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J Virol, May 1998, p. 4472-4477, Vol. 72, No. 5
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Immunogenicity and Protective Efficacy in Mice of
Influenza B Virus Vaccines Grown in Mammalian Cells or Embryonated
Chicken Eggs
I. V.
Alymova,1,2
S.
Kodihalli,1
E. A.
Govorkova,1,2
B.
Fanget,3
C.
Gerdil,3 and
R.
G.
Webster1,4,*
Department of Virology and Molecular Biology,
St. Jude Children's Research Hospital, Memphis, Tennessee
381051;
D. I. Ivanovsky Institute
of Virology, 123098 Moscow, Russia2;
Department of Pharmaceutical Development, Pasteur Merieux,
69280 Marcy l'Etoile, France3; and
Department of Pathology, University of Tennessee, Memphis,
Tennessee 381634
Received 3 November 1997/Accepted 2 February 1998
 |
ABSTRACT |
The immunogenicity and protective efficacy of formalin-inactivated
influenza B/Memphis/1/93 virus vaccines propagated exclusively in Vero
cells, MDCK cells, or embryonated chicken eggs (hereafter referred to
as eggs) were investigated. Mammalian cell-grown viruses differ from
the egg-grown variant at amino acid position 198 (Pro/Thr) in the
hemagglutinin gene. The level of neuraminidase activity was highest in
egg-grown virus, while MDCK and Vero cell-derived viruses possessed 70 and 90% less activity, respectively. After boosting, each of the
vaccines induced high levels of hemagglutinin-inhibiting, neuraminidase-inhibiting, and neutralizing antibodies that provided complete protection from MDCK-grown virus challenge. Mammalian cell-derived virus vaccines induced serum antibodies that were more
cross-reactive, while those induced by egg-grown virus vaccines were
more specific to the homologous antigen. Enzyme-linked immunospot analysis indicated that cell-grown virus vaccines induced high frequencies of immunoglobulin G (IgG)-producing cells directed against
both cell- and egg-grown virus antigens, whereas egg-grown virus
vaccine induced higher frequencies of IgG- and IgM-producing cells
reacting with homologous antigen and low levels of IgG-producing cells
reactive with cell-grown viruses. These studies indicate that influenza
B virus variants selected in different host systems can elicit
different immune responses, but these alterations had no detectable
influence on the protective efficacy of the vaccines with the
immunization protocol used in this study.
| |
TEXT |
Whole influenza virus vaccine
induces antibodies against both hemagglutinin (HA) and neuraminidase
(NA) surface glycoproteins, and these antibodies play a major role in
protection against challenge infection (5). Antibody against
HA neutralizes viral infectivity and prevents infection. Although the
immune response to HA overrides the NA immune responses in primed
subjects through intravirionic antigenic competition (9,
10), antibody to NA exerts its effect at later stages of
infection (11, 16).
Embryonated chicken eggs (hereafter referred to simply as eggs) have
been an extremely useful substrate for the propagation of influenza
viruses. However, it has been established that variant viruses are
selected when human influenza virus is first passaged in eggs, while
virus propagated exclusively in mammalian cell systems is structurally
and antigenically identical to the natural virus (13, 24,
26). The amino acid substitutions in the HA of egg-grown
influenza A and B viruses cluster around the receptor binding site
(12, 23, 25), which is located at the distal tip of the HA
molecule. Therefore, altered immune responses can be induced by
influenza virus vaccines produced in different host systems, and the
resulting small number of changes in the HA can influence the efficacy
of the vaccine (35). Recent studies have indicated that
viruses isolated and passaged in MDCK cells possess low NA activity
compared to that of egg-grown isolates (3). However, the
immunological consequences of these changes have not been elucidated.
Influenza A virus vaccines of the H1N1 and H3N2 subtypes that were
propagated in MDCK cells were more protective in animal models than
were their egg-grown counterparts (15, 35). A single amino
acid change in the HA decreased the efficacy of inactivated egg-grown
influenza A virus vaccines (17); reduced memory B-cell responsiveness at the challenge site accounted for this effect. The
significance of host cell-associated mutations for influenza B viruses
have been less well characterized. It was shown that egg-grown
influenza B viruses lost a potential glycosylation site in the HA at
amino acid positions 196 to 198, and this was associated with loss of
both infectivity and virulence for human volunteers (21,
36). Vaccination of mice with recombinant vaccinia viruses expressing either egg- or MDCK cell-derived HA genes from influenza B/England/222/82 virus can protect against challenge with influenza B
virus isolates exhibiting minor HA sequence differences (27, 28).
Continuous mammalian cell lines provide a realistic substitute for eggs
in vaccine production. Of the cell lines that might meet production
requirements, Vero and MDCK cells are the most promising. Vero cells
are a suitable host cell system for the cultivation of infectious
influenza A and B viruses (8). This cell line is certified
for vaccine production and is currently used for the preparation of
vaccines against poliomyelitis and rabies (20). MDCK cells
are generally used to grow influenza viruses, and they have recently
been used to produce safe and well-tolerated influenza virus subunit
vaccines (22).
In the present study we compare the serum and B-cell responsiveness
induced in BALB/c mice by influenza B/Memphis/1/93 virus vaccines
propagated in mammalian cells and in eggs. The protective efficacy of
vaccine preparations obtained from different host systems was
determined against challenge with MDCK cell-grown virus.
Characteristics of influenza B/Memphis/1/93 virus variants
grown in mammalian cells and chicken eggs.
Influenza
B/Memphis/1/93 virus from the original clinical sample was
cultured in Vero and MDCK cells in the presence of
L-1-tosylamide-2-phenylethyl chloromethyl ketone-treated
trypsin (1.0 µg/ml; Worthington Diagnostics, Freehold, N.J.) and in
eggs. Nucleotide sequences were obtained by the dideoxynucleotide
chain termination method (29) with the fmol DNA sequencing
system (Promega, Madison, Wis.) and end-labeled primers. Sequence
analysis of the HA1 regions of these variants revealed that those grown
in mammalian cells maintained a potential carbohydrate attachment site
at amino acid residues 196 through 198 (Asn-Lys-Thr). In contrast, the
egg-grown counterpart had a substitution at position 198, yielding the
sequence Asn-Lys-Pro and resulting in the loss of a glycosylation site.
This change occurred at the distal tip of the HA molecule, which is
homologous to antigenic site B of the influenza A virus HA molecule.
This raises the possibility that these variant viruses may differ
antigenically, but antigenic analysis with available polyclonal and
monoclonal antibodies failed to demonstrate any differences (results
not shown).
The NA activity of the concentrated viral preparations obtained in Vero
and MDCK cells and eggs was quantitated by the method of Warner and
O'Brien (33) with 1 mM
2'-(4-methylumbelliferyl)-
-D-N-acetylneuraminic acid (4-MUAc) (Sigma Chemical Co., St. Louis, Mo.) as the substrate and
by using the Warren method (34) with fetuin as the
substrate. The NA activity is reported as enzymatic activity per
milligram of total viral protein. To compare the viral enzymatic
activities, we considered the NA activity of egg-grown virus as 100%.
The NA activity of viruses propagated in MDCK cells was 56 to 70% lower than that of viruses grown in eggs, as determined with 4-MUAc and
fetuin, respectively. The NA activity of viruses produced in Vero cells
was reduced by 82 to 91% compared to that of the egg-grown variant, as
determined with the two substrates.
Serum antibodies in mice immunized with influenza
B/Memphis/1/93 virus vaccines.
Inactivated whole-virus
vaccine was prepared by treating purified virus preparations (40,000 hemagglutinating units/ml) with 0.025% formalin at 4°C for 3 days
(14). The immunogenicity of influenza B virus vaccines grown
in different host systems was determined in BALB/c mice (H-2d), which
were immunized subcutaneously with a dose of whole-virus vaccine
containing 5 µg of HA adsorbed to aluminum hydroxide adjuvant
(Rehydragel LV; Reheis Inc., Berkeley Heights, N.J.) (2).
Four weeks later, the mice received an intraperitoneal booster
inoculation of the same dose of vaccine without the adjuvant. One week
after receiving the boost, the mice were challenged intranasally with
200 50% mouse infectious doses of MDCK cell-grown virus. Blood samples
were obtained from anesthetized mice on days 14, 35, 42, and 55 postvaccination (p.v.). The level of serum antibodies was examined by
HA-inhibiting (HI), NA-inhibiting (NI), and neutralization assays. The
titers of HI antibodies produced within the first 2 weeks p.v. did not
differ between the experimental groups (Fig.
1). In all vaccine groups, booster
vaccination (7 days postboost, i.e., day 35 p.v.) of the mice
increased the titers of HI antibodies against all three antigens. One
week after challenge with MDCK cell-grown virus, the mice primed with
cell-grown virus vaccine produced higher levels of antibodies that were
cross-reactive with cell-grown virus antigens. Although post-challenge
antibodies induced by mice primed with egg-grown virus vaccine were
cross-reactive with both egg-grown and cell-grown virus antigens, they
were of lower magnitude than antibodies induced by cell-grown virus
vaccines. The Vero cell-grown virus vaccine induced at least as high HI
antibody response as did the egg-grown virus vaccine. The MDCK
cell-derived virus vaccine induced HI antibody titers that were
detectably higher than those of egg- or Vero cell-grown virus vaccine,
probably due to the homologous challenge.
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FIG. 1.
HI antibody titers in sera of mice immunized with
inactivated cell- or egg-grown influenza B/Memphis/1/93 virus vaccines.
BALB/c mice were immunized subcutaneously with the vaccines containing
5 µg of HA with aluminum hydroxide adjuvant and, four weeks later,
with vaccine containing HA alone. One week postboost, the mice were
challenged with 200 50% mouse infectious doses of MDCK cell-grown
virus. The data points represent the means from five mice for each time
point.
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Titers of anti-NA antibodies produced in response to vaccination with
influenza B/Memphis/1/93 virus isolated and grown in
Vero cells, MDCK
cells, or eggs are shown in Fig.
2. MDCK
cell-
and egg-derived virus vaccines induced moderate and comparable
levels of prechallenge anti-NA antibodies, ranging from 1.5 to
2.0 log
10 NI titer with homologous antigen. The challenge
appeared
to increase anti-NA antibody titers in all vaccine groups;
this
increase was most marked in the group of mice receiving Vero
cell-derived
virus vaccine. The levels of postchallenge antibodies
obtained
with Vero cell-grown virus vaccine were not distinguishable
from
those induced by egg-grown virus vaccine (2.1 versus 2.4 log
10).
However, with the small interval between boosting
and challenge
we cannot clearly discriminate between the responses
induced by
each. Anti-NA antibodies induced by Vero cell-grown virus
vaccine
cross-reacted with the egg- and MDCK cell-grown virus antigens
to similar titers, while immunization of mice with the egg-grown
virus
vaccine resulted in the induction of antibodies that were
less
efficacious against mammalian cell-grown virus vaccines.
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FIG. 2.
Serologic response of mice to immunization with
inactivated cell- or egg-grown influenza B/Memphis/1/93 virus as
measured by the NI test. The dilution of serum that inhibited 50% of
virus NA activity was taken as the antibody titer.
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The levels of neutralizing antibodies induced by influenza B virus
vaccines grown in different host systems are shown in Fig.
3. At 14 days p.v., they ranged from 1.8 to 2.5 log
10 in all vaccine
groups tested, and they
increased after booster vaccination. The
mammalian cell-grown virus
vaccines induced cross-reactive antibodies,
while those induced by
egg-grown virus vaccine were specific for
egg-grown virus and gave less
neutralization of cell-grown viruses.
After challenge there were no
anamnestic neutralizing antibody
responses, suggesting that the vaccine
dose of 10 µg of HA had
achieved titers similar to those in
postchallenge animals.
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FIG. 3.
Neutralizing antibody titers in sera of mice in response
to immunization with inactivated cell- or egg-grown influenza
B/Memphis/1/93 virus. The mean titer of antibody is expressed as the
reciprocal of the highest dilution of serum that neutralized 100 50%
tissue culture infective doses in virus-infected MDCK cells.
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Thus, cell-derived influenza B virus vaccines are able to induce levels
of anti-HA, anti-NA, and neutralizing antibodies comparable
to those
induced by egg-grown virus vaccines. It is important
to emphasize that
mammalian cell-derived virus vaccines induced
more-cross-reactive serum
antibodies, while those obtained in
the egg-grown group were more
specific to the homologous antigen.
B-cell response to glycoproteins of influenza B/Memphis/1/93
viruses.
B-cell responsiveness to purified surface glycoproteins
was evaluated by using a modified enzyme-linked immunospot assay
(6). An HA-NA-rich fraction was obtained by treating virus
preparations with the nonionic detergent n-octyl
-thioglucopyranoside as described previously (9). Mice (5 per group) were anesthetized and exsanguinated, and the superficial
cervical and mediastinal lymph nodes (cells from these lymph nodes were
pooled) and spleens were collected, at 14 and 32 days p.v. The tissues
were processed individually as previously described (1).
Antibody-forming cells (AFCs) of the immunoglobulin M (IgM) and IgG
isotypes were visualized by using alkaline phosphatase-conjugated goat
anti-mouse Ig isotype-specific reagents (Southern Biotechnology
Associates, Birmingham, Ala.) diluted in 5% bovine serum albumin
and developed with 5-bromo-4-chloro-3-indolylphosphate (Sigma). The
B-cell response was statistically analyzed by using the Kruskal-Wallis
test to determine whether there was an overall difference in B-cell
response among the three vaccine groups for a particular antigen. If
such a difference was seen, a post hoc analysis was performed to
compare the vaccine groups pairwise with the Wilcoxon-Mann-Whitney
test.
At 14 days p.v., all vaccine groups had low numbers of HA- and
NA-specific AFCs (median, 12 IgM and 38 IgG AFCs per 10
5
cells) in the lymph nodes and moderate (median, 3 IgM and 31
IgG AFCs
per 10
5 cells) numbers in the spleen. Although the number
of IgG-producing
AFCs in the lymph nodes increased after booster
vaccination, there
was no significant difference between the groups
when assayed
against glycoproteins, regardless of the host system in
which
the virus was propagated. However, the egg-grown virus vaccine
group appeared to have a significantly higher number of IgM-producing
AFCs (median = 42;
P = 0.008) reacting with
egg-grown virus antigen
in comparison to those of the MDCK (median = 8) and Vero (median
= 6) cell-grown virus vaccine groups. The
cell-grown virus vaccines
induced low numbers of IgM-producing cells
when assayed against
glycoproteins from all the host systems.
The number of IgG-producing AFCs did not differ significantly among the
three groups in reaction to the egg-grown virus antigen.
In mice
immunized with Vero cell-grown virus vaccine, the number
of
IgG-producing cells increased significantly when assayed against
homologous antigen (median = 899;
P = 0.008) in
comparison to
egg-grown (median = 370) or MDCK cell-grown antigen
(median =
360). In animals that were vaccinated with virus
produced in MDCK
cells (Fig.
4), the
profiles of AFC induction were similar to
those of mice that received
Vero-grown virus vaccine. Although
high numbers of AFCs of IgG isotype
(median = 363) reacting with
MDCK cell-grown virus antigen were
induced in mice immunized with
MDCK cell-grown virus vaccine in
comparison to the numbers induced
in mice immunized with egg-grown
virus vaccine (median = 160),
there was no significant difference
in the antibody-forming cells
induced by MDCK or Vero cell-grown
vaccine against MDCK cell-grown
virus antigen (
P = 1.0), suggesting that there are similarities
in the antigenicities of
these two cell-grown virus vaccines.
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FIG. 4.
B-cell responsiveness induced in mice by inactivated
influenza B/Memphis/1/93 virus vaccines grown in mammalian cells or in
eggs. Lymphoid tissues of immunized mice were collected on days 14 and
32 (5 days after boost) p.v. At each time point in the enzyme-linked
immunospot assay, freshly isolated cells from each tissue (mediastinal
lymph nodes and spleens) were sampled for Ig isotype determination
against glycoprotein preparations derived from cell- or egg-grown
B/Memphis/1/93 virus. The data shown are the means of five mice per
vaccine group. agn, antigen; LN, lymph nodes; SP, spleen.
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The profiles of the AFCs induced by the egg-grown virus (Fig.
4)
differed from those stimulated by vaccines that were produced
in
mammalian cell lines. The predominant AFC response was directed
against
the egg-grown virus antigen and was of the IgM isotype.
The egg-grown
virus vaccine group also induced high numbers (median
= 330) of
IgG-producing AFCs per 10
5 cells reacting with homologous
antigen. A significantly lower
(
P = 0.008) number of
IgG-producing cells were induced in the
egg-grown virus vaccine group
against virus grown in MDCK (median
= 160) or Vero (median = 163) cells compared to the number induced
against egg-grown virus
antigen (median = 330), suggesting that
AFCs within this group
were more specific for the homologous antigen.
Response of vaccinated mice to challenge with MDCK cell-grown
virus.
To evaluate vaccine efficacy following challenge with MDCK
cell-grown influenza B/Memphis/1/93 virus, 3 days postchallenge, the
lungs of eight mice in each vaccine group were processed separately by
the method described by Liang et al. (19). As exemplified by
our inability to recover infectious virus from any vaccinated animal,
all three vaccine preparations were considered to give complete
protection against challenge. All of the control (unvaccinated) mice
shed infectious virus (mean titer, 3.5 log10 50% tissue
culture infective doses/ml).
The findings presented in this study indicate that influenza B viruses
produced in Vero and MDCK cells are equally immunogenic
and induce
serum antibodies and B-cell responses against mammalian
cell-propagated
as well as egg-grown virus antigens. Mammalian
cell-derived virus
vaccines elicited more-cross-reactive serum
antibodies, while egg-grown
virus induced antibodies more specific
to the homologous antigen.
Despite their differing immunogenicities,
comparative studies showed
that all vaccines induced complete
protection in mice against MDCK
cell-grown virus challenge.
Our results regarding protective immunity are in agreement with a
previous study, which suggested that vaccination with egg-derived
influenza B viruses protected against variant viruses produced
in
mammalian cells (
27). However, recombinant vaccinia
virus-expressing
construct could affect the type of immune response,
presumably
inducing both cellular and humoral immunity. In contrast to
our
results, it has been reported that inactivated influenza A virus
vaccines containing single or multiple amino acid substitutions
in HA
due to growth in eggs have reduced capacity to protect against
challenge with native virus (
14,
17). The differences
between
these reports and the results of the present study may be due
to differences in the type of influenza antigen used (influenza
A or B)
and in the location of the amino acids involved in the
antigenic
region.
There are several possible reasons for the reduced reactivity pattern
of the serum antibodies and the B-cell responsiveness
with egg-grown
virus vaccine. The first possibility is that amino
acid changes in one
of the neutralization epitopes on the HA could
influence the response.
The influenza A antigenic site B (positions
174 to 209) is
immunodominant in BALB/c mice for both antibody
and helper T cells
(
4,
7,
30). Subtle conformational changes
induced by
sequences flanking epitopes have been shown to be sufficient
to alter
immunogenicity (
32). Based on the similarities in the
HA
structures of influenza A and B viruses (
18,
31), it is
possible that substitution at amino acid residues 196 to 198 in
egg-grown virus vaccine may have affected both the specificities
and
the levels of antibody-producing cells induced.
Secondly, a change in the glycosylation site could affect immune
recognition by either uncovering or hiding the epitope. Because
of the
extensive reciprocity of B-cell and T-cell recognition
of influenza
virus HA, B cells can selectively process and present
antigen to class
II-restricted helper T cells, thereby defining
the memory T- and B-cell
responses. Changes in the glycosylation
site could result in
differential antigen processing, resulting
in differences in the
spectrum of peptides generated for the stimulation
of helper T cells.
Thirdly, it is possible that the differences in immunogenicity may be
due to differences in the NAs of the variant viruses.
It is interesting
to note that the egg-grown virus, with the highest
NA activity, induced
serum antibodies that reacted weakly with
Vero cell-grown antigen,
which has the lowest NA activity. There
could be several reasons for
this pattern of reactivity. (i) In
the NI assay we used whole virus,
not subunits of NA. The anti-HA
antibodies in serum may have caused
stearic hindrance of NA activity.
(ii) The differences in the NA
activities of Vero cell- and egg-grown
viruses may be influenced by the
glycosylation site at amino acid
positions 196 to 198 in the HA. This
effect would be indirect
and might affect the affinity of binding of
the HA to sialic acid
and might require less NA for release. (iii) Vero
cell-grown virus
may require more antibodies to inhibit NA activity,
even though
it has the least NA activity. This possibility seems less
likely,
but if the NA on Vero cells differs in specificity, it may have
some validity.
The present study raises an interesting question: would challenge of
the egg-grown virus vaccine group with homologous virus
generate memory
AFC responsiveness and a serum antibody response
equivalent to that
seen with the MDCK cell-grown virus? Our earlier
study (
14)
indirectly addressed this issue and showed that MDCK
cell-grown virus
vaccine provided better protection than did an
egg-grown counterpart to
challenge with both MDCK cell- and egg-grown
virus. Overall, our
results suggested that the close antigenic
relatedness with prevalent
natural viruses may be responsible
for the greater immunogenicity of
MDCK and Vero cell-grown virus
candidate vaccines. These studies
support the notion that mammalian
cells (both Vero and MDCK cells) can
provide a host cell system
alternative to eggs for the preparation of
influenza B virus vaccines.
A dose-response study is needed to
determine if the higher levels
of cross-reactive antibody induced by
mammalian cell-grown virus
vaccine provides any advantage over
egg-grown influenza virus
vaccine.
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ACKNOWLEDGMENTS |
We thank Mikhail Matrosovich for valuable consultation, Amy L. B. Frazier for scientific editing, Deepthi A. Jayawardene for statistical analysis, and members of our laboratories (Scott Krauss, Larisa V. Gubareva, and Darwyn Kobasa) for helpful discussions.
This work was supported by a research grant from Pasteur Merieux with
core support from Cancer Center grant CA-21765 from the National
Institutes of Health and by the American Lebanese Syrian Associated
Charities (ALSAC).
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Virology and Molecular Biology, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38105. Phone: (901) 495-3400. Fax: (901)
523-2622. E-mail: robert.webster{at}stjude.org.
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J Virol, May 1998, p. 4472-4477, Vol. 72, No. 5
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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