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JOURNAL OF VIROLOGY, June 2008, p. 5161–5166 Vol. 82, No. 11
0022-538X/08/$08.00⫹0 doi:10.1128/JVI.02694-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Cross-Recognition of Avian H5N1 Influenza Virus by Human
Cytotoxic T-Lymphocyte Populations Directed to Human
Influenza A Virus
䌤
J. H. C. M. Kreijtz, G. de Mutsert, C. A. van Baalen, R. A. M. Fouchier,
A. D. M. E. Osterhaus, and G. F. Rimmelzwaan*
Department of Virology, Erasmus Medical Center, P.O. Box 2040, Rotterdam, The Netherlands
Received 19 December 2007/Accepted 4 March 2008
Since the number of human cases of infection with avian H5N1 influenza viruses is ever increasing, a
pandemic outbreak caused by these viruses is feared. Therefore, in addition to virus-specific antibodies, there
is considerable interest in immune correlates of protection against these viruses, which could be a target for
the development of more universal vaccines. After infection with seasonal influenza A viruses of the H3N2 and
H1N1 subtypes, individuals develop virus-specific cytotoxic T-lymphocyte responses, which are mainly directed
against the relatively conserved internal proteins of the virus, like the nucleoprotein (NP). Virus-specific
cytotoxic T lymphocytes (CTL) are known to contribute to protective immunity against infection, but knowledge
about the extent of cross-reactivity with avian H5N1 influenza viruses is sparse. In the present study, we
evaluated the cross-reactivity with H5N1 influenza viruses of polyclonal CTL obtained from a group of
well-defined HLA-typed study subjects. To this end, the recognition of synthetic peptides representing H5N1
analogues of known CTL epitopes was studied. In addition, the ability of CTL specific for seasonal H3N2
influenza virus to recognize the NP of H5N1 influenza virus or H5N1 virus-infected cells was tested. It was
concluded that, apart from some individual epitopes that displayed amino acid variation between H3N2 and
H5N1 influenza viruses, considerable cross-reactivity exists with H5N1 viruses. This preexisting cross-reactive
T-cell immunity in the human population may dampen the impact of a next pandemic.
Since the first documentation of bird-to-human transmis-
sions of highly pathogenic avian H5N1 influenza viruses, these
viruses have spread from Southeast Asia to other regions of the
world (2, 3, 22, 27). Since 2003 the number of human cases has
continued to increase; as of 28 February 2008, 369 human cases
have been reported, of which 234 were fatal (28). It is feared
that an H5N1 virus may cause the next influenza pandemic
when it is able to replicate in mammalian species by adaptation
through genetic reassortment or accumulation of point muta-
tions in relevant gene segments (11). Although neuraminidase
subtype 1 cross-reactive antibodies have been demonstrated in
human subjects, antibodies to H5 molecules are hardly existent
in the human population as a result of limited exposure to
H5N1 viruses, which contributes to a scenario for these viruses
to become pandemic (20). In general, the exposure history and
the immune status of the human population will influence the
size and the severity of pandemics (4, 8, 9, 14). The presence of
T-cell immunity induced by infection with human influenza
virus strains may provide some degree of cross-protective im-
munity against the H5N1 viruses. Cytotoxic T-lymphocyte
(CTL) responses are predominantly directed to internal viral
proteins, the nucleoprotein (NP) in particular (23, 29),
which is much more conserved than the surface hemagglu-
tinin and neuraminidase glycoproteins (5, 13, 15, 29). It has
been suggested that cross-reactive CD8
⫹
T cells may temper
the impact a pandemic potentially could have on the human
population (9, 14, 19). In humans, the presence of cross-
reactive CTL responses inversely correlated with the
amount of shedding of a heterosubtypic strain that was used
for experimental infection of study subjects (14). Although
preexisting CTL immunity against influenza virus may be of
importance in the face of the current H5N1 pandemic
threat, our knowledge of the cross-reactive nature of the
human CTL response is limited (9).
In the present study we tested the cross-reactivity of poly-
clonal virus-specific CD8
⫹
T-cell populations obtained from
well-defined HLA-typed study subjects with H5N1 virus. The
recognition of target cells pulsed with peptide variants, trans-
fected with the NP gene from a human or an avian influenza
virus, or infected with viruses of the H3N2 or H5N1 subtype
was tested.
It was concluded that the human CTL response displays a
high degree of cross-reactivity with avian H5N1 influenza vi-
ruses and could reduce morbidity and mortality during a pan-
demic caused by these H5N1 strains.
MATERIALS AND METHODS
Cells. Peripheral blood mononuclear cells (PBMC) were isolated from hepa-
rinized blood obtained from fifteen HLA-typed healthy blood donors (Sanquin
Bloodbank, Rotterdam, The Netherlands) by density gradient centrifugation
using lymphoprep (Axis-Shield PoC AS, Oslo, Norway) and then cryo-preserved
at ⫺135°C. Genetic subtyping was performed in the laboratory for Histocom-
patibility and Immunogenetics at the Sanquin Bloodbank using a commercial
typing system (Genovision, Vienna, Austria). Three groups of study subjects
were selected on the basis of their major histocompatibility complex class I
(MHC-I) alleles, for which influenza CTL epitopes were identified. Within
groups the subjects shared identical HLA-A and -B alleles; between groups there
* Corresponding author. Mailing address: Department of Virol-
ogy, Erasmus Medical Center, P.O. Box 2040, 3000 CA Rotterdam,
The Netherlands. Phone: 31 10 7044243. Fax: 31 10 7044760. E-
mail: g.rimmelzwaan@erasmusmc.nl.
䌤
Published ahead of print on 19 March 2008.
5161
were differences of one or two alleles. The groups were as follows: group I, HLA
A*0101, A*0201, B*0801, and B*3501; group II, HLA A*0101, A*0201, B*0801,
and B*2705 (2702); and group III, HLA A*0101, A*0301, B*0801, and B*3501
(3503) (1). Subject 15 was not tested since PBMC of this donor were no longer
available.
Peptides. Amino acid sequences of all known human influenza A virus CTL
epitopes were compared with their counterparts in H5N1 influenza viruses iso-
lated since 2003, which were obtained from the influenza sequence database (12).
All possible variants that could be identified in the H5N1 sequences are listed in
Table 1. A set of immunograde peptides representing immunodominant CTL
epitopes and the most prevalent analogues in H5N1 strains were synthesized and
analyzed by mass spectrometry and were found to be ⬎70% pure (Eurogentec,
Seraing, Belgium). Variant peptide analogues from the NP, which is the main
target for CTL responses, were synthesized when they had a prevalence in H5N1
strains of ⬎0.25%, with the exception of the NP
383-391
epitope since the G384K
mutation observed in H5N1 viruses was known to abrogate recognition by spe-
cific CTLs completely (26). For the remaining viral proteins, all variants with a
prevalence of ⬎2.25% were synthesized and tested. Only peptides that matched
the HLA alleles of the study subjects were considered.
Target cells. B-lymphoblastoid cell lines (BLCL) were established as described
previously (18) and used as target or stimulator cells. A total of 30,000 cells were
incubated in the absence or presence of 10 M peptide for1hat37°C, washed
once, and resuspended in RPMI 1640 medium (Cambrex, East Rutherford, NJ)
containing antibiotics, L-glutamine, and 10% fetal calf serum (R10F medium).
Cells of the BLCL were also infected at a multiplicity of infection of five 50%
tissue culture infective doses/cell (17) with influenza viruses A/Netherlands/18/94
(A/NL/18/94) (H3N2) or A/Vietnam/1194/04 (A/VN/1194/04) (H5N1), which
were propagated and titrated in MDCK cells using standard procedures. After an
incubation period of1hat37°C, the cells were washed and resuspended in R10F
medium and incubated for 16 to 18 h at 37°C prior to their use for the stimulation
of CD8
⫹
T cells. Infection rates were determined by an immunofluorescence
assay and were similar for both viruses (data not shown). The human influenza
virus A/NL/18/94 (H3N2) was used as a representative of seasonal influenza
viruses, while influenza virus A/VN/1194/04 was used as an example for H5N1
influenza virus.
T-cell clones. CD8
⫹
T-cell clones directed against the HLA-A1-restricted
NP
44–52
(CTELKLSDY) epitope, HLA-A3-restricted NP
265–273
(ILRGSVAHK)
epitope, HLA-B27-restricted NP
174–184
(RRSGAAGAAVK) epitope, and the
HLA-B*3501-restricted NP
418–426
(LPFEKSTVM) epitope were generated as
described previously (26).
In vitro expansion of influenza A virus-specific T-cell populations. PBMC
were stimulated with influenza virus A/NL/18/94-infected cells as previously
described (1). Eight days after stimulation, cells were harvested and used as
effector cells in an enzyme-linked immunospot (ELISPOT) or fluorescent anti-
gen-transfected target cell (FATT)-CTL assay. For the ELISPOT assays, CD8
⫹
T cells were purified from the in vitro expanded PBMC by magnetic bead cell
sorting (Miltenyi Biotec, Bergisch Gladbach, Germany). Typically, a purity of
⬎96% was obtained.
FATT-CTL assay. The NP genes of influenza viruses A/NL/18/94 and A/VN/
1194/04 without their stop codons were cloned into the plasmid pEGFP-N1
(Becton Dickinson, Alphen a/d Rijn, The Netherlands) in frame with the
open reading frame of the green fluorescent protein (GFP) as previously
described (25). Plasmid DNA was purified using a Genopure plasmid midi kit
(Roche, Woerden, The Netherlands). Nucleotide sequences of the recombi-
nant plasmids were confirmed using a Big Dye Terminator, version 3.1, cycle
sequencing kit (Applied Biosystems, Foster City, CA) and an ABI Prism 3100
Genetic Analyzer (Applied Biosystems). Primer and plasmid sequences are
available on request.
The plasmids were used in the FATT-CTL assay for the detection of lytic
activity of virus-specific CTLs as described previously (25). In brief, BLCL were
nucleofected using cell line nucleofector kit V (Amaxa Biosystems, Cologne,
Germany) with program T16 and subsequently incubated in R10F medium for
4 h at 37°C. Then, they were cocultured for another4hintriplicate with PBMC
cultures and in vitro expanded after stimulation with influenza virus A/NL/18/94
at various effector-to target (E:T) cell ratios. The number of viable GFP-positive
cells was measured using a FACSCalibur (Becton Dickinson). The percent nu-
cleoprotein-specific lysis was then calculated by the following formula: 100 ⫻
[(number of viable GFP-positive cells in the sample without effector ⫺number
of viable GFP-positive cells in the sample with effector)/number of viable GFP-
positive cells in the sample without effector].
Gamma interferon (IFN-␥) assay. ELISPOT assays were performed with in
vitro expanded CD8
⫹
T cells as effector cells and peptide-pulsed or virus-
infected HLA-matched BLCL as stimulator cells as described previously (1). The
number of spots was determined using an ELISPOT reader and image analysis
software (Aelvis, Sanquin Reagents, Amsterdam, The Netherlands), and the
average number was calculated of triplicate wells.
RESULTS
Comparison of amino acid sequences of known influenza A
virus CTL epitopes. The amino acid sequences of known hu-
man influenza A virus CTL epitopes were compared with the
corresponding sequences in approximately 900 H5N1 viruses
obtained from the influenza sequence database (12). As shown
in Fig. 1A, the epitope sequences were identical in ⬎95% of
the H5N1 viruses for the majority of the known epitopes an-
alyzed including PB1
591–599
,M1
13–21
,M1
128–135
, NS1
158–166
,
NP
44–52
,NP
146–154
,NP
174–184
,NP
265–273
,NP
380–388,
NP
381–388
,
and NP
383–391
. For some of the other epitopes the percentage
TABLE 1. Variant sequences of known CTL epitopes in H5N1 viruses
Sequence
name
Variant sequence of the CTL epitope at the indicated HLA allele
a
HLA-A1 PB1
(591–599)
HLA-A3 M1
(13–21)
HLA-A*0201
M1 (58–66)
HLA-B*3501
M1 (128–135)
HLA-A*0201
NS1 (122–130)
HLA-A*0201
NS1 (123–132)
HLA-B*44
NS1 (158–166)
HLA-A1 NP
(44–52)
HLA-A*6801
NP (91–99)
Epitope VSDGGPNLY SIIPSGPLK GILGFVFTL ASCMGLIY AIMDKNIIL IMDKNIILKA GEISPLPSL CTELKLSDY KTGGPIYKR
Variant
1I-------- -V------- -M------- ------S- -----T--- ----T----- ------H-- --------H -------R-
2-------P- --V------ --W------ -----V--- ----V----- -------F- --------Q -----V---
3------IP- F-------- -------T- ------T--- ---L----- ------N-- -----F-RG
4G-------- -T------- -----TV-- ----TV---- --------I ------T-- --------G
5-A------- -L------- -----A--- ----A----- -----S--- ---F-----
6ATS------ -T-----T- T-----T--- -------Y-
7--VR----Q -----D--- ----D----- A--------
8--VL----- --V--T--- -V--T----- -----I---
9S----T--- ----T-S---
10 -----T-S- --N-------
11 ---N----- -V--T-T-- -
12 --V- -T-T- -V----T---
13 --V- -- -T- -- --T-L- --
14 -----T-L- ----I-----
15 -----I--- -V--TV----
16 --V- -TV-- - -- -T--W- -
17 -----T--W ----T----G
a
Variant sequences of known CTL epitopes were ranked according to their relative prevalence. Anchor residues of the epitopes are underlined. Amino acid residue
positions are given in parentheses.
5162 KREIJTZ ET AL. J. VIROL.
of H5N1 viruses with identical sequences was variable and
ranged from 79% for epitope M1
58–66
to 4% for epitope
NS1
122–130
. For the epitopes NP
91–99
,NP
188–198
,NP
339–347
,
and NP
418–426
, no identical sequences were found in the H5N1
viruses. In order to identify the most prevalent variant se-
quences in H5N1 viruses, the number of individual variants
was analyzed (Fig. 1B). In some cases a single variant was
identified that accounted for almost all variant sequences ob-
served in H5N1 viruses (Table 1 and Fig. 1B). For other
epitopes multiple variants were identified, although for some
of these the number was low, and the number of major variants
was limited (12).
The recognition of known CTL epitopes and their avian
analogues. All subjects in group I (HLA A*0101, A*0201,
B*0801, and B*3501) displayed T-cell reactivity with the
epitopes NP
44–52
, NS1
122–130
,NP
418–426
, and M1
58–66
as they
are present in human influenza A viruses although the fre-
quency of specific CTLs varied between study subjects and the
peptides tested (Fig. 2A). In none of the subjects of this group
was reactivity observed with the peptide variants of epitopes
NP
44–52
and NS1
122–130
, obtained from H5N1 influenza vi-
ruses. Three out of four subjects responded to the NP
418–426
variant LPFERSTIM, and all subjects responded to the
M1
58–66
variant GMLGFVFTL. Of group II (HLA A*0101,
A*0201, B*0801, and B*2705 [2702]), most subjects responded
to the peptides representing epitopes from human influenza A
viruses (Fig. 2B) although the magnitudes of the responses
varied considerably. Only one subject in this group had an
appreciable response to the H5N1 analogue sequence of the
NS1
122–130
epitope. Four out of five subjects responded to the
H5N1 variants of the HLA B*2705-restricted NP
174–184
epitope whereas all five responded to the M1
58–66
variant. The
subjects of group III (HLA A*0101, A*0301, B*0801, and
B*3501 [3503]) responded to the original epitopes to various
extents. Some subjects were poor responders and hardly dis-
played CTL reactivity with some of these epitopes (Fig. 2C).
However, the in vitro expanded PBMC of subject 14 responded
strongly to the NP
418–426
and also reacted with both epitope
variants from H5N1 viruses, indicating that at least a fraction
of the CTL population was capable of cross-recognizing these
analogues. The same holds true for the NP
44–52
- and NP
265–273
-
specific CTL responses in this study subject. Clones were used
as positive and negative controls, and the clonal responses
supported the results obtained with the polyclonal populations
(data not shown).
FIG. 1. The presence of known CTL epitopes in H5N1 strains. The
percentage of H5N1 viruses with an epitope sequence identical to
human influenza viruses (white bars) is shown. The black bars indicate
the percentage of H5N1 viruses with one or more amino acid substi-
tutions in the epitope sequence. The absolute numbers of each variant
of an epitope are shown in panel B, where each color represents a
single variant (sequences can be found in Table 1). For this analysis
almost 900 H5N1 viruses for which sequence information was available
in the influenza sequence database (12) were analyzed.
TABLE 1—Continued
Variant sequence of the CTL epitope at the indicated HLA allele
a
HLA-B*1402
NP (146–154)
HLA-B*2705
NP (174–184)
HLA-A3
NP (188–198)
HLA-A3
NP (265–273)
HLA-B*3701
NP (339–347)
HLA-B*08
NP (380–388)
HLA-B*2702
NP (381–388)
HLA-B*2705
NP (383–391)
HLA-B*3501
NP (418–426)
TTYQRTRAL RRSGAAGAAVK TMVMELVRMIK ILRGSVAH EDLRVLSFI ELRSRYWAI LRSRYWAI SRYWAIRTR LPFEKSTVM
S-------- ---------I- ------I---- -----I--- -----S--- ----K---- ---K---- -K------- ----RA-I-
A-------- -I------L-- --A-------- -----M--- ----IS--- D-LGK---K -LGK---K ------K-- ----R--I-
AA------- --F-------- -----QI---- V-------- -----H--- ----H--- -------P- ----RAAI-
A--H----- Q----VI---- --------K ----RV-I-
AP------- --------P ----RA---
A-------V GK-- -KWMI
A-SQ----- --H------
--------G
VOL. 82, 2008 CROSS-RECOGNITION OF AVIAN H5N1 VIRUS BY HUMAN CTLs 5163
Cross-recognition of the NP derived from influenza virus
A/VN/1194/04. The capacity of polyclonal T-cell populations
directed to the human influenza virus A/NL/18/94 to cross-
react with the NP of influenza virus A/VN/1194/04 was as-
sessed in the FATT-CTL assay. PBMC from all study subjects
were stimulated with influenza virus A/NL/18/94 and allowed
to proliferate. As shown in Fig. 3, 2 out of the 15 subjects tested
(subjects 5 and 8) were low responders or nonresponders (Fig.
3) since no NP-specific lytic activity could be demonstrated. In
the remaining 13 subjects, lytic activity was observed against the
homologous NP. In most cases the PBMC cross-reacted with the
NP of influenza virus A/VN/1194/04 to a considerable extend
(Fig. 3). Only for subject 3 (group I) and subject 9 (group III) did
the influenza virus A/NL/18/94 NP-specific CTLs fail to recognize
the NP of influenza virus A/VN/1194/04 (Fig. 3).
Cross-recognition of BLCL infected with influenza virus
A/VN/1194/04. Next, we wished to assess the cross-reactive
nature of the whole repertoire of CD8
⫹
T lymphocytes specific
for the human influenza virus A/NL/18/94. To this end, PBMC
were stimulated with this virus, and after 8 days the CD8
⫹
cells
were isolated to obtain virus-specific polyclonal CTL populations.
These cells were used as effector cells in an IFN-␥ELISPOT
assay using MHC-I-matched BLCL infected with influenza virus
A/NL/18/94 or A/VN/1194/04 as stimulator cells.
As shown in Fig. 4, the in vitro expanded PBMC population
that recognized cells infected with influenza virus A/NL/18/94
also recognized cells infected with influenza virus A/VN/1194/
04. The average number of IFN-␥-positive spots per 10
4
cells
observed after stimulation with A/NL/18/94-infected cells was
151 (standard deviation, 58), and the number observed after
stimulation with A/VN/1194/04 was even slightly higher at 192
(standard deviation, 65) although this difference was not sta-
tistically significant.
DISCUSSION
In the present paper the cross-reactive nature of the human
influenza virus-specific CTL response was investigated. It was
concluded that a considerable portion of CTL populations
specific for the H3N2 influenza virus A/NL/18/94 cross-reacted
with the H5N1 strain A/VN/1194/04.
For most CTL epitopes, it was found that a vast majority of
the H5N1 strains contained epitope sequences identical to
those present in human influenza A viruses. This conservation
of epitopes is responsible for the cross-reactive nature of CTL
responses in humans against seasonal influenza A viruses of
the H3N2 and H1N1 subtypes. However, some variation in
these epitopes was observed also, and for a number of CTL
epitopes the H5N1 strains did not contain identical sequences.
Apart from the NP
174–184
and M1
58–66
epitope restricted by
HLA-B*2705 and HLA-A*0201, respectively, very little cross-
reaction was observed of polyclonal CTL populations with
variant peptides derived from H5N1 viruses.
However, as indicated above, most epitopes are relatively
conserved, including those located in the NP, which contrib-
uted to the cross-reactive nature of the NP-specific CTL re-
sponse. Most of the study subjects that responded to NP de-
rived from seasonal H3N2 influenza viruses also responded to
the NP derived from influenza virus A/VN/1194/04 (H5N1).
The polyclonal virus-specific T-cell populations of two of these
subjects failed, however, to cross-react with the NP of influenza
virus A/VN/1194/04 for reasons that are unclear. Possibly, the
most immunodominant responses in these subjects were di-
rected to CTL epitopes in the NP that were not conserved.
To account for the full repertoire of virus-specific CD8
⫹
T
lymphocytes, the reactivity with MHC-I-matched cells infected
with influenza virus A/NL/18/94 or A/VN/1194/04 was also
analyzed. In all cases A/VN/1194/04-infected target cells were
recognized to a similar extent as A/NL/18/94-infected cells,
indicating that the level of cross-reactivity of human CTL re-
sponses to seasonal H3N2 influenza viruses with H5N1 strains
is substantial.
Thus, apart from some individual epitopes that display
amino acid sequence variation between H3N2 and H5N1 in-
fluenza A viruses, the level of cross-reactivity is considerable
FIG. 2. Epitope-specific IFN-␥production by CTLs after stimulation with peptide-pulsed BLCL. The number of IFN-␥-producing cells per
10,000 CD8
⫹
T cells (5,000 cells for subject 3) from subjects from HLA groups I (A), II (B), and III (C) were measured by ELISPOT assay. CD8
⫹
T cells were isolated from PBMC populations expanded in vitro with influenza virus A/NL/18/94 and subsequently stimulated with peptide variants
as indicated. (*, peptide sequence of the known human influenza virus CTL epitopes).
5164 KREIJTZ ET AL. J. VIROL.
and does not seem to be influenced by the HLA phenotype of
the study subjects. The NP
383–391
epitope is present in H5N1
viruses but has disappeared from human H3N2 viruses during
decades of virus evolution (7, 26). Since a virus was used for
stimulation of the PBMC that does not contain this epitope,
this does not give a false sense of cross-reactivity in HLA-
B*2705-positive subjects. It is unknown whether the presence
of the NP
383–391
epitope interferes with the presentation of
other HLA-B*2705-restricted epitopes. Interference has been
observed only with the overlapping HLA-B*0801-restricted
NP
380–388
epitope (1, 24).
Although it is unknown to what extent preexisting T-cell
immunity can dampen the impact of a next influenza pan-
demic, it is speculated that the protective effect of cross-reac-
tive CTL responses has a beneficial effect on the outcome of
infection with new pandemic influenza virus strains. This spec-
ulation is supported by a number of different observations.
First, in animal models it has been shown that virus-specific
CTLs contribute to heterosubtypic immunity (6, 10, 16); sec-
ondly, it was found in 1957 that individuals that had experi-
enced documented infections with H1N1 influenza A viruses
were less likely to develop severe disease or succumb to infec-
tion with the pandemic strain of the H2N2 subtype (4). In this
respect it is of interest that during the current outbreak of
H5N1 infections in humans, younger individuals are especially
at risk for severe disease and a fatal outcome of infection (21).
It can be hypothesized that younger individuals are less likely
to have been exposed to seasonal influenza A viruses of the
H3N2 and H1N1 subtypes and thus have not mounted a (cross-
reactive) CTL response to an alternative subtype. However, it
cannot be excluded that confounding factors play a role in the
observed disproportionate age distribution of severe H5N1
human cases. Last but not least, the human CTL response
against epidemic strains is largely cross-reactive with H5N1
influenza virus strains, as was demonstrated in the present
study. Although these cross-reactive CTL populations may not
prevent infection with pandemic strains, they may contribute to
a certain degree of heterosubtypic immunity and facilitate a
more rapid clearance of the infection than in immunologically
naı¨ve individuals who lack cross-reactive T-cell populations.
This may determine the difference between life and death
during a pandemic outbreak. In addition, the induction of
cross-reactive CTL responses may be an attractive target for
the development of universal vaccines that could confer
broadly protective immunity against influenza viruses of vari-
ous subtypes.
FIG. 3. Recognition of NP derived from H3N2 and H5N1 influ-
enza virus by in vitro expanded PBMC specific for influenza virus
A/NL/18/94 (H3N2). The lytic activity of in vitro expanded PBMC was
tested with MHC-I matched BLCL nucleofected with NP-GFP coding
plasmid (NP of either influenza virus A/NL/18/94 [black circles] or
A/VN/1194/04 [open circles]) or empty control GFP plasmid (gray
circles). The NP-specific lytic activity was tested with PBMC of the
subjects from group I (subjects 1 to 4), II (subjects 5 to 9), and III
(subjects 10 to 14 and 16). E:T ratios were 0, 3.125, 6.25, 12.5, 25, and
50 for all subjects except numbers 13 and 14, for whom E:T ratios were
0, 0.3, 1, 3, 10 and 30. The lytic activities against control plasmid-
transfected target cells are not visible for subjects 13 and 14 as a result
of negative values for the percentages of specific lysis, which were
caused by slight increases in the number of GFP-positive viable cells.
Standard deviation of the means was ⬍10%.
FIG. 4. Recognition of influenza virus-infected BLCL by CTLs.
The number of IFN-␥-producing cells per 10,000 CD8
⫹
T cells was
measured by ELISPOT assay after stimulation with BLCL infected
with influenza virus A/NL/18/94 or A/VN/1194/04. Each symbol rep-
resents an individual subject from group I (A), II (B), or III (C).
Uninfected BLCL were used as negative controls. The horizontal
bars represent the average responses of all study subjects in groups
I, II, and III.
VOL. 82, 2008 CROSS-RECOGNITION OF AVIAN H5N1 VIRUS BY HUMAN CTLs 5165
ACKNOWLEDGMENTS
This study was conducted under the auspices of The Netherlands
Influenza Vaccine Research Center and financially supported in part
by The Netherlands Organization for Health Research and Develop-
ment (ZonMW; grant 91402008).
We thank T. Bestebroer and C. Baas for outstanding technical
assistance.
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