactive for HK/1073 HA.
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FIG. 3.
Comparison of serum antibody responses following
vaccination of mice with inactivated H9N2 vaccines. Twenty mice per
group were vaccinated i.m. with 10 µg of purified
formalin-inactivated H9N2 virus vaccine alone, vaccine with alum, or
alum alone. Three months later, serum from 10 mice per group were
collected and anti-HK/1073 HA IgG, IgA, and IgM antibodies were
detected by ELISA. Antibody responses are expressed as reciprocal
endpoint titers + standard deviations (SD).
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Since H9N2 viruses that possess HA antigenically similar to G1 and G9
viruses have continued to circulate in Asia, it was
important to
determine the cross-protective efficacy that a vaccine
of one H9
sublineage afforded against a virus of the other sublineage.
Infection
of mice with either live G1 or G9 group viruses resulted
in essentially
complete cross-protection against subsequent reinfection
with the
heterologous virus, despite a lack of detectable HI or
neutralizing
cross-reactive serum anti-H9 antibody. However, mice
infected with the
Ck/G9 virus had substantial titers of anti-HK/1073
HA (G1 group) serum
IgG, IgA, and IgM and mucosal IgG and IgA
antibody, suggesting that
these antibody responses may play a
role in cross-protection. Although
primary infection with one
H9N2 virus may have also induced anti-NA
antibody and a memory
cytotoxic T lymphocyte response that may
contribute to enhanced
viral clearance, it is unlikely that either of
these immune mechanisms
alone would result in a complete lack of virus
4 days after reinfection
with the heterologous virus (
3,
17). O'Neill et al. (
25)
have demonstrated that
infection with the H9N2 virus protected
C57BL/6 mice against lethal
challenge with an H5N1 virus. Such
heterosubtypic immunity is typically
the result of only a modest
reduction in lung virus titers measured on
or after day 5 p.i.
(
11).
Although the protective immunity induced by the HK/1073 inactivated
vaccine was more cross-reactive, the Ck/G9 vaccine induced
higher
titers of homologous antibody, particularly when coadministered
with
alum (Table
2). Ck/G9 virus also induced substantially higher
HI
antibody titers in infected mice compared with HK/1073, despite
the
fact that Ck/G9 replicated less efficiently in the lungs of
mice (Fig.
1). One reason for the observed differences in immunogenicity
and
antigenicity between the G1 and G9 group viruses may be the
differential glycosylation of their HA molecules. Sodium dodecyl
sulfate-polyacrylamide gel electrophoresis analysis of the viruses
used
in the present study revealed a higher molecular weight for
the HA1
polypeptides of G1 group viruses that was consistent with
the presence
of additional glycosylation sites predicted by nucleotide
sequence
analysis (
12) (data not shown). Oligosaccharide side
chains are known to affect both B- and T-cell recognition of the
HA
(
9,
16,
33). In addition, oligosaccharides at the distal
tip of the HA may reduce receptor binding (
7,
8), which
may, in turn, affect the uptake of virus by antigen-presenting
cells
(
10).
The optimal strategy for control of pandemic influenza is early
intervention with a vaccine produced, ideally, from the actual
pandemic
strain, or at least from a related strain that is a close
antigenic
match. Our results suggest that the HK/1073 vaccine
induced antibody
that was more broadly cross-reactive as well
as more cross-protective
against the heterologous H9 virus than
that induced by the Ck/G9
vaccine. The HK/1073 virus has the further
advantage of growing to high
titers in embryonated eggs and may
eliminate the need for preparation
of a high-growth reassortant
for human vaccine preparation.
Furthermore, unlike the poor immunogenicity
of H5 vaccine in animals
(
23,
30), a single dose of inactivated
H9 vaccine was
sufficient to induce homologous serum HI antibody
titers that were
comparable to the HI antibody response in infected
mice (Table
2) and
provided essentially complete homologous protection.
Nevertheless, the
cross-protective efficacy of both H9N2 vaccines
was improved by the
coadministration of alum. It will be important
to determine whether H9
vaccine candidates are similarly immunogenic
in humans and whether
inactivated vaccines prepared from an HK/1073
virus can induce titers
of cross-reactive HI antibody that may
be considered adequate for
protection in
humans.
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ACKNOWLEDGMENTS |
We thank Wilina Lim, Government Virus Unit, Department of Health,
Hong Kong, for providing the human influenza A H9N2 virus isolates;
Robert Webster, Department of Virology and Molecular Biology, St. Jude
Children's Research Hospital, Memphis, Tenn., and Alan Hay, Division
of Virology, National Institute for Medical Research, Mill Hill,
London, for providing avian and swine H9N2 influenza virus isolates,
respectively; Yumiko Matsuoka and Jing Huang for assistance in sequence
analyses; Thomas Rowe for preparation of figures and technical support;
and Nancy Cox and Kanta Subbarao for critical reviews of the manuscript.
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FOOTNOTES |
*
Corresponding author. Mailing address: Influenza
Branch, Mailstop G-16, DVRD, NCID, Centers for Disease Control and
Prevention, 1600 Clifton Rd., N.E., Atlanta, GA 30333. Phone: (404)
639-4966. Fax: (404) 639-2334. E-mail: JKatz{at}cdc.gov.
Present address: USDA/ARS/Southeast Poultry Research Laboratory,
Athens, GA.
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Journal of Virology, May 2001, p. 4896-4901, Vol. 75, No. 10
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.10.4896-4901.2001
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