Pathogenicity and immunogenicity of influenza viruses with genes from the 1918 pandemic
Swayne, and Christopher F. Basler
Terrence M. Tumpey, Adolfo García-Sastre, Jeffery K. Taubenberger, Peter Palese, David E.
2004;101;3166-3171; originally published online Feb 12, 2004;
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Pathogenicity and immunogenicity of influenza
viruses with genes from the 1918 pandemic virus
Terrence M. Tumpey*†, Adolfo Garcı ´a-Sastre‡, Jeffery K. Taubenberger§, Peter Palese‡, David E. Swayne*,
and Christopher F. Basler‡
*Southeast Poultry Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Athens, GA 30605;‡Department of Microbiology,
Mount Sinai School of Medicine, New York, NY 10029; and§Division of Molecular Pathology, Department of Cellular Pathology and Genetics, Armed Forces
Institute of Pathology, Rockville, MD 20850
Contributed by Peter Palese, December 16, 2003
The 1918 influenza A H1N1 virus caused the worst pandemic of
influenza ever recorded. To better understand the pathogenesis and
immunity to the 1918 pandemic virus, we generated recombinant
influenza viruses possessing two to five genes of the 1918 influenza
virus. Recombinant influenza viruses possessing the hemagglutinin
protein (NP) genes or any recombinant virus possessing both the HA
and NA genes of the 1918 influenza virus were highly lethal for mice.
Antigenic analysis by hemagglutination inhibition (HI) tests with
ferret and chicken H1N1 antisera demonstrated that the 1918 recom-
binant viruses antigenically most resembled A?Swine?Iowa?30 (Sw?
Iowa?30) virus but differed from H1N1 viruses isolated since 1930. HI
and virus neutralizing (VN) antibodies to 1918 recombinant and
Sw?Iowa?30 viruses in human sera were present among individuals
intramuscular immunization of the homologous or Sw?Iowa?30-
inactivated vaccine developed HI and VN antibodies to the 1918
recombinant virus and were completely protected against lethal
challenge. Mice that received A?PR?8?34, A?Texas?36?91, or A?New
Caledonia?20?99 H1N1 vaccines displayed partial protection from
lethal challenge. In contrast, control-vaccinated mice were not pro-
tected against lethal challenge and displayed high virus titers in
respiratory tissues. Partial vaccine protection mediated by baculo-
virus-expressed recombinant HA vaccines suggest common cross-
reactive epitopes on the H1 HA. These data suggest a strategy of
vaccination that would be effective against a reemergent 1918 or
high mortality rate, especially among young adults, was not ob-
served during later influenza pandemics of 1957 and 1968 (5, 6). It
was estimated that ?30% of the world’s population was clinically
infected during the 1918 pandemic (7). Sequence analysis of the
molecular characterization and phylogenetic analysis of this strain.
The complete coding sequences of the 1918 nonstructural (NS),
hemagglutinin (HA), neuraminidase (NA), and matrix (M) genes
have been determined (8–14); however, the sequences of these
genes did not reveal features that could account for its high
virulence. The sequence analysis combined with the laboratory
method of reverse genetics has allowed for the generation of
recombinant viruses containing one or more 1918 influenza virus
genes entirely from cloned cDNAs (14–16). This technology was
effective against a reemergent 1918 influenza virus. We found that
a recombinant virus possessing the 1918 M segment was inhibited
effectively both in tissue culture and in vivo by the M2 ion-channel
inhibitors amantadine and rimantadine (15). Moreover, a recom-
binant virus bearing the surface glycoproteins, HA and NA, of the
1918 pandemic influenza virus (1918 HA?NA:WSN) with the
remaining genes of influenza A?WSN?33 virus was found to be
sensitive in vitro and in vivo to the NA inhibitors zanamivir and
oseltamivir. The 1918 HA?NA:WSN virus had a high virulence
uring 1918 and 1919, the ‘‘Spanish’’ influenza pandemic killed
up to forty million people worldwide (1–4). The exceptionally
phenotype on intranasal (i.n.) infection in mice without prior
virus with both the HA and NA of the A?New Caledonia?20?99
1918 HA?NA:WSN or parental WSN virus (15).
The HA and NA transmembrane glycoproteins are the major
viral surface antigens that define an influenza virus strain and are
important virulence factors in birds and mice (17–21). These
glycoproteins evolve simultaneously creating balanced HA–NA
functional interactions important for efficient replication of influ-
enza A viruses (22). Indeed, our previous observations demon-
strated that the 1918 HA and NA proteins appear to be compatible
with each other as recombinant viruses possessing either the 1918
HA or 1918 NA individually led to attenuation in mice (15). The
HA is also the principal target of the host’s immune system and
protective immunity provided by current influenza vaccines is
largely based on the induction of strain-specific IgG neutralizing
antibodies directed against the HA. Major antigenic changes
through HA and NA gene reassortment have occurred to create
new human pandemic viruses that possess the ability to evade
existing immunity. Although evidence suggests that the 1957 Asian
and 1968 Hong Kong pandemic strains emerged after genetic
the origin of the 1918 pandemic virus has not been precisely
elucidated. Phylogenetic and sequence analysis placed the 1918
viral HA within the mammalian group of influenza A viruses and
having a close genetic relationship with the oldest available swine
influenza strain, A?Swine?Iowa?30 (Sw?Iowa?30, H1N1).
The basis for the exceptional virulence of the 1918 pandemic
virus has remained elusive because no influenza virus isolates from
the period have been available for study. In the present investiga-
tion, we generated recombinant influenza viruses possessing from
report here that these recombinant viruses replicated efficiently in
mouse lungs, without prior adaptation and were highly lethal for
BALB?c mice. These results indicate that the mouse model of
influenza virus infection might give insights into the pathogenicity
of the 1918 virus. We also have antigenically characterized these
recombinant viruses and identified vaccine strategies capable of
inducing protective immunity against viruses with antigenic deter-
1918-like viruses (24).
Abbreviations: eID50, egg 50% infectious dose; HA, hemagglutinin; HI, hemagglutination
inhibition; i.n., intranasal(ly); M, matrix; MDCK, Madin–Darby canine kidney; NA, neur-
aminidase; New Cal?99, influenza A?New Caledonia?20?99 (H1N1) virus; NP, nucleopro-
tein; NS, nonstructural; p.c., postchallenge; p.i., postinfection; Sw?Iowa?30, influenza
A?Swine?Iowa?30 (H1N1) virus; rHA, recombinant HA; WSN, influenza A?WSN?33 (H1N1)
virus; VN, virus neutralization.
†To whom correspondence should be addressed at: Influenza Branch, Division of Viral and
Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control
and Prevention, Mailstop G-16, 1600 Clifton Road Northeast, Atlanta, GA 30333. E-mail:
© 2004 by The National Academy of Sciences of the USA
March 2, 2004 ?
vol. 101 ?
Materials and Methods
Generation of 1918 HA, NA, NP, NS, and M cDNAs and Recombinant
Viruses. The 1918 HA, NA, NP, M, and NS cDNAs were con-
structed by PCR, using overlapping deoxyoligonucleotides corre-
sponding to the published sequence of the influenza A?South
Carolina?1?18 (H1N1) virus HA (12) ORF, the influenza A?Bre-
vig Mission?1?18 (BM?1?18, H1N1) virus NA ORF (11), the
BM?1?18 virus NP ORF (A. H. Reid, R. Lourens, T. A. Janczew-
ski, T. G. Fanning, and J.K.T., unpublished work), the influenza
BM?1?18 virus M ORF (8), or the influenza BM?1?18 virus NS
ORF (14). The noncoding regions of each segment are identical to
H1N1 virus. Primer sequences and PCR reaction conditions are
available on request. Recombinant viruses were generated by using
the reverse genetics system of Fodor et al. (25), following the
methods of Basler et al. (14). Generation of viruses possessing 1918
genes in a WSN background was performed under biosafety level
3 Ag containment (26) to ensure the safety of laboratory workers,
work with live virus was also performed under these high contain-
ment conditions. The identity of the 1918 influenza virus genes
in the recombinant viruses was confirmed by RT-PCR and
Infection of Mice. Male BALB?c mice, 6–7 weeks old (Simonsen
Laboratories, Gilroy, CA), were anesthetized with ketamine-
xylazine (1.98 and 0.198 mg per mouse, respectively), and 50 ?l of
infectious virus diluted in PBS was inoculated i.n. LD50titers were
determined by inoculating groups of three mice i.n. with serial
the highest inoculating dose (106plaque-forming units) for deter-
mination of weight loss and virus titers in lungs. Whole lungs were
removed on day 4 postinfection (p.i.) and homogenized in 1 ml of
10-day-old embryonated eggs (15). Egg 50% infectious dose
(eID50?ml) titers were calculated by the method of Reed and
Human Serum Samples. For the first serology test, nine human sera
were obtained from randomly chosen volunteers (age range 36–93
years) in March of 2002 and stored at ?70°C before influenza
hemagglutination inhibition (HI) and virus neutralization (VN)
analysis. In the second serology test, serum samples were obtained
from individuals pre- and postvaccination. All subjects were organ
transplant patients who received vaccine as a prophylactic measure
before the 2001 influenza season. A total of 15 subjects, born
between 1936 and 1956, received inactivated New Cal?99 vaccine
or placebo control.
Vaccine Preparation. Viruses used as vaccines were concentrated
from allantoic fluid and purified by equilibrium density centrifu-
gation through a 30–60% linear sucrose gradient as described (28).
For inactivation, purified whole viruses were adjusted to a protein
concentration of 1 mg?ml and treated with 0.025% formalin at 4°C
for 3 days. The vaccine doses given throughout are expressed as
amounts of total protein measured by Bradford assay (Bio-Rad).
Immunization of Mice. Groups of BALB?c mice (n ? 13) were
injected i.m. with a single dose of 10 ?g of formalin-inactivated
vaccine as described (29). Vaccines were suspended in sterile PBS,
and a volume of 0.1 ml was injected in the left hind leg. Mock
control mice received PBS in place of H1N1 vaccine. Subtype
control mice received a similar dose of X-31 (which possesses the
surface glycoprotein genes of A?Aichi?2?68 [H3N2] and the in-
ternal protein genes of A?Puerto Rico?8?34) vaccine virus (30).
Baculovirus-expressed recombinant HA (rHA) protein, corre-
sponding to the HA of A?Texas?36?91 (Tx?91), New Cal?99, or
A?Panama?2007?99 (H3N2) virus, was acquired from Protein
Sciences (Meriden, CT). Three weeks after vaccination, sera from
nine individual mice per group were collected for antibody studies.
Antibody Assays. All sera were initially diluted 1:10 in receptor-
in Madin–Darby canine kidney (MDCK) cell cultures. Antigenic
analysis of the H1N1 viruses was performed with reference H1N1
virus stocks and corresponding p.i. ferret antisera, generously
Atlanta). Chicken antisera were generated by infecting animals i.v.
with 106eID50of virus in a 0.2-ml volume, followed by a s.c. boost
3 weeks later.
Viral Challenge. Three weeks after vaccination, mice were chal-
lenged i.n. with 100 LD50of 1918 HA?NA:WSN or 1918 HA?NA?
M?NP?NS:WSN virus in a volume of 50 ?l. After infection, nine
mice were monitored daily for disease signs for 14 days p.i. To
evaluate protection of the nose, lung, and brains from infection,
tissue samples of four mice per group were removed on day 5 p.i.
and titrated for virus infectivity as described above.
Construction and Characterization of Recombinant Viruses with 1918
Influenza Virus Genes.Genesencodingthe1918pandemicinfluenza
virus were reconstructed from deoxyoligonucleotides and corre-
viral segments not derived from the 1918 influenza virus were
derived from the mouse-adapted WSN virus. Recombinant influ-
enza viruses were created expressing two to five genes of the 1918
embryonated eggs (7.2–8.8 log10 eID50?ml). The 1918 HA?
NA:WSN virus (15) was included in these pathotyping studies for
M, NS, or NP genes. The mean virus titers in the lungs were
determined on day 4 p.i., when titers were maximal. All four 1918
caused lethal disease without prior host adaptation (Table 1). The
LD50titers and percent weight loss observed in mice infected with
each of the 1918 recombinant viruses were not significantly differ-
ent from each other or mice infected with the parental WSN virus
(Table 1). Mice infected with lethal doses of the 1918 recombinant
or WSN virus began to lose weight 2 days after infection and died
5–9 days p.i.
Table 1. Properties of recombinant influenza viruses used in
Parental WSN virus
2.2 ? 107
2.1 ? 107
1.4 ? 108
2.1 ? 107
1.4 ? 108
6.7 ? 0.2
7.3 ? 0.1
7.9 ? 0.2
7.3 ? 0.2
7.4 ? 0.2
*All viral genomic segments were derived from the WSN virus unless other-
†Titer of virus stocks prepared on MDCK cells.
‡Average percent weight loss on day 4 p.i. (five mice per group).
§Average lung titers of four mice on day 4 p.i. expressed as eID50?ml ? SE.
¶Expressed as the log10plaque-forming units required to give 1 LD50.
Tumpey et al.
March 2, 2004 ?
vol. 101 ?
no. 9 ?
Antigenic Reactivity of Selected Viruses in HI Test with Ferret and
Chicken Serum. Antigenic characterization by HI, using p.i. ferret
and chicken antisera, was performed with the 1918 HA?NA
recombinant virus and reference variants representing early and
recent H1N1 viruses. The ferret antisera revealed that the 1918
HA?NA recombinant virus was antigenically most related to
Sw?Iowa?30 virus but distinct from all other influenza A (H1N1)
viruses examined (Table 2). Similarly, chicken H1N1 antisera
confirmed the antigenic cross-reactivity observed among the 1918
and Sw?Iowa?30 strains, with progressive diminution of inhibition
with subsequent H1N1 strains (Table 3). There was low reactivity
to the 1918 HA?NA viruses with ferret and chicken antisera to
WS?33, PR?8?34, and Tx?91 viruses.
Serological Reactivity of Human Sera with 1918 HA?NA Recombinant
Virus. Sera from nine humans ranging from 36 to 93 years of age
were tested for HI activity against Sw?Iowa?30, PR?8?34, New
Cal?99, or 1918 HA?NA recombinant virus. A virus neutralization
assay was also included, because with some influenza viruses it
provides greater sensitivity than the commonly used HI test (33).
Individuals born before 1918 have the highest levels of HI and VN
antibodies to the 1918 HA?NA recombinant virus but no reactivity
antibody in these human sera was generated by natural infection.
Two (serum C and E) of three individuals born between 1928 and
1933 possessed HI and VN antibodies to 1918 HA but also
Individuals born after 1962 (G and H) had HI and VN antibody
titers of ?20 antibodies to 1918 HA. Overall, the four individuals
(A–C and E) with detectable HI and neutralization antibodies to
the 1918 HA?NA virus also possessed reactivity to Sw?Iowa?30
virus, indicating the antigenic relatedness of the two viruses. In a
induction of cross-reactive antibodies to 1918 HA?NA virus was
investigated. Volunteers, born between 1936 and 1956, were ad-
ministered influenza vaccine following standard vaccination pro-
cedures, and serum was collected from 15 subjects before and after
Individuals with paired serum samples were tested for HI activity
to New Cal?99, 1918 HA, and Sw?Iowa?30 viruses. Before vacci-
(?20) against all three viruses tested. Sera collected three weeks
postvaccination showed increases in HI antibody titers to New
5). Although, the HI antibody response to Sw?Iowa?30 and 1918
HA antigens was considerably lower in comparison to New Cal?99
virus antigen, HI titer increases to these viruses were also observed
after New Cal?99 vaccination.
Protective Efficacy of H1N1 Vaccines. The mouse model was used to
evaluate a strategy of vaccination against the lethal 1918 recombi-
nant virus. In the first vaccine experiment, we tested the ability of
three inactivated H1N1 vaccines to induce protection against the
lethal 1918 HA?NA recombinant virus. Vaccinated mice received
10 ?g of formalin-inactivated whole H1N1 or control (H3N2)
vaccine, and 3 weeks after inoculation mice received a lethal i.n.
challenge with 100 LD50of 1918 HA?NA:WSN recombinant virus.
The extent of vaccine efficacy was measured as (i) weight loss and
in the upper respiratory tract (nose), lower respiratory tract (lung),
and brain tissue of individual mice. Immunization with PR?8?34 or
homologous 1918 HA?NA:WSN virus vaccine protected the mice
against lethal virus challenge, whereas 75% of New Cal?99-
vaccinated mice were protected (Fig. 1A). Although PR?8?34- and
New Cal?99-vaccinated mice were mostly protected against death,
they all had significant weight loss (data not shown) and listlessness
the first week of infection; these were taken as signs of morbidity.
Lethal H1N1 challenge of H3N2-vaccinated or unvaccinated
(mock) control mice resulted in a progressive loss of body weight
from day 2 p.c. and death after virus challenge. Furthermore,
control mice had high titers of virus in the lung and nose tissue at
day 5 p.c. (Fig. 1B). Infectious virus was also present in the brain
Table 2. HI reactions of H1N1 virus variants with ferret antisera
HI titer with ferret antisera
1918 HASw?Ia?30WS?33PR?8?34USSR?77Chili?83Tx?91New Cal?99
dilution of sera inhibiting agglutination of 0.5% chicken erythrocytes by 4 hemagglutination units of virus.
Table 3. HI reactions of H1N1 virus variants with p.i. chicken antisera
HI titer with chicken antisera
1918 HA Sw?Ia?30 WS?33 PR?8?34USSR?77 Chili?83Tx?91New Cal?99
Serum samples from chickens infected with indicated H1N1 viruses. HI titers represent reciprocal of highest
dilution of sera inhibiting agglutination of 0.5% chicken erythrocytes by 4 hemagglutination units of virus.
www.pnas.org?cgi?doi?10.1073?pnas.0308391100Tumpey et al.
tissue of 2 of 4 mock-control and X-31-vaccinated mice, but the
titers of virus were considerably lower in comparison to respiratory
tissues. Protection against infection was incomplete in mice vacci-
nated with either New Cal?99 or PR?8?34 vaccine, although these
mice displayed significant reductions (16- and 500-fold, respec-
tively) in lung virus titers compared to the unvaccinated control
mice. In contrast, the 1918 HA?NA:WSN homologous vaccine
In a second vaccine study, mice received 10 ?g of inactivated
H1N1 vaccines or purified H1 rHA proteins generated in insect
cells by using the recombinant baculovirus system (34). Three
weeks after inoculation, mice received a lethal challenge with 100
LD50of 1918 HA?NA?M?NP?NS:WSN recombinant virus. Mice
that received homologous 1918 HA?NA vaccine virus or Sw?
Iowa?30 whole virus vaccine were completely protected from
death, whereas 50–90% of mice administered PR?8?34, New
Cal?99, or Tx?91 whole virus vaccine survived the lethal challenge
(Fig. 2A). Despite the degree of resistance to lethal virus challenge
observed in PR?8?34-, New Cal?99-, or Tx?91-immunized mice,
the lung and nose virus titers at 5 days p.c. were 200- to 50,000-fold
higher than virus titers observed in Sw?Iowa?30-vaccinated mice.
Infectious virus was undetectable in brain and respiratory tissues of
mice administered homologous 1918 HA?NA or Sw?Iowa?30
succumbed to the lethal H1N1 infection and had high titers in the
respiratory tissues and low titers in brain tissues at 5 days p.c. Mice
were vaccinated with rHA from H1N1 viruses to determine
whether the partial cross-protection induced by these vaccines was
due to anti-HA immunity. An influenza recombinant protein
to the HA of Tx?91 or New Cal?99 virus resulted in 50–60%
mice corresponding to the rHA-immunized groups had high titers
of infectious virus in the respiratory tissues (Fig. 2B).
The prechallenge antibody responses to the 1918 HA?NA re-
Table 4. VN and HI antibody responses to H1N1 viruses detected
in human sera
Neutralization†and HI‡antibody titer
*Serum samples from individuals ranging from 36 to 93 years of age.
†Reciprocal dilution endpoint in VN titers.
‡Reciprocal dilution in HI titration (in parentheses).
Table 5. HI antibody responses to H1N1 viruses before and after A?New Cal?20?99 vaccination
HI antibody titer†
1, 16, 18, 21, and 50 were from placebo controls.
†Reciprocal dilution in HI titration.
mice received a single i.m. inoculation of H1N1 or H3N2 (X-31) vaccine. Control
mice received PBS in place of vaccine. Twenty-two days after vaccination, mice
lung, nose, and brain tissue were determined (B). Virus endpoint titers are
expressed as mean log10eID50?ml.
Tumpey et al.
March 2, 2004 ?
vol. 101 ?
no. 9 ?
combinant virus was measured in individual serum samples col-
mice achieving an HI titer of ?40 to the homologous virus (29).
Each inactivated or rHA vaccine elicited HI titers of 40 or greater
contrast, the PR?8?34, Tx?91, and New Cal?99 vaccines failed to
induce HI and VN antibodies to the 1918 HA?NA:WSN recom-
greatest antigenic similarity to the 1918 influenza virus.
not revealed any obvious features that could account for its high
virulence thus far. By contrast, the analysis in mice of recombinant
WSN viruses containing two to five genes derived from the 1918
virus point to a critical role of the 1918 HA and NA genes in
virulence, at least in the mouse model. Our previous observations
demonstrated that replacement of the HA and NA genes of WSN
virus by those of the 1918 virus did not decrease virulence in mice,
an unusual outcome due to the absence of previous mouse adap-
tation of these genes (15). To understand the contribution of
additional 1918 genes in the mouse model of influenza virus
pathogenicity, recombinant viruses possessing one to three addi-
tional (M, NS, and NP) 1918 genes were generated. Each of the
recombinant viruses possessing two to five genes of the 1918
pandemic virus replicated efficiently in mouse respiratory tissues
and were highly lethal for this species. Introduction into the 1918
genes did not significantly increase the virulence of this virus. The
ability of the 1918 recombinant viruses to cause lethal disease in
mice without prior adaptation was remarkable given that the 1918
genes were derived directly from sequences corresponding to a
human virus. Generally, prior adaptation is required before influ-
enza A viruses can replicate efficiently and induce disease in mice
(35). Exceptions include some of the H5N1 viruses, which were
highly virulent in mice without prior host adaptation, isolated in
Hong Kong during 1997 (36–38).
Previously, we have described that the introduction of the NS1
gene of the 1918 virus into a WSN virus background results in
attenuation in mice, suggesting that the 1918 NS1 protein is not
adapted to the mouse host (14). It is therefore interesting that the
additional introduction of the HA, NA, and M genes of 1918
overcomes the loss of pathogenicity in mice associated with the
1918 NS gene alone in a WSN background. Because the
1918NS:WSN virus is less virulent than the 1918 HA?NA?M?
NS:WSN virus, these results point to the 1918 HA?NA genes, and
perhaps M, as responsible for increased pathogenicity in mice.
viruses was the presence of infectious virus in the brain tissues of
mice after lethal i.n. virus challenge. The mouse-adapted WSN
virus, which is recognized as a neurovirulent strain (39), could also
be recovered from mouse brain tissue after i.n. immunization on
days 4 and 5 p.i. (data not shown). It has been demonstrated
previously that the NA gene of WSN virus plays a critical role in its
neurovirulence, most likely by facilitating HA cleavage without the
requirement of exogenous trypsin (17). Like the WSN strain, the
trypsin to grow in MDCK cells. By contrast, the control H1N1
recombinant virus with both the HA and NA of the New Cal?99
(New Cal HA?NA:WSN) required exogenous trypsin in MDCK
cells (data not shown). Interestingly, the 1918 NA protein does not
contain the ?146 mutation associated with this feature in WSN
(11). Thus, the genetic basis by which the 1918 constructs share
these features with WSN is not known.
The key prevention strategy to reduce influenza pandemic-
associated morbidity and mortality will be the implementation of
inactivated influenza virus vaccines effective against the pandemic
strain. There are no influenza vaccines currently available that
could efficiently be used as prophylactic measures if a 1918-like
virus reemerges. Therefore, we sought to identify candidate im-
munogens and evaluate the vaccine efficacy of these immunogens
against the highly pathogenic 1918 HA?NA recombinant viruses.
virus vaccine exhibited significant resistance against subsequent
challenge with a 1918 HA?NA recombinant virus. Because pro-
duction of a 1918 recombinant influenza vaccine would be com-
plicated by the higher levels of biosafety containment required, we
selected an antigenically related nonpathogenic virus that could be
by the biosafety level 2 virus, Sw?Iowa?30 vaccine was similar to
that observed in mice that received homologous (1918 HA?
NA:WSN) virus vaccine. Sw?Iowa?30-immune mice were pro-
tected against mortality and significant weight loss and had unde-
tectable virus in respiratory tissues on day 5 p.i. The high level of
protection induced by Sw?Iowa?30 virus vaccine correlated with
detectable HI and virus neutralizing antibodies measured in vitro.
Because mouse models are useful as preliminary screens for
candidate vaccines and may not be the definitive model for vaccine
efficacy in humans, we also carried out HI tests with a panel of p.i.
ferret antisera generated against seven influenza A (H1N1) viruses
and the 1918 HA?NA:WSN recombinant virus. Ferrets are used to
illustrate that the HA gene of the 1918 virus was most similar to
Sw?Iowa?30 H1N1 virus. The relationship to swine influenza was
until now based partly on historical accounts documenting wide-
spread severe influenza-like disease outbreaks in swine during the
limited variation in antigenic sites (43, 44) compared to human
H1N1 viruses, very few genetic changes might be expected of the
that survivors of the 1918 influenza pandemic had antibodies that
vaccination, mice were challenged i.n. with 100 LD50of 1918 HA?NA?M?NS?
later, and virus titers in individual lung, nose, and brain tissue were determined
(B). An asterisk indicates that the H1N1-vaccinated group was significantly (P ?
0.05) different from the control groups by ANOVA.
Sw?Iowa?30 vaccine provides protection against lethal challenge with
www.pnas.org?cgi?doi?10.1073?pnas.0308391100Tumpey et al.
neutralized both the 1918 HA?NA:WSN and Sw?Iowa?30 virus.
This finding was consistent with archeoserological data demon-
strating that survivors of the 1918 pandemic had antibodies that
neutralized classic swine influenza virus (3, 45, 46). The Sw?
Iowa?30 and 1918 HAs were found to be easily distinguished from
this time period (Tables 2 and 3). Previous HA protein sequence
comparisons between 1918, Sw?Iowa?30, and PR?8?34 viruses
support the results of our antigenic analysis (1, 9, 12). There are
twenty-two amino acid differences in the HA protein between the
1918 and Sw?Iowa?30 viruses. Only four of these amino acid
(12). The rate of genetic drift in the HA1 segments of the H1N1
human influenza viruses along with the acquisition of glycosylation
sites to mask antigenic sites (47) most likely accounts for the
antigenic variation observed between the 1918 HA and other
human HIN1 viruses isolated since 1933.
Although PR?8?34, Tx?91, and New Cal?99 viruses differed
antigenically from the 1918 HA, vaccines prepared from these
H1N1 viruses were able to provide some degree of protection
protection afforded by these vaccines cannot be explained by the
presence of detectable HI or neutralizing antibodies. However, the
of virus neutralization in vivo (48). Thus, other factors in vivo may
influence or enhance antibody activity, such as Fc or complement
receptor expressing cells types may facilitate the opsonization of
virus (49, 50). Although there are multiple cross-reactive viral
protection observed in PR?8?34-, Tx?91-, and New Cal?99-
vaccinated mice is largely due to anti-HA immunity. This hypoth-
derived from the 1981 and 1999 H1 influenza strains.
Because the genetic structure of the 1918 ‘‘Spanish’’ influenza
virus is becoming fully known, questions arise regarding the patho-
genesis, antigenicity, and immunity to the pandemic virus. The
generation of 1918 recombinant influenza A viruses that are
pathogenic in mice provides a reliable model system to test vaccine
candidates and identify the viral genes associated with pathogenic-
ity. This study helps to further define the pathogenic nature of this
virus, the antigenic characteristics, and vaccine strategies to the
1918 pandemic influenza virus. The identification of effective
vaccine strategies should provide additional prophylaxis for labo-
ratory workers and the public if a virus emerged through natural or
some other means.
This work was partially supported by grants from the National Institutes of
Health to P.P., C.F.B., and J.K.T. P.P. is a senior fellow of the Ellison
Medical Foundation New Scholar in Global Infectious Diseases. C.F.B. is
a New Scholar of the Ellison Medical Foundation Program in Global
Infectious Diseases. J.K.T. was supported by National Institutes of Health
Grant 5R01 AI0506919-02. This work was also supported by U.S. Depart-
ment of Agriculture?Agricultural Research Service Current Research
Information System Project Number 6612-32000-022-93.
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