This information is current as
Influenza Virus-like Particle Vaccination
against Influenza A Virus Infection following
Protective CD8 T Cell-Mediated Immunity
Emily A. Hemann, Sang-Moo Kang and Kevin L. Legge
http://www.jimmunol.org/content/191/5/2486
doi: 10.4049/jimmunol.1300954
July 2013;
J Immunol 2013; 191:2486-2494; Prepublished online 24
Material
Supplementary
4.DC1.html
http://www.jimmunol.org/content/suppl/2013/07/24/jimmunol.130095
References
http://www.jimmunol.org/content/191/5/2486.full#ref-list-1
This article cites 63 articles, 30 of which you can access for free at:
Subscriptions
http://jimmunol.org/subscriptions
Information about subscribing to The Journal of Immunology is online at:
Permissions
http://www.aai.org/ji/copyright.html
Submit copyright permission requests at:
Email Alerts
http://jimmunol.org/cgi/alerts/etoc
Receive free email-alerts when new articles cite this article. Sign up at:
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Immunologists, Inc. All rights reserved.
Copyright ¨Ï 2013 by The American Association of
9650 Rockville Pike, Bethesda, MD 20814-3994.
The American Association of Immunologists, Inc.,
The Journal of Immunology is published twice each month by
Downloaded from http://www.jimmunol.org/ at Univ of Houston-Clear Lake Neumann Lib on October 9, 2013
The Journal of Immunology
Protective CD8 T Cell.Mediated Immunity against Influenza
A Virus Infection following Influenza Virus.like Particle
Vaccination
Emily A. Hemann,* Sang-Moo Kang,¢Ó and Kevin L. Legge*,¢Ô
The development of influenza A virus (IAV) vaccines capable of inducing cytotoxic CD8 T cell responses could potentially provide
superior, long-term protection against multiple, heterologous strains of IAV. Although prior studies demonstrated the effectiveness
of baculovirus-derived virus-like particle (VLP) vaccination in generating Ab-mediated protection, the role that CD8 T cell
immunity plays in overall VLP-mediated protection is less-well understood. In this article, we demonstrate that intranasal
vaccination of mice with a VLP containing the hemagglutinin and matrix 1 proteins of IAV/PR/8/34 leads to a significant increase
in hemagglutinin 533.specific CD8 T cells in the lungs and protection following subsequent homologous challenge with IAV. VLPmediated
protection was significantly reduced by CD8 T cell depletion, indicating a critical role for CD8 T cells in protective
immunity. Importantly, our results show that VLP vaccine.induced CD8 T cell.mediated protection is not limited to homologous
IAV strains. VLP vaccination leads to an increase in protection following heterosubtypic challenge with a strain of IAV that avoids
vaccine-induced neutralizing Abs but contains conserved, immunodominant CD8 T cell epitopes. Overall, our results demonstrate
the ability of influenza protein.containing VLPs to prime IAV-specific CD8 T cell responses that contribute to protection from
homo- and heterosubtypic IAV infections. These results further suggest that vaccination strategies focused on the development of
cross-protective CD8 T cell responses may contribute to the development of ¡°universal¡± IAV vaccines. The Journal of Immunology,
2013, 191: 2486.2494.
I nfluenza A virus (IAV) causes significant seasonal illness,
leading to ¡200,000 hospitalizations and 36,000 deaths annually
in the United States during nonpandemic years (1, 2).
This high rate of severe illness, along with the constant threat of
a pandemic influenza outbreak, has renewed interest in developing
novel influenza vaccination strategies. Following primary IAV
infection, the development of a cytotoxic, influenza-specific CD8
T cell response is important in terminating the acute infection and
contributes to long-term immunity (3, 4). The current influenza
subunit vaccine leads to the production of influenza-neutralizing
Abs (5). However, whether a robust influenza-specific CD8 T cell
response is generated following vaccination remains unclear (5,
6). Importantly, as the virus drifts, neutralizing Abs induced by
either previous vaccination or IAV infection can rapidly become
ineffective as the result of constant Ab-mediated selective pressure
on IAV¡¯s hemagglutinin (HA) and neuraminidase (NA) (7).
Conversely, CD8 T cell responses to IAV are often generated
against influenza-derived proteins/epitopes that are more highly
conserved and, thus, are able to contribute to heterosubtypic
protection (8, 9). Therefore, the development of an IAV vaccine
capable of inducing a cytotoxic CD8 T cell response could potentially
provide superior, long-term protection against multiple,
heterologous strains of IAV (10, 11).
Recently, influenza virus-like particles (VLPs) were developed
from recombinant baculoviruses containing influenza virus proteins,
such as HA and/or NA, on the surface and matrix 1 protein
(M1) as a core of the VLP. Influenza VLPs resemble influenza
virions in both size and morphology (12). These influenza VLPs
are an attractive vaccination candidate because they are replication
deficient (i.e., they contain no IAV genomic material) and,
therefore, could be administered to the young and elderly populations
that exhibit the highest risk for complications from seasonal
influenza virus infection. Influenza VLPs induce potent Ab
responses and provide heterosubtypic cross-protection from morbidity
and mortality during lethal IAV challenge following a single,
intranasal (i.n.) treatment (12.17). However, the immune
component mediating heterosubtypic protection following influenza
VLP vaccination remains unknown. Given this cross-protection,
VLP-induced CD8 T cell responses may contribute to protection
from subsequent infections. Indeed, one in vitro study (17) highlighted
the ability of dendritic cells (DCs) pulsed with influenza
VLPs to stimulate human influenza.specific CD8 T cells. However,
whether i.n.-administered influenza VLPs can induce influenzaspecific
CD8 T cell responses in vivo remains unknown. Further,
the role of VLP-induced CD8 T cell immunity in mediating
protection following homo- and heterosubtypic IAV challenges
remains to be elucidated.
In this study, we examine the development and contribution of
influenza VLP-induced CD8 T cells to IAV immunity following
a single, i.n. vaccination with VLPs containing HA and M1 of
*Department of Pathology and Interdisciplinary Graduate Program in Immunology,
University of Iowa, Iowa City, IA 52242; ¢ÓCenter for Inflammation, Immunity, and
Infection, and Department of Biology, Georgia State University, Atlanta, GA 30303;
and ¢ÔDepartment of Microbiology, University of Iowa, Iowa City, IA 52242
Received for publication April 10, 2013. Accepted for publication June 29, 2013.
This work was supported by National Institutes of Health Grants AI071085 (to K.L.L.)
and T32 AI007533 (to E.A.H.) and by the University of Iowa Department of
Pathology.
Address correspondence and reprint requests to Dr. Kevin L. Legge, Department of
Pathology, University of Iowa, 1028ML, 200 Hawkins Drive, Iowa City, IA 52242.
E-mail address: kevin-legge@uiowa.edu
The online version of this article contains supplemental material.
Abbreviations used in this article: CL-4, clone-4; DC, dendritic cell; dLN, lungdraining
lymph node; HA, hemagglutinin; IAV, influenza A virus; i.n., intranasal
(ly); M1, matrix 1 protein; ma, mouse adapted; NA, neuraminidase; NP, nucleocapsid
protein; Penh, enhanced pause; p.i., postinfection; TCIU, tissue culture infectious
unit; VLP, virus-like particle; WT, wild-type.
Copyright 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1300954
A/PR/8/34. Our findings demonstrate a small, but significant, increase
in HA533-specific CD8 T cells immediately following influenza
VLP administration, which is sustained for $1 mo. Our results
also indicate HA533-specific CD8 T cells primed by influenza VLP
vaccination are increased in the lungs upon subsequent IAV
challenge. These vaccine-induced CD8 T cells are crucial in
providing protection from mortality during lethal, homologous
IAV challenge because mice depleted of CD8 T cells 30 d following
vaccination succumb to the challenge infection. Further
support for vaccine-induced CD8 T cells in mediating protection
is demonstrated by the ability of VLP-induced, HA533-specific
CD8 T cells to aid in protection from high-dose, heterosubtypic
IAV challenge. Together, our findings highlight the potential use
of influenza VLPs to induce effective, cross-protective CD8
T cells that can contribute to protective immunity during particularly
severe seasonal and pandemic outbreaks of influenza virus
infections.
Materials and Methods
Mice
Six- to eight-week-old wild-type (WT) BALB/c mice were obtained from
The National Cancer Institute (Frederick, MD). Clone 4 (CL-4) CD90.1+
mice containing TCR-transgenic T cells specific for the HA533/HA529
epitope of A/PR/8/34 or A/Japan/305/57 were obtained from The Jackson
Laboratory (Bar Harbor, ME). All mice were housed and maintained in the
specific pathogen.free animal care facility at the University of Iowa. All
experiments were performed in accordance with regulatory standards and
guidelines and were approved by the Institutional Animal Care and Use
Committee of the University of Iowa.
VLP vaccination
VLPs containing HA and M1 of mouse-adapted (ma) A/PR/8/34 were
produced as described previously (12). Mice were anesthetized i.n. with
isoflurane and administered 2.5 mg VLPs in PBS or 50 ml PBS (as
a control).
Influenza virus infection
Mouse-adapted IAVs A/PR/8/34 (maH1N1), A/HK/1/68 (maH3N2), and
A/Japan/305/57 (maH2N2) were prepared from stocks, as described previously
(18). Mice were anesthetized with isoflurane and infected i.n. with
5.5 3 104 tissue culture infectious units (TCIU) of virus. For day-8
analysis, mice were challenged with 5.5 3 103 TCIU A/PR/8/34.
MHC class I tetramers
MHC class I tetramers HA533.541 [H-2K(d)/IYSTVASSL] and nucleocapsid
protein (NP)147.155 [H-2K(d)/TYQRTRALV] were obtained from
the National Institute of Allergy and Infectious Disease MHC Tetramer
Core Facility (Atlanta, GA).
Surface staining
Single-cell suspensions of lungs, lung-draining LNs (dLNs; i.e., mediastinal
and peribronchial), and spleens were prepared by pressing the tissues
through wire mesh screens, plating 1 3 106 cells/well in a 96-well plate,
and blocking with 2 ml rat serum in FACS buffer for 30 min at 4¡ÆC.
Following blocking, cells were incubated with FACS buffer containing
rat anti-mouse CD8a conjugated to FITC (53-6.7), rat anti-mouse CD3¥å
conjugated to PerCP Cy5.5 (145-2C11) purchased from BD, or HA533 or
NP147 tetramers conjugated to allophycocyanin and PE for 1 h at 4¡ÆC. Cells
were fixed in FACS Lysis Buffer (BD), per the manufacturer¡¯s instructions,
and resuspended in PBS. Data were acquired on a BD FACSCanto II and
analyzed with FlowJo software (TreeStar).
Intracellular cytokine staining
Intracellular cytokine staining was performed as previously described (19).
Briefly, 5 3 105 cells from lung single-cell suspensions were incubated for
6 h with HA533.541 or NP147.155 peptides in complete media containing
recombinant human IL-2 (Novartis) and brefeldin A (Sigma). Following
incubation, cells were surface stained with anti-mouse CD8a and antimouse
CD3¥å mAbs. Following fixation, cells were permeabilized by incubation
for 30 min at 4¡ÆC in FACS buffer containing 0.5% saponin
(ACROS) and subsequently stained with rat anti-mouse IFN-g mAb
(XMG1.2) conjugated to allophycocyanin (BD) for 30 min at 4¡ÆC in FACS
buffer containing 0.5% saponin. Data were acquired on a BD FACSCanto
II and analyzed with FlowJo software (TreeStar).
Purification and adoptive transfer of CL-4 T cells
Single-cell suspensions of spleens from CL-4 CD90.1+ mice (containing
TCR-transgenic T cells specific for the HA533/HA529 epitope of A/PR/8/34
and A/Japan/305/57, respectively) were labeled with CD8a MicroBeads
and purified using LS columns, according to the manufacturer¡¯s instructions
(Miltenyi Biotec). A total of 5 3 105 purified donor CD90.1+/CD8a+
CL-4 cells was labeled with 2.5 mM CFSE (Invitrogen) and adoptively
transferred i.v. into BALB/c CD90.2+ host mice (20). Mice were vaccinated
with VLPs 24 h later. Three days following VLP vaccination, dLNs
were collected, and proliferation was determined by analyzing CFSE dilution
in CD3¥å+/CD8a+/CD90.1+ cells.
CD8a depletion
An anti-CD8a.depletion strategy described to reduce splenic IAV-specific
memory CD8 T cells, but allow naive T cell repopulation, was modified to
ensure depletion of HA533-specific memory CD8 T cells within the lungs
and dLNs (21). Briefly, mice were treated with anti-CD8a mAb (clone
2.43) i.p. (250 mg) and i.n. (100 mg) 30 d following VLP vaccination or
sublethal (0.1 LD50) A/PR/8/34 challenge. The repopulation of naive CD8
T cells in the spleen, lungs, and dLNs was monitored for 14 wk. In subsequent
experiments, all memory CD8 T cell.depleted mice were used 14
wk after anti-CD8a Ab treatment (i.e., a time point when naive CD8
T cells had re-established and stabilized). Control mice were given
equivalent volumes of PBS i.n. and i.p.
Pulmonary virus titer
Lungs were harvested on days 2, 4, 6, and 8 postinfection (p.i.), homogenized,
and snap-frozen. Pulmonary virus titers were determined as previously
described (22). Briefly, 5 3 105 MDCK cells in Iscove¡¯s complete
medium containing 50 mg/ml gentamicin and 100 U/ml penicillin/100 mg/
ml streptomycin (all from Life Technologies) were infected with 10-fold
serial dilutions of lung homogenates, as well as stock IAV (A/PR/8/34) as
a control, and incubated at 37¡ÆC. Twenty-four hours later, medium was
removed from all wells and replaced with Iscove¡¯s complete media containing
0.0002% TPCK-Trypsin (Worthington), 50 mg/ml gentamicin (Life
Technologies), and 100 U/ml penicillin/100 mg/ml streptomycin (Life
Technologies). Three days later, 0.5% chicken RBCs (Colorado Veterinary
Products) were mixed 1:1 with supernatant from each of the cell culture
wells, and agglutination was measured after incubating for 1 h at room
temperature. Subsequently, TCIU were calculated using the Reed.Muench
method.
Measurement of airway resistance
A whole-body plethysmograph (Buxco Electronics) was used to measure
enhanced pause (Penh), an indicator of airway resistance, during respiration
(23). Penh values for each mouse were recorded daily following IAV infection.
Day-0 baseline Penh values were recorded prior to infection.
Statistical analysis
Data were compiled in graphical format using Prism software (GraphPad
Software, San Diego, CA). Error bars represent the SEM. Statistical significance
was determined using unpaired, two-tailed Student t tests, logrank
tests, or ANOVAs followed by the Bonferroni posttest.
Results
Influenza VLP vaccination leads to an increase in
HA533-specific CD8 T cells in the lungs following vaccination
We first determined whether vaccine-specific CD8 T cells accumulate
in the lungs 7 and 14 d following a single, i.n. VLP vaccination.
Given the expected low frequency of Ag-specific CD8
T cells present at baseline and following vaccination, we used
a dual tetramer.labeling strategy to identify influenza-specific
CD8 T cells that had expanded following vaccination (Fig. 1A)
(24.26). No change in the frequency or numbers of influenzaspecific
lung CD8 T cells was observed on day 7 postvaccination
(data not shown). The frequency and number of HA533-specific
CD8 T cells were increased significantly in the lungs on day
The Journal of Immunology 2487
14 postvaccination compared with mice that were given PBS i.n.
as a control (Fig. 1). Further, this expansion on day 14 post.i.n.
vaccination was specific for the IAV proteins contained within the
VLPs, because no change in the NP147-specific CD8 T cell population
was observed when comparing vaccinated mice with
controls (data not shown). (Note: IAV NP is not present in the
VLP preparation used in these studies.) We also observed no
changes in the total number of CD8 T cells present in the lungs at
day 7 or 14 following VLP vaccination (Supplemental Fig. 1, data
not shown). We observed no increase in the frequency or total
numbers of HA533-specific CD8 T cells in the dLNs at day 7 or 14
following vaccination (data not shown). Together, the above
results indicate that the expansion of HA533-specific CD8 T cells
was specific to the HA protein contained within the VLP and was
not an unknown, nonspecific effect of influenza VLP vaccination
on total, or influenza-specific, CD8 T cell expansion.
Influenza VLP vaccination leads to proliferation of
HA533-specific CD8 T cells in dLNs
Given that we observed an increase in HA533-specific CD8 T cells
only in the lungs, but not dLNs, following VLP vaccination, we
next sought to determine the location where these T cells were
primed. During infection with influenza virus, naive CD8 T cells
are primed in dLNs and then migrate to the site of infection
(i.e., lungs) to kill virally infected cells (19, 27.30). Although the
dLN is the initial site of expansion, activated CD8 T cells continue
to proliferate in the lung during IAV infection (31). Thus, analysis
of dLNs at day 7 postvaccination may have been too late to observe
vaccine-induced CD8 T cell expansion (20). Further, recent
studies demonstrated that influenza-specific CD8 T cells can be
primed and expand in the lungs of mice lacking dLNs, spleen, and
Peyer¡¯s patches when challenged with IAV (32). Therefore, it was
determined whether HA533-specific CD8 T cells were activated
and expanded in dLNs or lung tissue. To monitor this expansion,
5 3 105 CFSE-labeled, naive CL-4, CD90.1+ CD8 T cells (transgenic
CD8 T cells specific for the HA533 epitope) were adoptively
transferred into host WT (CD90.2+) mice. The host mice were
vaccinated 24 h later with either HA/M1 containing VLPs or
a control VLP containing only M1. At 2.5 and 3 d following
vaccination, the dLNs, lungs, and spleens were harvested and
analyzed to determine CL-4 proliferation. Compared with M1-
VLP controls (in which we observed limited CFSE dilution in
donor CL-4 cells), HA/M1-VLP vaccination induced substantial
proliferation of HA533-specific CD8 T cells in the dLNs by day 2.5
postvaccination (Fig. 2, left panels). On day 3 postvaccination,
a greater frequency of CL-4 cells was CFSElo/2 in the dLNs of
mice treated with HA/M1-VLP versus M1-VLP. Further, limited
divided CL-4 cells were present in the lungs, but not in the
spleen, at day 2.5 postvaccination (Fig. 2, upper panels). However,
by day 3, divided CL-4 cells had begun to accumulate in
the lungs of mice vaccinated with HA/M1-VLP (Fig. 2, lower
middle panel), suggesting that these cells are trafficking to the
site of vaccination. These results imply that activation and early
division of influenza VLP-specific CD8 T cells initially occur in
dLNs following i.n. influenza HA/M1-VLP administration and
that these influenza-specific cells subsequently migrate to the
lung tissue.
HA533-specific CD8 T cells are increased in dLNs and lungs
following lethal IAV challenge 30 d post.HA/M1-VLP
vaccination
Following infection with influenza virus, IAV-specific, long-lived
memory CD8 T cells are generated and can reside in both the
local lung tissue and in secondary lymphoid organs (33.41). These
local, tissue-resident memory cells are key in early (days 0.3 p.i.)
control of viral infection, because they are present at the site of
initial Ag encounter and are able to respond rapidly to mediate
protective immunity (35, 36). At later times following infection
(day 5 p.i. and beyond), memory T cells that reside in dLNs during
the steady state dominate the response. These dLN-resident
memory CD8 T cells have a greater proliferative and cytotoxic
capacity compared with local, tissue-resident CD8 T cells (38).
To determine whether CD8 VLP.induced T cell.mediated
protection occurs following challenge, we examined the frequency
and numbers of the influenza-specific CD8 T cells in both the
lungs and dLNs prior to challenge, as well as at various times
postchallenge. In the dLNs, we observed no significant difference
between VLP-vaccinated or control mice with regard to the frequency
or numbers of HA533-specific CD8 T cells at any of the
times analyzed postchallenge (data not shown). Similarly, at
30 d following VLP vaccination (i.e., day 0 of challenge), no
difference in the number of HA533-specific CD8 T cells present in
the lungs was detected between the two groups (Fig. 3A, 3B).
However, as early as 1 d following lethal IAV challenge, we observed
a significant increase in HA533-specific CD8 T cells in the
lungs that continued through at least day 8 postchallenge (Fig. 3).
In contrast, we observed no significant differences in the NP147-
specific CD8 T cell response (i.e., an epitope not contained within
FIGURE 1. VLP vaccination boosts the HA533
+ CD8 T cell response in
the lungs. BALB/c mice were treated i.n. with 2.5 mg VLPs or 50 ml PBS
as a control. On day 14 postvaccination, lungs were harvested, and the
frequency and total numbers of HA533
+ CD8 T cells were determined by
flow cytometry. (A) Representative tetramer gating of CD8a+/CD3¥å+ lung
samples on day 14 post.VLP vaccination or PBS administration. (B)
Cumulative frequency and total numbers of HA533
+ CD8 T cells. The data
are the mean values 6 SEM of two independent experiments (n = 9 mice/
group). Data were analyzed using the Student t test.
2488 VLP VACCINATION INDUCES IAV-SPECIFIC CD8 T CELL RESPONSES
the VLPs) in the lungs and dLNs of VLP-vaccinated versus non-
VLP.primed mice (Supplemental Fig. 2, data not shown).
HA533-specific CD8 T cells generated during VLP vaccination
provide protection during lethal IAV challenge
Influenza-specific CD8 T cells generated following IAV challenge
are crucial in the clearance of virus from the host during primary
IAV infections (3, 4). However, neutralizing anti-H1 Ab responses
are important in providing sterilizing immunity following subsequent
homologous IAV challenge (42, 43). To evaluate whether
CD8 T cells primed during influenza VLP vaccination were required
for protection against subsequent lethal influenza virus
infection, we first determined the efficiency of anti-CD8a depletion
in a model generating a robust CD8 T cell response. We
FIGURE 2. VLP treatment leads
to proliferation of IAV-specific CD8
T cells in dLNs. CD90.2+ BALB/c
mice were given 5 3 105 MACSpurified,
CFSE-labeled CL-4 CD90.1+
cells i.v. and treated i.n. with 2.5 mg
of VLP (open graph), PBS (data not
shown), or 2.5 mg of VLP containing
only M1 (shaded graph) 24 h later.
dLNs, lungs, and spleens were collected
2.5 and 3 d following vaccination
and analyzed via flow cytometry
for proliferation (i.e., CFSE dilution).
Data are representative of two independent
experiments (n = 3.6 mice
pooled/group).
FIGURE 3. Kinetics of the HA533-specific CD8 T cell response primed by VLP vaccination following lethal IAV challenge. BALB/c mice were treated
i.n. with 2.5 mg VLPs or 50 ml PBS as a control. Thirty days later, all mice were infected with 5 LD50 of A/PR/8/34, with the exception of mice that were to
be analyzed on day 8 p.i., which were challenged with 1 LD50 dose to ensure the survival of control animals at this time point. (A and B) Lungs were
harvested at the indicated time points prior to infection (day 0) or p.i., and HA533-specific CD8a+/CD3¥å+ cells were identified by flow cytometry with
HA533-tetramers. (A) Representative tetramer gating of CD8a+/CD3¥å+-gated lung samples on days 0 and 8 post.IAV challenge. (B) The frequency and total
numbers of HA533-specific CD8a+/CD3¥å+ cells. (C and D) Lungs were harvested on day 8 p.i., and single-cell homogenates and HA533 cells were measured
by intracellular cytokine staining. (C) Representative IFN-g gating of CD8a+/CD3¥å+-gated lungs on day 8 post.IAV challenge. (D) The frequency and total
numbers of IFN-g+CD3+CD8+ T cells. The data are the mean values 6 SEM from three independent experiments (n = 6.13 mice/group for lungs). Data
were analyzed using two-way ANOVA followed by the Bonferroni posttest. *p , 0.05, **p , 0.01.
The Journal of Immunology 2489
challenged mice with a sublethal dose of IAV, depleted CD8a+
cells 30 d later, and monitored the total and HA533-specific CD8
T cell response. Forty-eight hours following depletion, virtually no
CD8 T cells remained in the blood, dLNs, lungs, or spleen of mice
(Supplemental Fig. 3). Fourteen weeks following CD8a depletion,
total CD8 T cell recovery had plateaued in the blood, dLNs, lungs,
and spleen of mice (Supplemental Fig. 3). Further, when HA533-
specific CD8 T cells were examined, we observed that anti-CD8a
administration also depleted these cells. However, there was no
significant increase in the HA533-specific cells within the recovered,
total CD8 T cell population, suggesting that IAV-induced
CD8 T cell immunity had been ablated (Supplemental Fig. 4).
After determining the efficiency of our depletion strategy, HA/
M1-VLP.vaccinated mice were challenged 14 wk following CD8
T cell depletion with a lethal dose of A/PR/8/34. Although mice
that were vaccinated and challenged without CD8 depletion survived
following lethal IAV challenge, only 60% survival was observed
when vaccinated mice were depleted of CD8 T cells and
then lethally challenged with IAV (Fig. 4B). Further, we observed
a slight, but not significant, increase in weight loss and Penh in
vaccinated mice that had been depleted of CD8 T cells and allowed
to recover total CD8 T cell numbers prior to lethal challenge
compared with mice that were vaccinated and challenged with IAV
(Fig. 4C, 4D). These data indicate that HA533-specific CD8 T cells
generated following VLP vaccination are required to mediate
complete protection from lethal IAV challenge. It is likely that the
remaining protection observed after removal of memory CD8
T cells is due to the IAV-specific Ab previously demonstrated to be
induced following VLP vaccination (16).
Influenza VLPs provide protection from high-dose,
heterosubtypic IAV challenge
Influenza-specific CD8 T cells were demonstrated to provide crossprotection
against heterosubtypic strains of influenza virus in cases
in which previously generated neutralizing Abs may not be able to
control IAV infection (11, 44, 45). To determine whether CD8
T cells primed following i.n. influenza VLP administration could
mediate a similar protection from heterosubtypic IAV challenge,
groups of mice were vaccinated with HA/M1-VLPs and challenged
30 d later with homo- or heterosubtypic strains of IAV.
When mice were challenged with a lethal dose of homosubtypic
maH1N1 (A/PR/8/34) following influenza VLP vaccination,
86.6% of the mice survived the challenge and had significantly
reduced morbidity compared with PBS-treated controls (Fig. 5A).
We also observed significantly lower virus titers on days 2 and 4
post.IAV infection in mice that were administered influenza VLPs
i.n. compared with PBS-treated controls (Fig. 5A). To determine
whether influenza VLP vaccine.induced CD8 T cells play a role
in mediating protection during heterosubtypic IAV infection, we
next challenged mice vaccinated i.n. with VLPs containing HA
and M1 from A/PR/8/34 with maH2N2 (A/Japan/305/57). Challenge
with A/Japan/305/57 avoids neutralizing Ab responses directed
against HA, but a modified, cross-reactive version of the
HA533 epitope is still present within A/Japan/305/57 (Table I)
(46). Following challenge with maH2N2, 50% of control mice
survived infection, whereas significantly more mice (90%) survived
infection when they were given A/PR/8/34 HA/M1-VLPs
30 d prior to challenge (Fig. 5B). Interestingly, despite this difference
in lethality, we observed no difference in morbidity or
virus titer at days 2 and 4 p.i. (Fig. 5B). To ensure that these
results were specific to the conserved CD8 T cell response generated
by vaccination and not due to a highly conserved component
of the immune response or another nonspecific effect
mediated by VLP vaccination, we also challenged HA/M1 (H1)-
vaccinated mice withmaH3N2 (A/HK/1/68). Challengewith A/HK/
1/68 avoids neutralizing Ab responses along with the majority of
memory CD4 and CD8 T cell responses that could have been
generated following i.n. VLP administration specific forHA and M1
of A/PR/8/34 (8). Further, the HA533 epitope is altered in the
maH3N2 strain, such that the peptide is no longer recognized by
CD8 T cells (Table I) (47). As expected, all of the vaccinated and
controlmice succumbed to infection, and we observed no difference
in weight loss or virus titer in the lungs of these mice (Fig. 5C).
To further investigate differences that could account for the
survival of influenza VLP-vaccinated mice challenged with
maH2N2, even though no early difference in viral titer was
detected, we challenged mice and monitored their airway resistance
daily for 8 d using a whole-body plethysmograph. We observed
significantly higher Penh values in mice that were not vaccinated
and challenged with maH1N1 compared with vaccinated mice on
days 3.8 p.i. (Fig. 5D). In contrast, we observed no difference in
Penh when mice were challenged with maH3N2 following i.n.
FIGURE 4. CD8 T cells primed by VLP vaccination are required for
complete protection during IAV infection. (A) BALB/c mice were treated
i.n. with 2.5 mg VLP in 50 ml PBS or 50 ml PBS as a control. Thirty days
later, mice were treated i.n. and i.p. with anti-CD8a. Fourteen weeks later,
mice were challenged with 5 LD50 A/PR/8/34. Mortality (B), morbidity
(C), and airway resistance (D) were monitored daily for 14 d (n = 4.5
mice/group).
2490 VLP VACCINATION INDUCES IAV-SPECIFIC CD8 T CELL RESPONSES
VLP vaccination or PBS administration (Fig. 5F, lower right
panel). When mice were challenged with maH2N2 following VLP
vaccination, a significant reduction in Penh was observed starting
on day 6 p.i. compared with PBS-treated controls (Fig. 5E). This
reduction in Penh correlates with the period of time during which
unvaccinated mice challenged with maH2N2 succumbed to infection
(Fig. 5B).
Because Penh was reduced significantly at day 6 postheterosubtypic
challenge in H1 influenza VLP.vaccinated mice,
we also determined lung viral titers at days 6 and 8 p.i. At both
days 6 and 8 after heterosubtypic IAV challenge, virus titers were
significantly reduced in mice that had been vaccinated compared
with PBS controls (Fig. 6A). The lack of early control of infection,
but reduced Penh and virus titers at day 6 following infection,
suggests that early Ab-mediated clearance of virus does not lead
to increased survival in HA/M1-VLP.vaccinated mice challenged
with maH2N2. Given this, we sought to determine whether A/PR/
8/34 HA/M1-VLP vaccination led to a significant increase in
protective HA533-specific CD8 T cells following A/Japan/305/57
challenge. We did not detect any significant difference in HA533-
specific CD8 T cells in the lungs on day 2 p.i. between mice that
were vaccinated with HA/M1-VLPs or control M1-VLPs (data not
shown). This finding correlates with the lack of difference in Penh
and virus titers observed at day 2 postchallenge. However, we
observed a significant increase in both the frequency and number
of HA533-specific CD8 T cells in the lungs of HA/M1-VLP.vaccinated
mice versus M1-VLP.vaccinated mice at day 6 postchallenge
(Fig. 6B). Together, the results from Figs. 5 and 6
strongly suggest that the cross-reactive HA533-specific CD8
T cells primed by HA/M1-VLP vaccination are key to mediating
protection during maH2N2 (A/Japan/305/57) challenge.
Discussion
Vaccination continues to be the best defense against influenza virus
infection. However, it remains unclear whether current subunit
vaccination strategies induce influenza-specific CD8 T cell
responses (17). Our findings suggest that HA533-specific CD8
T cells generated following influenza VLP vaccination are important
in providing full protection during lethal IAV challenge,
because mice depleted of CD8 T cells displayed reduced survival
and increased airway resistance compared with nondepleted,
vaccinated mice (Fig. 4). Although a characterization of the full
breadth of the immune response generated following influenza
VLP vaccination has not been completed, recent work demonstrated
the ability of influenza VLPs to generate Abs specific to the
stalk domain of A/PR/8/34 HA (48). These HA stalk Abs were
FIGURE 5. VLP vaccination leads to protection from mortality during heterosubtypic IAV infection. BALB/c mice were treated i.n. with 2.5 mg VLP or
50 ml PBS as a control. Thirty days later, mice were challenged with 5 LD50 PR/8 [(A), maH1N1] or equivalent TCIU (¡5.5 3 104 TCIU/mouse) doses of
A/Japan/305/57 [(B), maH2N2] or A/HK/1/68 [(C), maH3N2], and morbidity (middle panels) and mortality (left panels) were measured daily for 14 d.
Lung homogenates (n = 3/group) were also collected on days 2 and 4 p.i. to determine viral titers (right panels). Airway resistance following challenge with
maH1N1 (D), maH2N2 (E), or maH3N2 (F) was measured using a whole-body plethysmograph daily for 8 d (n = 6 mice/group for maH1N1 and maH2N2;
n = 3 mice/group for maH3N2). The morbidity and mortality data are pooled from three independent experiments (n = 10.15 mice/group). Titer data were
analyzed using the Student t test. Morbidity data were analyzed using two-way ANOVA followed by the Bonferroni posttest. Mortality data were analyzed
using the log-rank test. *p , 0.05, ***p , 0.001.
Table I. The HA533 epitope
IAV Strain Epitope Amino Acid Sequence
A/PR/8/34 (maH1N1) HA533.541 IYSTVASSL
A/JAPAN/305/57 (maH2N2) HA529.537 IYATVAGSL
A/HK/1/168 (maH3N2) None (aa 533.541 of HA) ISFAISCFL
Bold residues differ from the A/PR/8/34 amino acid sequence.
The Journal of Immunology 2491
found to broadly neutralize groups 1 and 2 influenza virus and
were suggested as a universal influenza vaccination candidate (49,
50). However, reduced airway resistance and virus titers following
maH2N2 heterosubtypic challenge in HA/M1-VLP.vaccinated
mice does not occur until day 6 p.i., when we detected significantly
greater HA533-specific CD8 T cell responses in the lungs
(Figs. 5B, 6). This delayed protection and lack of reduction in
early viral titers suggest that stalk-specific Ab-mediated protection
is likely not responsible for the protection observed following
VLP vaccination and heterosubtypic maH2N2 challenge. These
findings, along with a loss of protection observed during homologous
IAV challenge when mice have been vaccinated and subsequently
depleted of IAV-reactive CD8 T cells (Fig. 4), suggest
that investigation of IAV vaccine candidates that elicit CD8 T cell
responses will also be important in developing broadly crossreactive
IAV vaccines.
Our study demonstrates that influenza VLPs drive the proliferation
of naive, HA533-specific CD8 T cells in dLNs as early as
2.5 d postvaccination (Fig. 2). Although influenza VLPs were
administered in this study without adjuvant, recent publications
highlighted the wide range of baculovirus and Sf9 host cell proteins
present within the VLPs (48, 51). Both WT baculovirus and
baculovirus-derived influenza VLPs administered i.n. led to similar
mRNA upregulation of IFN-b and Mx1 in the lungs 6 h
postadministration (48). Although this indicates that baculovirus
particles alone prime the system to respond, our results still suggest
the protective effect that we observed is specific to the influenza
proteins present in the virus-like particles. HA/M1-VLPs
induced the proliferation of transgenic, transferred HA533-specific
CD8 T cells (CL-4 cells) in dLNs, whereas influenza VLPs containing
only M1 did not (Fig. 2).
The ability of influenza VLP vaccination to prime CD8 T cell
responses that play a role in protection during lethal and heterosubtypic
IAV infections is surprising, especially considering that
these VLPs are nonreplicative. During IAVinfection, dLN-resident
CD8a+ DCs and migratory CD103+ DCs (the known crosspresenting
DC populations) are the primary DC subsets responsible
for presentation of IAVAgs to CD8 T cells within dLNs (27.
29, 52). Although acquisition of Ag by these DC subsets can occur
via direct (infection) or indirect (cross-presentation or trogocytosis)
methods, data demonstrated that cross-presentation alone is
sufficient to induce functional CD8 T cell responses during IAV
infection in vivo (53.55). Our results support the idea that crosspresentation
alone is sufficient to induce functional and protective
CD8 T cell responses following influenza VLP vaccination because
the influenza VLPs used in these studies have no internal,
IAV genomic material and cannot replicate. Interestingly, DC
activation and cross-presenting ability have been linked to type I
IFN responses. Natural influenza virus infection is known to induce
type I IFN within the lungs, and type I IFN has been well
documented to boost the ability of DCs to activate naive,
influenza-specific CD8 T cell responses (56.60). At this time it
remains unclear which mechanisms ¡°license¡± cross-presentation
during VLP vaccination (61). Whether experimental induction of
type I IFN at the time of influenza VLP vaccination would improve
the ability of DCs in activating IAV-specific CD8 T cell
responses remains to be elucidated. Furthermore, it will be important
to determine the APC subsets (presumably CD103+ DCs)
priming this VLP-specific CD8 T cell response, as well as whether
these APCs are similar to those responsible for activating CD8
T cells during IAV infection and live attenuated influenza vaccination.
Determining the differences in the ability of DC subsets to
regulate CD8 T cell responses following different methods of i.n.
IAV vaccination will allow for the development of IAV vaccines
that are better able to induce broadly cross-protective CD8 T cell
responses.
Vaccines that generate broadly cross-protective CD8 T cell
responses may aid in the development of human IAV vaccines
in the future. Phase I and II clinical trials testing a baculovirusderived
VLP containing HA, NA, and M1 of A/California/04/
2009 (H1N1) and A/Indonesia/05/2005 (H5N1) independently
demonstrated that these vaccines are safe and elicit Ab responses
(62, 63). However, it remains unknown whether these vaccinations
elicit CD8 T cell responses in humans. The Immune Epitope
Database and Analysis Resource identifies 456 positive T cell
epitopes within the M1 protein of various IAV strains contained
in the database (http://www.iedb.org). Therefore, in humans, the
M1 and HA proteins may elicit cross-reactive CD8 T cell populations,
which could mediate protection from homo- and heterologous
IAV infection in addition to HA-and M1-specific Abs. In
contrast to humans, there are no identified T cell epitopes contained
within the M1 protein of A/PR/8/34 in BALB/c mice
(http://www.iedb.org). Given this, it is not surprising that M1
VLPs alone did not mediate protection from lethality in our
BALB/c murine model (data not shown). In the future it will be
important to determine in human trials whether CD8 T cell
responses are generated in response to VLP vaccination in addi-
FIGURE 6. VLP vaccination leads to a reduction in virus titer and
a significant increase in HA533-specific CD8 T cells at later time points
following heterosubtypic maH2N2 challenge. BALB/c mice were treated
i.n. with 2.5 mg VLP or 50 ml PBS as a control. Thirty days later, mice
were challenged with ¡5.5 3 104 TCIU A/Japan/305/57. (A) Mice were
sacrificed, and lungs (n = 3/group) were collected on days 6 and 8 p.i. to
determine viral titers. 2/3 and 0/3 indicate the number of mice with detectable
IAV titers in the lungs at day 8 p.i. (B) Lungs were harvested at
day 6 post.A/Japan/305/57 infection, and tetramer-specific CD8a+/CD3¥å+
cells were identified by flow cytometry (n = 4 mice/group). Data were
analyzed using the Student t test.
2492 VLP VACCINATION INDUCES IAV-SPECIFIC CD8 T CELL RESPONSES
tion to Ab titers, as well as whether these CD8 T cells are capable
of killing virally infected cells.
In summary, to our knowledge, the results presented in this
article demonstrate for the first time that i.n. vaccination with
influenza VLPs containing HA and M1 leads to proliferation of
HA533-specific CD8 T cells in dLNs and subsequent accumulation
of these T cells in the lungs. These HA533-specific CD8 T cells
rapidly accumulate in the lungs following IAV challenge and are
crucial in mediating complete protection from IAV challenge.
Furthermore, influenza VLP vaccination induces heterosubtypic
protection correlated with a late (day 6 p.i. and beyond) decrease
in airway resistance and Penh, as well as a significant increase in
vaccine-induced CD8 T cell responses. These results support
continued investigation into influenza vaccines capable of generating
protective CD8 T cell responses and the potential use of
influenza VLPs as a cross-protective IAV vaccine.
Acknowledgments
We thank Dr. Jae-Min Song for technical assistance. We also thank Drs.
John Harty, Steven Varga, and Thomas Waldschmidt for critical evaluation
of this manuscript.
Disclosures
The authors have no financial conflicts of interest.
References
1. Thompson, W. W., D. K. Shay, E. Weintraub, L. Brammer, C. B. Bridges,
N. J. Cox, and K. Fukuda. 2004. Influenza-associated hospitalizations in the
United States. JAMA 292: 1333.1340.
2. Thompson, W. W., D. K. Shay, E. Weintraub, L. Brammer, N. Cox,
L. J. Anderson, and K. Fukuda. 2003. Mortality associated with influenza and
respiratory syncytial virus in the United States. JAMA 289: 179.186.
3. Topham, D. J., R. A. Tripp, and P. C. Doherty. 1997. CD8+ T cells clear influenza
virus by perforin or Fas-dependent processes. J. Immunol. 159: 5197.
5200.
4. Bender, B. S., T. Croghan, L. Zhang, and P. A. Small, Jr. 1992. Transgenic mice
lacking class I major histocompatibility complex-restricted T cells have delayed
viral clearance and increased mortality after influenza virus challenge. J. Exp.
Med. 175: 1143.1145.
5. Bodewes, R., P. L. Fraaij, J. H. Kreijtz, M. M. Geelhoed-Mieras, R. A. Fouchier,
A. D. Osterhaus, and G. F. Rimmelzwaan. 2012. Annual influenza vaccination
affects the development of heterosubtypic immunity. Vaccine 30: 7407.7410.
6. Combadiere, B., A. Vogt, B. Mahe, D. Costagliola, S. Hadam, O. Bonduelle,
W. Sterry, S. Staszewski, H. Schaefer, S. van der Werf, et al. 2010. Preferential
amplification of CD8 effector-T cells after transcutaneous application of
an inactivated influenza vaccine: a randomized phase I trial. PLoS ONE 5:
e10818.
7. Blackburne, B. P., A. J. Hay, and R. A. Goldstein. 2008. Changing selective
pressure during antigenic changes in human influenza H3. PLoS Pathog. 4:
e1000058.
8. Effros, R. B., P. C. Doherty, W. Gerhard, and J. Bennink. 1977. Generation of
both cross-reactive and virus-specific T-cell populations after immunization with
serologically distinct influenza A viruses. J. Exp. Med. 145: 557.568.
9. Yap, K. L., G. L. Ada, and I. F. McKenzie. 1978. Transfer of specific cytotoxic
T lymphocytes protects mice inoculated with influenza virus. Nature 273: 238.
239.
10. Brown, L. E., and A. Kelso. 2009. Prospects for an influenza vaccine that induces
cross-protective cytotoxic T lymphocytes. Immunol. Cell Biol. 87: 300.308.
11. Hillaire, M. L., A. D. Osterhaus, and G. F. Rimmelzwaan. 2011. Induction of
virus-specific cytotoxic T lymphocytes as a basis for the development of broadly
protective influenza vaccines. J. Biomed. Biotechnol. 2011: 939860.
12. Quan, F. S., C. Huang, R. W. Compans, and S. M. Kang. 2007. Virus-like particle
vaccine induces protective immunity against homologous and heterologous
strains of influenza virus. J. Virol. 81: 3514.3524.
13. Bright, R. A., D. M. Carter, S. Daniluk, F. R. Toapanta, A. Ahmad, V. Gavrilov,
M. Massare, P. Pushko, N. Mytle, T. Rowe, et al. 2007. Influenza virus-like
particles elicit broader immune responses than whole virion inactivated influenza
virus or recombinant hemagglutinin. Vaccine 25: 3871.3878.
14. Quan, F. S., M. C. Kim, B. J. Lee, J. M. Song, R. W. Compans, and S. M. Kang.
2012. Influenza M1 VLPs containing neuraminidase induce heterosubtypic
cross-protection. Virology 430: 127.135.
15. Quan, F. S., D. Steinhauer, C. Huang, T. M. Ross, R. W. Compans, and
S. M. Kang. 2008. A bivalent influenza VLP vaccine confers complete inhibition
of virus replication in lungs. Vaccine 26: 3352.3361.
16. Quan, F. S., D. G. Yoo, J. M. Song, J. D. Clements, R. W. Compans, and
S. M. Kang. 2009. Kinetics of immune responses to influenza virus-like particles
and dose-dependence of protection with a single vaccination. J. Virol. 83: 4489.
4497.
17. Song, H., V. Wittman, A. Byers, T. Tapia, B. Zhou, W. Warren, P. Heaton, and
K. Connolly. 2010. In vitro stimulation of human influenza-specific CD8+ T cells
by dendritic cells pulsed with an influenza virus-like particle (VLP) vaccine.
Vaccine 28: 5524.5532.
18. Legge, K. L., and T. J. Braciale. 2005. Lymph node dendritic cells control
CD8+ T cell responses through regulated FasL expression. Immunity 23:
649.659.
19. Legge, K. L., and T. J. Braciale. 2003. Accelerated migration of respiratory
dendritic cells to the regional lymph nodes is limited to the early phase of
pulmonary infection. Immunity 18: 265.277.
20. McGill, J., N. Van Rooijen, and K. L. Legge. 2008. Protective influenza-specific
CD8 T cell responses require interactions with dendritic cells in the lungs. J.
Exp. Med. 205: 1635.1646.
21. Wlodarczyk, M. F., A. R. Kraft, H. D. Chen, L. L. Kenney, and L. K. Selin. 2013.
Anti-IFN-gamma and peptide-tolerization therapies inhibit acute lung injury
induced by cross-reactive influenza A-specific memory T cells. J. Immunol. 190:
2736.2746.
22. Langlois, R. A., D. K. Meyerholz, R. A. Coleman, R. T. Cook, T. J. Waldschmidt,
and K. L. Legge. 2010. Oseltamivir treatment prevents the increased influenza
virus disease severity and lethality occurring in chronic ethanol consuming mice.
Alcohol. Clin. Exp. Res. 34: 1425.1431.
23. Castilow, E. M., K. L. Legge, and S. M. Varga. 2008. Cutting edge: Eosinophils
do not contribute to respiratory syncytial virus vaccine-enhanced disease. J.
Immunol. 181: 6692.6696.
24. Stetson, D. B., M. Mohrs, V. Mallet-Designe, L. Teyton, and R. M. Locksley.
2002. Rapid expansion and IL-4 expression by Leishmania-specific naive helper
T cells in vivo. Immunity 17: 191.200.
25. Moon, J. J., H. H. Chu, M. Pepper, S. J. McSorley, S. C. Jameson, R. M. Kedl,
and M. K. Jenkins. 2007. Naive CD4(+) T cell frequency varies for different
epitopes and predicts repertoire diversity and response magnitude. Immunity 27:
203.213.
26. Obar, J. J., K. M. Khanna, and L. Lefrancois. 2008. Endogenous naive CD8+
T cell precursor frequency regulates primary and memory responses to infection.
Immunity 28: 859.869.
27. Belz, G. T., C. M. Smith, L. Kleinert, P. Reading, A. Brooks, K. Shortman,
F. R. Carbone, and W. R. Heath. 2004. Distinct migrating and nonmigrating
dendritic cell populations are involved in MHC class I-restricted antigen presentation
after lung infection with virus. Proc. Natl. Acad. Sci. USA 101: 8670.
8675.
28. GeurtsvanKessel, C. H., M. A. Willart, L. S. van Rijt, F. Muskens, M. Kool,
C. Baas, K. Thielemans, C. Bennett, B. E. Clausen, H. C. Hoogsteden, et al.
2008. Clearance of influenza virus from the lung depends on migratory langerin+
CD11b- but not plasmacytoid dendritic cells. J. Exp. Med. 205: 1621.
1634.
29. Kim, T. S., and T. J. Braciale. 2009. Respiratory dendritic cell subsets differ in
their capacity to support the induction of virus-specific cytotoxic CD8+ T cell
responses. PLoS ONE 4: e4204.
30. Brincks, E. L., A. Katewa, T. A. Kucaba, T. S. Griffith, and K. L. Legge. 2008.
CD8 T cells utilize TRAIL to control influenza virus infection. J. Immunol. 181:
4918.4925.
31. McGill, J., and K. L. Legge. 2009. Cutting edge: contribution of lung-resident
T cell proliferation to the overall magnitude of the antigen-specific CD8 T cell
response in the lungs following murine influenza virus infection. J. Immunol.
183: 4177.4181.
32. Moyron-Quiroz, J. E., J. Rangel-Moreno, K. Kusser, L. Hartson, F. Sprague,
S. Goodrich, D. L. Woodland, F. E. Lund, and T. D. Randall. 2004. Role of
inducible bronchus associated lymphoid tissue (iBALT) in respiratory immunity.
Nat. Med. 10: 927.934.
33. Marshall, D. R., S. J. Turner, G. T. Belz, S. Wingo, S. Andreansky,
M. Y. Sangster, J. M. Riberdy, T. Liu, M. Tan, and P. C. Doherty. 2001. Measuring
the diaspora for virus-specific CD8+ T cells. Proc. Natl. Acad. Sci. USA
98: 6313.6318.
34. Kohlmeier, J. E., S. C. Miller, and D. L. Woodland. 2007. Cutting edge: Antigen
is not required for the activation and maintenance of virus-specific memory CD8
+ T cells in the lung airways. J. Immunol. 178: 4721.4725.
35. Hogan, R. J., E. J. Usherwood, W. Zhong, A. A. Roberts, R. W. Dutton,
A. G. Harmsen, and D. L. Woodland. 2001. Activated antigen-specific CD8+
T cells persist in the lungs following recovery from respiratory virus infections.
J. Immunol. 166: 1813.1822.
36. Roberts, A. D., and D. L. Woodland. 2004. Cutting edge: effector memory CD8+
T cells play a prominent role in recall responses to secondary viral infection in
the lung. J. Immunol. 172: 6533.6537.
37. Sallusto, F., D. Lenig, R. Forster, M. Lipp, and A. Lanzavecchia. 1999. Two
subsets of memory T lymphocytes with distinct homing potentials and effector
functions. Nature 401: 708.712.
38. Wherry, E. J., V. Teichgraber, T. C. Becker, D. Masopust, S. M. Kaech, R. Antia,
U. H. von Andrian, and R. Ahmed. 2003. Lineage relationship and protective
immunity of memory CD8 T cell subsets. Nat. Immunol. 4: 225.234.
39. Kaech, S. M., and R. Ahmed. 2001. Memory CD8+ T cell differentiation: initial
antigen encounter triggers a developmental program in naive cells. Nat. Immunol.
2: 415.422.
40. Topham, D. J., M. R. Castrucci, F. S. Wingo, G. T. Belz, and P. C. Doherty. 2001.
The role of antigen in the localization of naive, acutely activated, and memory
CD8(+) T cells to the lung during influenza pneumonia. J. Immunol. 167: 6983.
6990.
The Journal of Immunology 2493
41. Yewdell, J. W., and J. R. Bennink. 1999. Immunodominance in major histocompatibility
complex class I-restricted T lymphocyte responses. Annu. Rev.
Immunol. 17: 51.88.
42. Virelizier, J. L. 1975. Host defenses against influenza virus: the role of antihemagglutinin
antibody. J. Immunol. 115: 434.439.
43. Krause, J. C., T. Tsibane, T. M. Tumpey, C. J. Huffman, R. Albrecht, D. L. Blum,
I. Ramos, A. Fernandez-Sesma, K. M. Edwards, A. Garcia-Sastre, et al. 2012.
Human monoclonal antibodies to pandemic 1957 H2N2 and pandemic 1968
H3N2 influenza viruses. J. Virol. 86: 6334.6340.
44. Epstein, S. L., and G. E. Price. 2010. Cross-protective immunity to influenza A
viruses. Expert Rev. Vaccines 9: 1325.1341.
45. Grebe, K. M., J. W. Yewdell, and J. R. Bennink. 2008. Heterosubtypic immunity
to influenza A virus: where do we stand? Microbes Infect. 10: 1024.1029.
46. Ream, R. M., J. Sun, and T. J. Braciale. 2010. Stimulation of naive CD8+ T cells
by a variant viral epitope induces activation and enhanced apoptosis. J. Immunol.
184: 2401.2409.
47. Hufford, M. M., T. S. Kim, J. Sun, and T. J. Braciale. 2011. Antiviral CD8+
T cell effector activities in situ are regulated by target cell type. J. Exp. Med.
208: 167.180.
48. Margine, I., L. Martinez-Gil, Y. Y. Chou, and F. Krammer. 2012. Residual
baculovirus in insect cell-derived influenza virus-like particle preparations enhances
immunogenicity. PLoS ONE 7: e51559.
49. Corti, D., J. Voss, S. J. Gamblin, G. Codoni, A. Macagno, D. Jarrossay,
S. G. Vachieri, D. Pinna, A. Minola, F. Vanzetta, et al. 2011. A neutralizing
antibody selected from plasma cells that binds to group 1 and group 2 influenza
A hemagglutinins. Science 333: 850.856.
50. Ekiert, D. C., R. H. Friesen, G. Bhabha, T. Kwaks, M. Jongeneelen, W. Yu,
C. Ophorst, F. Cox, H. J. Korse, B. Brandenburg, et al. 2011. A highly conserved
neutralizing epitope on group 2 influenza A viruses. Science 333: 843.850.
51. Song, J. M., C. W. Choi, S. O. Kwon, R. W. Compans, S. M. Kang, and
S. I. Kim. 2011. Proteomic characterization of influenza H5N1 virus-like particles
and their protective immunogenicity. J. Proteome Res. 10: 3450.3459.
52. Ballesteros-Tato, A., B. Leon, F. E. Lund, and T. D. Randall. 2010. Temporal
changes in dendritic cell subsets, cross-priming and costimulation via CD70
control CD8(+) T cell responses to influenza. Nat. Immunol. 11: 216.224.
53. Langlois, R. A., A. Varble, M. A. Chua, A. Garcia-Sastre, and B. R. Tenoever.
2012. Hematopoietic-specific targeting of influenza A virus reveals replication
requirements for induction of antiviral immune responses. Proc. Natl. Acad. Sci.
USA 109: 12117.12122.
54. den Haan, J. M., S. M. Lehar, and M. J. Bevan. 2000. CD8(+) but not CD8(2)
dendritic cells cross-prime cytotoxic T cells in vivo. J. Exp. Med. 192: 1685.
1696.
55. Norbury, C. C., M. F. Princiotta, I. Bacik, R. R. Brutkiewicz, P. Wood, T. Elliott,
J. R. Bennink, and J. W. Yewdell. 2001. Multiple antigen-specific processing
pathways for activating naive CD8+ T cells in vivo. J. Immunol. 166: 4355.4362.
56. Cella, M., M. Salio, Y. Sakakibara, H. Langen, I. Julkunen, and A. Lanzavecchia.
1999. Maturation, activation, and protection of dendritic cells induced by
double-stranded RNA. J. Exp. Med. 189: 821.829.
57. Lopez, C. B., A. Garcia-Sastre, B. R. Williams, and T. M. Moran. 2003. Type I
interferon induction pathway, but not released interferon, participates in the
maturation of dendritic cells induced by negative-strand RNA viruses. J. Infect.
Dis. 187: 1126.1136.
58. Moltedo, B., W. Li, J. S. Yount, and T. M. Moran. 2011. Unique type I interferon
responses determine the functional fate of migratory lung dendritic cells during
influenza virus infection. PLoS Pathog. 7: e1002345.
59. Beignon, A. S., M. Skoberne, and N. Bhardwaj. 2003. Type I interferons promote
cross-priming: more functions for old cytokines. Nat. Immunol. 4: 939.941.
60. Diamond, M. S., M. Kinder, H. Matsushita, M. Mashayekhi, G. P. Dunn,
J. M. Archambault, H. Lee, C. D. Arthur, J. M. White, U. Kalinke, et al. 2011.
Type I interferon is selectively required by dendritic cells for immune rejection
of tumors. J. Exp. Med. 208: 1989.2003.
61. Dresch, C., Y. Leverrier, J. Marvel, and K. Shortman. 2012. Development of
antigen cross-presentation capacity in dendritic cells. Trends Immunol. 33: 381.
388.
62. Khurana, S., J. Wu, N. Verma, S. Verma, R. Raghunandan, J. Manischewitz,
L. R. King, E. Kpamegan, S. Pincus, G. Smith, et al. 2011. H5N1 virus-like
particle vaccine elicits cross-reactive neutralizing antibodies that preferentially
bind to the oligomeric form of influenza virus hemagglutinin in humans. J. Virol.
85: 10945.10954.
63. Lopez-Macias, C., E. Ferat-Osorio, A. Tenorio-Calvo, A. Isibasi, J. Talavera,
O. Arteaga-Ruiz, L. Arriaga-Pizano, S. P. Hickman, M. Allende, K. Lenhard,
et al. 2011. Safety and immunogenicity of a virus-like particle pandemic influenza
A (H1N1) 2009 vaccine in a blinded, randomized, placebo-controlled trial
of adults in Mexico. Vaccine 29: 7826.7834.
No comments:
Post a Comment