Baculovirus-infected insect cells expressing peptide-MHC complexes elicit protective antitumor immunity.
ABSTRACT Evaluation of T cell responses to tumor- and pathogen-derived peptides in preclinical models is necessary to define the characteristics of efficacious peptide vaccines. We show in this study that vaccination with insect cells infected with baculoviruses expressing MHC class I linked to tumor peptide mimotopes results in expansion of functional peptide-specific CD8+ T cells that protect mice from tumor challenge. Specific peptide mimotopes selected from peptide-MHC libraries encoded by baculoviruses can be tested using this vaccine approach. Unlike other vaccine strategies, this vaccine has the following advantages: peptides that are difficult to solublize can be easily characterized, bona fide peptides without synthesis artifacts are presented, and additional adjuvants are not required to generate peptide-specific responses. Priming of antitumor responses occurs within 3 days of vaccination and is optimal 1 wk after a second injection. After vaccination, the Ag-specific T cell response is similar in animals primed with either soluble or membrane-bound Ag, and CD11c+ dendritic cells increase expression of maturation markers and stimulate proliferation of specific T cells ex vivo. Thus, the mechanism of Ag presentation induced by this vaccine is consistent with cross-priming by dendritic cells. This straightforward approach will facilitate future analyses of T cells elicited by peptide mimotopes.
- SourceAvailable from: Heiko Weyd[Show abstract] [Hide abstract]
ABSTRACT: Prevention of an immune response against self-antigens derived from apoptotic cells is essential to preclude autoimmune and chronic inflammatory diseases. Here, we describe apoptosis induced externalization of endogenous cytosolic annexin 1 initiating an anti-inflammatory effector mechanism that suppresses the immune response against antigens of apoptotic cells. Cytosolic annexin 1 rapidly translocated to the apoptotic cell surface and inhibited dendritic cell (DC) activation induced by Toll like receptors (TLR). Annexin 1-inhibited DC showed strongly reduced secretion of pro-inflammatory cytokines (e.g. TNF and IL-12) and costimulatory surface molecules (e.g. CD40 and CD86), while anti-inflammatory mediators like PD-L1 remained unchanged. T cells stimulated by such DC lacked secretion of interferon-γ (IFN-γ) and TNF but retained IL-10 secretion. In mice, annexin 1 prevented the development of inflammatory DC and suppressed the cellular immune response against the model antigen ovalbumin (OVA) expressed in apoptotic cells. Furthermore, annexin 1 on apoptotic cells compromised OVA-specific tumor vaccination and impaired rejection of an OVA-expressing tumor. Thus, our results provide a molecular mechanism for the suppressive activity of apoptotic cells on the immune response towards apoptotic cell-derived self-antigens. This process may play an important role in prevention of autoimmune diseases and of the immune response against cancer.PLoS ONE 04/2013; 8(4):e62449. · 3.53 Impact Factor
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ABSTRACT: Vaccines that incorporate peptide mimics of tumor antigens, or mimotope vaccines, are commonly used in cancer immunotherapy and function by eliciting increased numbers of T cells that cross-react with the native tumor antigen. Unfortunately, they often elicit T cells that do not cross-react or have low affinity for the tumor antigen. Using a high affinity tumor-specific T cell clone we identified a panel of mimotope vaccines for the dominant peptide antigen from a mouse colon tumor that elicits a range of tumor protection following vaccination. The TCR from this high affinity T cell clone was rarely identified in ex vivo evaluation of tumor-specific T cells elicited by mimotope vaccination. Conversely, a low affinity clone found in the tumor and following immunization was frequently identified. Using peptide libraries, we determined if this frequently identified TCR improved the discovery of efficacious mimotopes. We demonstrated that the representative TCR identified more protective mimotopes than the high affinity TCR. These results suggest that targeting a dominant fraction of tumor-specific T cells generates potent immunity and that consideration of the available T cell repertoire is necessary for targeted T cell therapy. These results have important implications when optimizing mimotope vaccines for cancer immunotherapy.Journal of Biological Chemistry 10/2013; · 4.60 Impact Factor
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ABSTRACT: Vaccination with antigens expressed by tumors is one strategy for stimulating enhanced T cell responses against tumors. However, these peptide vaccines rarely result in efficient expansion of tumor-specific T cells or responses that protect against tumor growth. Mimotopes, or peptide mimics of tumor antigens, elicit increased numbers of T cells that cross-react with the native tumor antigen, resulting in potent anti-tumor responses. Unfortunately, mimotopes may also elicit cells that do not cross-react or have low affinity for tumor antigen. We previously showed that one such mimotope of the dominant MHC class I tumor antigen of a mouse colon carcinoma cell-line stimulates a tumor-specific T cell clone and elicits antigen-specific cells in vivo, yet protects poorly against tumor growth. We hypothesized that boosting the mimotope vaccine with the native tumor antigen would focus the T cell response elicited by the mimotope towards high affinity, tumor-specific T cells. We show that priming T cells with the mimotope, followed by a native tumor-antigen boost improves tumor immunity, compared to T cells elicited by the same prime with a mimotope boost. Our data suggest that the improved tumor immunity results from the expansion of mimotope-elicited tumor-specific T cells that have increased avidity for the tumor antigen. The enhanced T cells are phenotypically distinct and enriched for T cell receptors previously correlated with improved anti-tumor immunity. These results suggest that incorporation of native antigen into clinical mimotope vaccine regimens may improve the efficacy of anti-tumor T cell responses.Cancer Research 11/2012; · 9.28 Impact Factor
Baculovirus-Infected Insect Cells Expressing Peptide-MHC
Complexes Elicit Protective Antitumor Immunity1
Kimberly R. Jordan,* Rachel H. McMahan,* Jason Z. Oh,* Matthew R. Pipeling,†
Drew M. Pardoll,†Ross M. Kedl,* John W. Kappler,*‡§and Jill E. Slansky2*
Evaluation of T cell responses to tumor- and pathogen-derived peptides in preclinical models is necessary to define the charac-
teristics of efficacious peptide vaccines. We show in this study that vaccination with insect cells infected with baculoviruses
expressing MHC class I linked to tumor peptide mimotopes results in expansion of functional peptide-specific CD8?T cells that
protect mice from tumor challenge. Specific peptide mimotopes selected from peptide-MHC libraries encoded by baculoviruses
can be tested using this vaccine approach. Unlike other vaccine strategies, this vaccine has the following advantages: peptides that
are difficult to solublize can be easily characterized, bona fide peptides without synthesis artifacts are presented, and additional
adjuvants are not required to generate peptide-specific responses. Priming of antitumor responses occurs within 3 days of vac-
cination and is optimal 1 wk after a second injection. After vaccination, the Ag-specific T cell response is similar in animals primed
with either soluble or membrane-bound Ag, and CD11c?dendritic cells increase expression of maturation markers and stimulate
proliferation of specific T cells ex vivo. Thus, the mechanism of Ag presentation induced by this vaccine is consistent with
cross-priming by dendritic cells. This straightforward approach will facilitate future analyses of T cells elicited by peptide
mimotopes. The Journal of Immunology, 2008, 180: 188–197.
ificity and, in combination with the appropriate adjuvants, generate
immune responses to pathogens and cancers. Peptides in combi-
nation therapies may be key in the next generation of vaccines.
Many studies have used insect cells infected with baculoviruses
(BV)3for production of proteins used in vaccines (1–3). Due to the
large viral genome and strong promoters, BV vectors accommo-
date large gene inserts (?1 kb) and produce high yields of mam-
malian proteins (4). Furthermore, posttranslational modifications,
such as glycosylation and phosphorylation, in insect cells are sim-
ilar to mammalian processes, allowing expression of proteins that
biochemically resemble those of mammalian origins (5). For ex-
ample, Spodoptera frugiperda (Sf9) and High Five insect cells
infected with rBV produce soluble immunogenic viral proteins and
viral-like particles from HIV and foot and mouth disease virus for
use in vaccines (6, 7). Both serological (8) and cellular responses
dentification of MHC class I- and MHC class II-binding
epitopes have expedited the use of peptides in immunother-
apy. Peptide vaccine strategies target T cells with fine spec-
(2) are elicited by purified HIV proteins produced by BV-infected
insect cells, which protect animals against subsequent viral chal-
lenge. Although vaccination with protein produced by BV-infected
insect cells induces Ag-specific immune responses, this vacci-
nation strategy requires protein purification and appropriate
Because the BV polyhedron promoter is not active (9–11) and
BV cannot replicate in mammalian cells (12), injection of rBV
may provide effective and safe means for delivery of vaccines.
rBV expressing immunogenic proteins linked to the transmem-
brane domain of gp64 for viral surface expression elicit Ag-specific
responses (13–15). However, rBV are inactivated by complement
proteins in vivo (16) and may be damaged during purification pro-
cesses, particularly by ultracentrifugation (17).
Injection of insect cells infected with rBV is an attractive
method for vaccine delivery because it combines benefits from
both the protein and viral vaccines. It has been shown that these
vaccines elicit humoral immune responses to surface-expressed
viral Ags (18). For example, vaccination with infected insect cells
expressing foot and mouth disease virus Ag elicits seroneutralizing
Abs, resulting in protection from viral challenge (7). The recom-
binant proteins are produced in culture, where complement pro-
teins do not interfere with Ag production. In addition, the Ag can
be quantified before injection, and preparation of infected insect
cells for vaccination requires only low-speed centrifugation. Thus,
we hypothesized that vaccination with insect cells infected with
BV-encoding tumor-specific Ags would be a promising technique
for priming specific CD8?T cell responses.
Peptide-MHC complexes and peptide-MHC libraries used for
the discovery of novel peptide Ags are successfully produced
by insect cells infected with rBV (19–23). These peptide librar-
ies are screened for binding to soluble TCR and activation of T
cells in vitro before testing the peptides in vivo. Peptides pro-
duced in the BV peptide-MHC library are soluble and not easily
oxidized, which permits screening of all amino acid residues,
including cysteine and tryptophan. In theory, peptide epitopes
*University of Colorado Denver and Health Sciences Center, Denver, CO 80206;
†Johns Hopkins University, Sidney Kimmel Cancer Center, Baltimore, MD
21231;‡National Jewish Medical and Research Center, Denver, CO 80206; and
§Howard Hughes Medical Institute, Denver, CO 80206
Received for publication May 16, 2007. Accepted for publication October 27, 2007.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by National Cancer Institute Grant CA109560 and a seed
grant from the American Cancer Society Institutional Research grant to the University
of Colorado Cancer Center (to J.E.S.). K.R.J., R.H.M., and J.Z.O. were supported in
part by the Cancer Research Institute Predoctoral Emphasis Pathway in Tumor Im-
2Address correspondence and reprint requests to Dr. Jill E. Slansky, National Jewish
Medical and Research Center, 1400 Jackson Street, Denver, CO 80206. E-mail
3Abbreviations used in this paper: BV, baculovirus; ?-gal, ?-galactosidase; ?2m,
?2-microglobulin; CT, colorectal tumor; DC, dendritic cell; MFI, mean fluorescence
intensity; Sf9, Spodoptera frugiperda; Tg, transgenic.
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
The Journal of Immunology
or peptide mimotopes identified using this library system may reg-
ulate the T cell response and thus the disease progression in au-
toimmunity, cancer, and infectious diseases (reviewed in Ref. 24).
We are using BV peptide-MHC libraries to identify novel can-
cer mimotopes, or mimics of tumor peptides, which stabilize the
peptide-MHC/TCR complex and elicit T cells that cross-react with
the tumor Ag (23). Like the peptide mimotopes we have identified
(27), most mimotopes used in clinical trials of cancer vaccines
have alterations in the MHC-anchor residues (reviewed in Ref.
25). We are characterizing mimotopes that improve antitumor im-
munity to the CT26 mouse colon carcinoma, specifically to the
immunodominant tumor Ag AH1 (gp70423–431) (26), restricted by
the MHC class I molecule H-2Ld. We previously showed that an-
titumor activity is improved by vaccinating with mimotope-li-
posome complexes (27) or mimotope-loaded dendritic cells
(DCs) (28). Because some mimotopes are insoluble in water,
sensitive to oxidation, and cannot be characterized using these
methods, we developed a vaccine using infected insect cells
expressing peptide-MHC molecules.
We demonstrate in this study that vaccination with insect cells
infected with rBV-encoding peptide-MHC complexes generates
peptide-specific cytotoxic T cell responses, and, when the appro-
priate peptide is used, protects mice from subsequent tumor chal-
lenge. Our results indicate that the infected insect cells activate
APCs in vivo, which effectively present the expressed peptides to
T cells. This vaccination strategy is advantageous for the following
reasons: it ensures the bone fide peptide is presented, it does not
require adjuvants in addition to those produced by BV and insect
cells, it greatly reduces the cost of in vivo studies by eliminating
the need to synthetically generate peptides, and it expedites the
direct evaluation of peptides identified in BV peptide-MHC
Materials and Methods
Sf9 and High Five insect cells (22) (Invitrogen Life Technologies) and
CT26 tumor cells (29) were cultured, as described. Splenocytes from vac-
cinated mice were expanded in vitro with AH1 peptide and IL-2, as de-
scribed (27). DCs were prepared from collagenase-digested spleens or mes-
enteric lymph nodes for flow cytometric analyses or in vitro proliferation
assays, as described (200 ?g/ml collagenase D (Roche) and 40 ?g/ml
DNase-I (Sigma-Aldrich)) (30). Mesenteric lymph node cells were har-
vested 0 (unvaccinated), 2, 8, 16, 24, or 48 h after vaccination with infected
insect cells for flow cytometric analyses. CD11c?cells were isolated for in
vitro proliferation assays 24 h after vaccination using a biotinylated
CD11c-specific mAb and anti-biotin microbeads. Labeled cells were
separated using LS MidiMacs columns, according to the manufacturer’s
protocol (Miltenyi Biotec).
CT26-specific TCR transgenic (CT-TCR Tg) mice expressing the TCR
from the V?8.3/V?4.11 T cell clone (28) were generated by inserting the
TCR ? and ? genes into shuttle vectors (31), which were subsequently
injected into embryos of (SJL ? B6)F1at the University of Pennsylvania
Transgenic Facility, and backcrossed to BALB/c 12 generations. Because
of low TCR expression, these mice were bred onto a RAG2-deficient back-
ground (C.12956(B6)-RAG2tm1fwaN12; Taconic Farms). Six- to eight-wk-
old female BALB/c were purchased from the National Cancer Institute/
Charles River Laboratories. All animal protocols were reviewed and
approved by the Institutional Animal Care and Use Committee of National
Jewish Medical and Research Center.
Sequence encoding H-2Ldwith either a peptide tag for biotinylation by the
enzyme BirA (LdBirA (27)) or the transmembrane domain from gp64
(LdTM (22, 32)) was inserted into a modified pBacp10pH BV expression
vector downstream of the p10 promoter (20). Sequence encoding mouse
?2-microglobulin (?2m) with covalently linked peptides (AH1, SPSYVY
HQF (26); 39, MNKYAYHML (27); 15, MPKYAYHML (27); ?-galac-
tosidase (?-gal), TPHGAGRIL (33); WMF, SPTYAYWMF (23)) was in-
serted downstream of the pH promoter. The constructions were introduced
into BV using the standard homologous recombination method (22). AH1
peptide-loaded LdBirA used for the ELISA standard was purified from
supernatants of infected High Five insect cells over an affinity column
using an Ab specific for H-2Ld(28.14.8s; American Type Culture Collec-
tion). Protein-containing fractions were concentrated and separated on a
Superdex-200 sizing column. AH1 peptide (Macromolecular Resources)
was added to the 57-kDa fraction in 5-fold molar excess. Fluorescent tet-
ramer was prepared, as described (27).
Abs and staining reagents
Soluble TCR was constructed by inserting the TCR-encoding V region
gene fragments from CT-Ig (28) into a modified pBacp10pH BV expres-
sion vector (23). CT-TCR-soluble protein was purified from supernatants
of infected High Five insect cells over an affinity column using an Ab
specific for TCR C? (HAM-597; American Type Culture Collection) and
a Superdex-200 sizing column. Purified CT-TCR was multimerized with a
biotinylated anti-TCR C?-specific Ab (ADO-304) and streptavidin-AF647
(Invitrogen Life Technologies), as previously described (22). Abs specific
for H-2Ld(28.14.8s), CD80 (16-10A1; eBioscience), CD86 (GL1; BD
Pharmigen), MHC class II (M5/114.15.2; BD Pharmingen), CD11c (N418;
BD Pharmingen), CD11b (M1/70; eBioscience), CD8? (2.43; American
Type Culture Collection), V?8.3 (CT-8C1; BD Pharmingen), IFN-?
(XMG1.2; eBioscience), and the compounds 7-aminoactinomycin D (Sig-
ma-Aldrich) and CFSE (Invitrogen Life Technologies) were used for flow
cytometric analyses. Mice were depleted with i.p. injections of Ab specific
for CD8? (53.6.72; American Type Culture Collection) 3 days (500 ?g)
and 1 day (250 ?g) before vaccination. Depletion was maintained with
weekly injections of 250 ?g of Ab and was confirmed by flow cytometry
(?99.8% depletion before vaccination and ?79% depletion before tumor
challenge; data not shown).
Infection of Sf9 insect cells
A total of 3 ? 107Sf9 insect cells was cultured in T175 flasks in complete
Grace’s Insect medium (Invitrogen Life Technologies) containing 10%
FCS (Atlanta Biologicals), 1% F-68 detergent (Invitrogen Life Technolo-
gies), and 1% antibiotic-antimycotic (Invitrogen Life Technologies). The
BV titer was determined using a limiting dilution assay. When a multi-
plicity of infection of 2 U/cell is used for each infection, consistent infec-
tion efficiencies and insect cell death rates (20% by day 3) are obtained.
Infected Sf9 insect cells were incubated for 3 days, harvested by centrif-
ugation at 1000 ? g for 5 min, and washed three times with HBSS
Infected Sf9 insect cells were resuspended in HBSS, and 5 ? 106cells
were injected i.p. on days 0 and 7. The number of responding Ag-specific
T cells is similar following i.v. and s.c. injection. Splenocytes or PBMCs
were harvested for flow cytometric analyses on days 10, 14, and 17. Sta-
tistical analyses were performed with Prism version 4.0 (GraphPad), using
unpaired two-tailed Student’s t test. A p value of ?0.05 was considered
Immunoprecipitation of H-2Ldfrom whole cell lysates
Whole cell lysates were prepared by incubating infected Sf9 insect cells in
lysis buffer and protease inhibitors, as previously described (34), at a con-
centration of 1 ? 107cells/ml for 4 h at 4°C. H-2Ldwas immunoprecipi-
tated from whole cell lysates with the 28.14.8s Ab and protein A-Sepharose
beads (35). Precipitated proteins were separated by SDS-PAGE (5–20%
Tris-HCl; Bio-Rad) under reducing conditions using a standard protocol
and stained with Coomassie blue.
In vivo killing assays
Mice were vaccinated with 39-LdTM-infected or uninfected insect cells on
days 0 and 7. Thirty days after the last vaccination, target cells (BALB/c
splenocytes) were incubated with either ?-gal or AH1 peptide (10 ?g/ml)
for 2 h at room temperature. Cells were washed and labeled with 0.2 ?M
or 2 ?M CFSE, respectively, and injected i.v. Splenocytes were harvested
20 h later, and the number of CFSE?cells in each peak was determined
(percentage of specific killing ? 1 – percentage of survival; percentage of
survival ? (number of AH1 targets remaining)/(number of ?-gal targets
remaining)). Groups were compared using Prism version 4.0 (GraphPad),
by unpaired two-tailed Student’s t test. A p value of ?0.05 was considered
189 The Journal of Immunology
For in vivo proliferation assays, mice were vaccinated, as described above,
with either 39-LdTM-infected or uninfected insect cells on day ?7, ?3, or
?1. A total of 1 ? 107splenocytes from CT-TCR Tg mice was labeled
with 10 ?M CFSE and transferred into vaccinated mice on day 0. Three
days later, CFSE dilution of transferred V?8.3?CD8?splenocytes was
determined by flow cytometry. Background proliferation was determined
in mice vaccinated with uninfected insect cells.
For in vitro proliferation assays, 5 ? 105CFSE-labeled splenocytes
(labeled as above) from CT-TCR Tg mice were incubated in 96-well plates
at 37°C with increasing concentrations of soluble peptide or 1 ? 105
CD11c?splenocytes preincubated with 100 ?g/ml peptide or from vacci-
nated mice in complete medium (27). Cells were harvested 3 days later,
and CFSE dilution of 7-aminoactinomycin D?CD8?cells was analyzed
by flow cytometry. In vitro proliferation assays using a T cell clone ex-
pressing the CT-TCR were performed, as previously described (27). The T
cell clone was incubated at a 5:1 ratio with insect cells that express ICAM
and B7 costimulatory molecules (22) and infected with BV-encoding
For tumor protection experiments, mice were injected i.p. with 5 ? 106
peptide-LdTM-infected Sf9 insect cells on days –14 and –7. On day 0, mice
were injected s.c. in the left hind flank with 5 ? 104CT26 tumor cells (26).
Tumor-free survival was assessed by palpation of the injection site. Once
tumors were palpable, they always proceeded to 100 mm2without shrink-
ing. When a tumor reached 100 mm2, the mouse was no longer considered
tumor free, as indicated on the Kaplin-Meier plot, and it was sacrificed. All
mice are represented in the Kaplin-Meier plot. Tumor-free survival was
analyzed by Kaplan-Meier survival plots, and statistical significance was
analyzed with Prism version 4.0 (GraphPad), using the log rank test.
For tumor treatment experiments, mice were injected with 5 ? 104
CT26 tumor cells and vaccinated with 5 ? 106infected insect cells 2, 5, 8,
11, and 14 days later. Tumors were measured every 2 days, and groups
were compared statistically on individual days using Prism version 4.0
(GraphPad) with unpaired two-tailed Student’s t test. A p value of ?0.05
was considered statistically significant. Differences in tumor size of mice
injected with 39-LdTM relative to unvaccinated or ?-gal-LdTM were sta-
tistically significant after day 9. The average tumor size of the indicated
number of mice is plotted.
BV-infected insect cells express peptide-LdTM
To ensure that insect cells infected with BV produce Ag recog-
nized by cognate TCR, we generated BV-encoding peptide-Ld
molecules. The transmembrane domain of BV gp64 was inserted
downstream of H-2Ldfor surface expression (LdTM) (6, 21–23),
and peptides were tethered to the ?2m molecule via a glycine-rich
linker. We inserted either the CT26 tumor Ag (the AH1 peptide) or
the negative control ?-gal peptide, which binds to H-2Ld, but is not
recognized by the AH1-specific TCR (CT-TCR). To ensure that
the cognate TCR recognizes peptides produced in insect cells with
similar relative affinity as synthetic peptides, we also generated
BV-encoding H-2Ldcovalently linked to previously studied pep-
tide mimotopes with changes in the MHC-binding residues of dif-
ferent affinities (27). The CT-TCR binds to mimotope 39-Ldwith
an intermediate affinity and to mimotope 15-Ldwith a high affinity,
referring to the peptide-MHC/TCR interaction, as determined by
surface plasmon resonance (27).
Three days after infection with BV, insect cells were stained
with Abs specific for H-2Ldbound to ?2m and peptide (28.14.8s)
and soluble TCR multimer (predicted octamer, CT-TCR) (23). As
shown previously, the amount of MHC expression on the surface
of the insect cells correlates with the extent of viral infection (22)
and is consistent between experiments. Thus, TCR staining within
a given intensity of MHC staining, represented by the thin gate in
Fig. 1a, can be compared between samples because the insect cells
are infected similarly.
As expected, similar amounts of H-2Ldprotein were detected on
the surface of insect cells infected with BV encoding all of the
peptide-LdTM constructions. ?-gal-LdTM was detected with the
H-2LdAb, but not with the CT-TCR, demonstrating specificity of
the CT-TCR reagent (Fig. 1a). Importantly, as shown with other
BV-encoded peptide-MHC complexes (22), the CT-TCR fluores-
cence intensity directly correlated with its affinity for the peptide-
MHC molecules (Fig. 1b). Specifically, 15-LdTM stained most
intensely with CT-TCR, followed by 39-LdTM and AH1-LdTM.
These results show that peptide-Ldcomplexes produced by in-
sect cells are processed and folded to resemble those produced in
mammalian cells. Furthermore, the binding properties of the co-
valently linked peptides directly correlate with the binding prop-
erties of soluble peptides, suggesting that insect cell-produced pep-
tide-Ldcomplexes bind Ag-specific T cells. Finally, these results
confirm the results of Crawford et al. (22), as follows: binding
affinity of the peptide-MHC/TCR interaction can be readily ana-
lyzed using these BV constructions.
BV-infected insect cells stimulate peptide-specific immune
responses in vivo
We hypothesized that the infected insect cells provide both an
Ag-specific signal and adjuvant from the combination of BV and
foreign insect cells. To determine whether infected insect cells
induce antitumor responses in vivo, we analyzed AH1-specific
CD8?T cells following injection of these cells. To produce this
vaccine, we infected Sf9 insect cells for 3 days, harvested and
washed the cells, then injected them i.p. Although the insect cells
were washed, remaining free virus and dead insect cells were also
We previously showed that vaccination with the intermediate
affinity mimotope 39 in liposomes elicits tumor-specific T cells
and protects ?50% of mice from tumor growth (27). Although the
high-affinity mimotope 15 elicits tumor-specific T cells, it does not
protect mice from tumor growth. Thus, for simplicity we used
insect cells infected with 39-LdTM throughout the following ex-
periments. As shown in Fig. 2a, AH1-specific (tumor-specific), but
not ?-gal-specific, T cells were elicited after injection of insect
indicated BV were stained with a mAb specific for H-2Ld(28.14.8s) and fluorescent CT-TCR and analyzed by flow cytometry. To control for the amount
of BV infection, an MHC?gate, the thin box, was used to determine the relative mean fluorescence intensity (MFI) of CT-TCR staining in b. b, Insect
cells infected with rBV were analyzed as in a. The MFI of the CT-TCR at a specific MHC expression level were 172, 95.3, 6.99, and 1.18 for 15-LdTM,
39-LdTM, AH1-LdTM, and ?-gal-LdTM, respectively. These data are representative of six independent experiments.
Insect cells infected with rBV express functional peptide-H-2Ldcomplexes that reflect their relative avidity. a, Insect cells infected with the
190INFECTED INSECT CELLS ELICIT PROTECTIVE ANTITUMOR IMMUNITY
cells infected with BV-encoding mimotope 39, as determined by
tetramer staining. AH1-specific T cells were not elicited with the
?-gal peptide vaccine, the negative control.
We next determined the optimal number of insect cells needed
to prime the T cell response and calculated the corresponding
amount of peptide-Ldproduced in infected insect cells by ELISA.
Mice were injected with increasing numbers of 39-LdTM-infected
Sf9 insect cells on days 0 and 7. Splenocytes were harvested on
day 14 and cultured for 1 wk. Insect cells infected with 39-LdTM
elicited CD8?AH1-Ldtet?(tumor-specific) T cells in a dose-
dependent manner (Fig. 2a). This increase was most evident 7 days
after the second injection (day 14), as determined by the average
frequency of tetramer-positive PBMCs on days 10, 14, and 17
(Fig. 2b). Although there are stochastic differences in the fre-
quency of AH1-Ldtet?T cells, the increase in the percentage of
these T cells at day 14 is significantly different from day 17. Sig-
nificant expansion of AH1-Ldtet?T cells was not detectable be-
fore the second injection (data not shown). There was minimal
background ?-gal-tetramer staining in these experiments (?0.2%,
Fig. 2a), and no adverse side effects were observed in the
To determine the amount of Ag delivered by this vaccine, we
compared the amount of H-2Ldprotein in the infected insect cell
vaccine to a standard curve of purified H-2Ldby ELISA using
conformation-specific Abs. The vaccine was prepared for the
ELISA as it was for injection. We calculated that insect cells in-
fected with 39-LdTM BV produce ?2 pg, or 2 ? 107peptide-
MHC molecules/cell (data not shown). Vaccination with 5 ? 106
infected insect cells therefore delivers 10 ?g (SD ? 1.82 ?g, n ?
3) of peptide-MHC complexes or 200 ng of peptide. This vacci-
nation strategy delivers more MHC molecules than an exosome-
based vaccine in which mice are vaccinated with up to 1 ? 1010
molecules of MHC/mouse (36) and ?10 ?g of peptide used in the
peptide-liposome vaccine (27).
We next determined whether vaccine-elicited T cells were func-
tional in an in vivo killing assay (Fig. 2, c and d). Splenocytes from
BALB/c mice were incubated with AH1 or ?-gal peptides and
labeled with a high or low concentration of CFSE, respectively.
These labeled splenocytes were transferred into vaccinated
BALB/c mice 30 days following the second injection of 39-LdTM-
infected insect cells. AH1 peptide-loaded target cells were specifically
eliminated in mice vaccinated with 39-LdTM-infected insect cells, but
not with uninfected insect cells (Fig. 2d). The number of ?-gal-loaded
targets remained similar in both samples. These results indicate that
39-LdTM-infected insect cells elicit effector T cells that specifically
kill Ag-loaded target cells in vivo. Thus, the same viral constructions
can be used for in vivo and in vitro analyses.
Persistence of peptide-LdTM Ag in vivo
To analyze T cell responses to this vaccine and ultimately to de-
sign mimotopes to tumor-associated Ags, we derived Tg mice that
express the TCR from the CT-T cell clone (28), a clone that rec-
ognizes the AH1 peptide restricted by H-2Ld. Like many other T
cells that recognize tumors, this T cell clone recognizes a self Ag,
and therefore is subject to negative selection in the thymus and
peripheral tolerance after leaving the thymus. We developed this
new Tg mouse model, rather than using an established Tg strain,
such as the OT-1 mice, to better mimic T cell tolerance encoun-
tered by tumor vaccines. We backcrossed the transgenes onto
BALB/c mice for 12 generations and then crossed the transgenes
onto RAG2-deficient mice. Approximately 90% of the T cells in
the thymus of these Tg mice are coreceptor negative (data not
shown), indicating the following: 1) strong negative selection of
the T cells during development; 2) a developmental block in the T
cells at the double-negative stage, because the CT-TCR is specific
for a self peptide derived from an endogenous retroviral gene prod-
uct, gp70; and/or 3) the Ig enhancer used to drive gene expression
is suboptimal (31).
indicated number (top) of 39-LdTM- or ?-gal-LdTM-infected insect cells on days 0 and 7. Splenocytes were harvested on day 14 and cultured for 1 wk
with AH1 peptide and IL-2. Quadrants were set using ?-gal-Ldtet-stained cells (left); the percentages of CD8?AH1-Ldtet?cells are indicated. b, Mice
were vaccinated with 5 ? 10639-LdTM-infected insect cells, and the frequency of CD8?AH1-Ldtet?cells in the blood was determined by flow cytometry
on the indicated days (x-axis). The bars indicate the average percentage of CD8?AH1-Ldtet?T cells minus background ?-gal-Ldtet staining (average
percentages ? SD were 1.7 ? 0.9, 4.2 ? 0.7, 1.4 ? 0.4 for days 10, 14, and 17, respectively; ?, p ? 0.0178, using unpaired two-tailed Student’s t test).
c, Splenocytes were labeled with CFSE and incubated with either the ?-gal peptide (left peak) or AH1 peptide (right peak), and equal numbers of cells were
transferred into mice vaccinated with the indicated insect cells. Splenocytes were harvested 20 h later, and the number of CFSE?cells in each peak as
indicated was determined by flow cytometry. d, The percentage of specific killing was determined as in c for multiple mice. The bars indicate the average
percentage of specific killing (39-LdTM, 45.9 ? 7.9, n ? 8; uninfected, 0.4 ? 1.6, n ? 6; ??, p ? 0.0004 using unpaired two-tailed Student’s t test).
Vaccination with insect cells infected with BV-encoding peptide-LdTM stimulates specific CD8?T cells. a, Mice were vaccinated with the
191 The Journal of Immunology
As in other models of self tolerance, some T cells escape
negative selection and are found in the periphery (37). Some of
the peripheral V?8.3?T cells from the Tg mice express CD8
molecules, and these T cells are functional, as determined by
tetramer binding and other assays (Fig. 3). The coreceptor-neg-
ative cells bind a V?8.3 Ab (Fig. 3b), but they do not all bind
to AH1-Ldtet (Fig. 3a), suggesting that T cells lacking core-
ceptor may require a higher affinity peptide to form a complex.
Consistent with this possibility, more coreceptor-negative T
cells bind 39-Ldtet than AH1-Ldtet (data not shown). Eighty to
90% of the CD8?T cells proliferated when incubated with 10
nM peptide 39 (Fig. 3d). Few of the T cells express CD4 mol-
ecules, and the remaining T cells are coreceptor negative
(CD4?/CD8?, Fig. 3b). The CT-TCR Tg RAG mouse produced
functional Ag-specific T cells, as determined by production of
IFN-? and proliferation to a range of peptide concentrations
(Fig. 3, c and d). Thus, we determined that these T cells may be
used to monitor Ag-specific T cell responses in adoptive trans-
fer assays and other assays to assess tumor-specific T cell
Ag persistence, or t1/2, directly correlates to the potency of a
vaccine (38). To determine the persistence of Ag following in-
jection of the BV-infected insect cell vaccine, we analyzed pro-
liferation of transferred splenocytes from CT-TCR Tg RAG
mice. BALB/c mice were vaccinated with 39-LdTM-infected or
uninfected insect cells, and 1 ? 107CFSE-labeled Tg spleno-
cytes were transferred i.v. 1, 3, or 7 days after vaccination.
Splenocytes were harvested 72 h after transfer, and proliferation
of CD8?/V?8.3?/CFSE?cells was determined by flow cyto-
metric analysis (Fig. 3e). Vaccination with 39-LdTM-infected
insect cells 1 day before transfer induced proliferation of a
significant number of Tg T cells (37%). Proliferation in mice
vaccinated 3 days before transfer was reduced (16%), although
it was significantly higher than in mice vaccinated with unin-
fected insect cells. Vaccination 7 days before transfer did not
induce proliferation of Tg T cells, indicating that most of the Ag
is cleared between 3 and 7 days after vaccination. The persis-
tence of infected insect cells is similar to vaccination with Ag
and IFA, which are cleared 6–8 days following injection (39).
Infected insect cells do not directly stimulate T cells in vivo
Although rBV that express both peptides and MHC molecules is
convenient and effective for both in vitro and in vivo analyses,
we wanted to determine whether Ag is presented directly by
insect cells, cross-presented by APCs, or presented using a
novel mechanism. To determine whether insect cells directly
TCR Tg T cells for at least 3 days after vaccination. a,
T cells from the CT-TCR Tg mice on a BALB/c RAG2
knockout genetic background were characterized.
Splenocytes from CT-TCR Tg RAG mice were gated
on MHC class II?cells and stained with a CD8 Ab and
AH1-Ldtet or ?-gal-Ldtet. b, Splenocytes from a CT-
TCR?mouse were gated on CD3?MHC class II?
cells, and the staining with Abs to CD8, CD4, and
V?8.3 is shown. c, T cells from the CT-TCR Tg RAG
mice produce IFN-? when stimulated with the AH1
peptide. Tg mice were injected twice (days 0 and 7)
with irradiated CT26 tumor cells. Splenocytes were
stimulated ex vivo (day 14) with AH1 or ?-gal peptides
and stained with Abs specific for V?8.3, CD8, and
IFN-?. Data shown are gated on V?8.3?splenocytes;
percentages indicate the percentages of V?8.3?/CD8?/
IFN-??cells. d, T cells from the CT-TCR Tg RAG
mice proliferate in response to the AH1 peptide and
peptide 39. CFSE-labeled splenocytes from CT-TCR
Tg mice were incubated with increasing concentrations
of synthesized peptide in vitro. Percentage of prolifer-
ation was determined by CFSE dilution of CD8?T
cells. e, BALB/c mice were injected with 5 ? 10639-
LdTM-infected (black lines) or uninfected (dashed
lines) insect cells 7, 3, or 1 days before transfer of 1 ?
107CFSE-labeled CT-TCR Tg RAG splenocytes. Pro-
liferation of Tg T cells was compared on the indicated
days using unpaired two-tailed Student’s t test (day –1,
p ? 0.0014; day –3, p ? 0.0106; day –7, p ? 0.0884).
The percentages shown are the average proliferation in
two mice vaccinated with 39-LdTM-infected insect
cells minus the background proliferation observed in
mice vaccinated with uninfected insect cells.
Peptide 39 is presented to transferred
192 INFECTED INSECT CELLS ELICIT PROTECTIVE ANTITUMOR IMMUNITY
present peptide-Ldto T cells in vivo, we compared vaccination
with insect cells expressing membrane-bound peptide-Ld(39-
LdTM) with nonmembrane-bound peptide 39 (39-LdBirA and
peptide-?2m). The BV-encoding 39-LdBirA is identical with
39-LdTM, but encodes a BirA peptide tag rather than the trans-
membrane domain of gp64. The sequence encoding H-2Ldwas
removed from these BV to produce 39-?2m. Insect cells in-
fected with membrane- and nonmembrane-bound peptide 39
produce a similar amount of protein, as detected by immuno-
precipitation (Fig. 4a) and ELISA (data not shown) of whole
insect cell lysates 3 days after infection. Although 39-?2m mol-
ecules may be detectable in whole cell lysates (Fig. 4a), they
are not detectable by immunoprecipitation or ELISA because
these assays are specific for H-2Ld. As expected, both 39-Ld-
BirA and 39-?2m are not detectable on the surface of infected
insect cells with the H-2LdAb (Fig. 4b), confirming that these
infected insect cells do not present MHC-restricted Ags.
If insect cells present Ags directly to T cells, we would ex-
pect Ag-specific responses to the insect cells expressing the
membrane-bound Ag and not to the insect cells expressing non-
membrane-bound Ag. If Ags are cross-presented by APCs, then
we would expect little difference between the specific T cell
response to insect cells expressing membrane- and nonmem-
brane-bound Ag. We vaccinated mice with insect cells infected
with BV-expressing 39-LdTM, 39-LdBirA, or 39-?2m, and an-
alyzed the frequency of CD8?AH1-Ldtet?T cells in the
spleen. Insect cells infected with membrane- or nonmembrane-
bound peptide induced a similar number of AH1-specific T cells
(Fig. 4c). In addition, surface expression on insect cells infected
with 39-LdTM that also express costimulatory molecules
ICAM-1 and B7.1, facilitators of direct Ag presentation and T
cell priming, did not increase the number of responding AH1-
specific T cells (data not shown). These results suggest that
infected insect cells do not present Ag directly to T cells, but
are processed by APCs, which present the peptides in the con-
text of H-2Ldto CD8?T cells.
Vaccination with infected insect cells induces maturation of
To determine whether the insect cell vaccine activates APCs as
expected, we examined surface markers on DCs after vaccina-
tion. Mice were vaccinated with 39-LdTM-infected insect cells,
and DCs from the draining mesenteric lymph nodes were char-
acterized over time. DCs were stained with Abs against the DC
subset markers CD11c, CD8, and CD11b, and the maturation
markers MHC class II, CD80, CD86, and CD70. We observed
an increase in the expression of MHC class II and the costimu-
latory molecules CD80 and CD86 in both DC populations (Fig.
5a). Expression of CD70, a molecule expressed by activated
DCs that was recently shown to be necessary for optimal T cell
stimulation (40), also increased (Fig. 5a). Similar results were
obtained for DC populations in the spleen (data not shown).
Although the vaccine stimulated both CD8?CD11c?and
CD11b?CD11c?cells, up-regulation of the costimulatory mol-
ecules CD80 and CD86 was more pronounced in the CD8?
CD11c?subset, suggesting that these cells respond more vig-
orously to the vaccine. The infected insect cell vaccine induces
maturation of DCs, particularly the CD11c?CD8?DCs, con-
sistent with a function in cross-presentation (41).
DCs cross-present insect cell-derived peptides
To confirm that Ags from infected insect cell vaccines can be
cross-presented by DCs, we determined whether DCs isolated
from mice vaccinated with 39-LdTM-infected insect cells in-
duce proliferation of CT-TCR Tg T cells ex vivo. Mice were
vaccinated with 39-LdTM- or ?-gal-LdTM-infected insect cells,
and spleens were harvested 24 h later. Purified CD11c?spleno-
cytes from vaccinated mice (average 88% CD11c?MHC class II?)
were incubated with CFSE-labeled Tg T cells for 3 days ex vivo. Like
Tg T cells incubated with DCs exogenously loaded with peptide 39,
Tg T cells proliferated when incubated with CD11c?cells from mice
vaccinated with 39-LdTM-infected insect cells (Fig. 5b, black lines).
insect cells expressing soluble or membrane-bound 39-H-2Ld, separated by SDS-PAGE under reducing conditions, and stained with Coomassie blue. Lanes
1 and 2, Controls of purified monomeric LdBirA and 28.14.8s Ab, respectively. Lanes 3, 6, and 8, Whole cell lysates; lanes 4, 5, and 7, immunoprecipitated
lysates from insect cells infected with the indicated BV encoding 39-LdTM, 39-LdBirA, or 39-?2m (no H-2Ld), respectively. b, Insect cells were infected
with the indicated BV and stained with the 28.14.8s Ab for H-2Ldsurface expression. The long dashed line represents H-2Ldstaining of uninfected insect
cells. c, Splenocytes from mice vaccinated with insect cells infected with the indicated BV on days 0 and 7 were harvested on day 14 and analyzed by flow
cytometry for CD8?AH1-Ldtet?T cells. Quadrants were set using ?-gal-Ldtet-stained cells (left), and the percentages shown are the average CD8?
AH1-Ldtet?cells from two mice minus the background ?-gal-Ldtet staining (right three panels).
Infected insect cells do not directly present peptide-H-2Ldto T cells in vivo. a, H-2Ldwas immunoprecipitated from whole cell lysates of
193The Journal of Immunology
Tg T cells did not proliferate when incubated with DCs exogenously
loaded with ?-gal peptide or DCs from mice vaccinated with unin-
fected insect cells (Fig. 5b, dashed lines). These results indicate that
peptides from infected insect cells may be cross-presented by DCs in
the spleen within 24 h of vaccination.
BV-infected insect cells induce protective antitumor immunity
We previously showed that vaccination with peptide 39 protects
mice from tumor challenge (27). To ensure that vaccination with
peptides produced in insect cells elicits similar antitumor re-
sponses as synthetic peptides, we tested the vaccine in tumor pro-
tection and therapeutic assays. Mice were vaccinated with 39-
LdTM-, AH1-LdTM-, or ?-gal-LdTM-infected insect cells 14 and
7 days before s.c. challenge with 5 ? 104CT26 tumor cells. The
timing of this tumor challenge correlates with the peak of the ex-
pansion of AH1-Ldtet?T cells 14 days after the initial vaccination
(Fig. 2b). Tumor growth was monitored for 60 days by palpation
of the injection site. As indicated on the Kaplan-Meier plot, mice
were sacrificed when their tumors reached 100 mm2. Vaccination
with 39-LdTM-infected insect cells protected the majority of mice
from subsequent CT26 tumor challenge, whereas vaccination with
?-gal-LdTM-infected, AH1-LdTM-infected, or uninfected insect
cells failed to protect against tumor development (Fig. 6a and data
not shown). As expected, vaccination with the high-affinity mimotope
15 protected significantly fewer mice from tumor formation than the
intermediate-affinity mimotope 39. The response to infected insect
cells depends on the presence of CD8?T cells, because CD8 Ab
depletion of mice vaccinated with 39-LdTM-infected insect cells re-
sults in tumor growth in all mice tested (Fig. 6a).
We next determined the therapeutic efficacy of vaccination with
infected insect cells. We injected mice with 5 ? 104tumor cells
and vaccinated 2, 4, 7, 10, and 14 days later with 39-LdTM-in-
fected, ?-gal-LdTM-infected, or uninfected insect cells (Fig. 6b).
We observed a statistically significant delay in tumor growth in
mice injected with 39-LdTM-infected insect cells, although no
mice remained tumor free. These results show that Ag-specific T
cells, elicited by the infected insect cell vaccine, slowed the growth
of the tumor, but did not eliminate it.
In vivo assessment of a water-insoluble mimotope with high
affinity in the peptide-MHC/TCR interaction identified in
a BV peptide library
Finally, we determined whether peptides identified in the BV pep-
tide library could be analyzed in vivo using this method. The syn-
thetic peptide designated WMF (SPTYAYWMF) (23), a mimo-
tope of the AH1 Ag, was identified in a BV peptide library with
substitutions in the MHC-contact residues. This peptide is insolu-
ble in water and is difficult to synthesize, indicated by the heter-
ogeneity of HPLC and mass spectrometry profiles (data not
shown). However, the WMF peptide produced in infected insect
cells binds to CT-TCR with high affinity relative to peptide 39
(Fig. 7a) and stimulates a corresponding amount of proliferation of
the CT-T cell clone (Fig. 7b). These experiments demonstrate that
the WMF peptide-H-2Ldcomplex is produced in insect cells and
specifically binds to both soluble CT-TCR molecules and T cells
DCs. a, Mice were vaccinated with 39-LdTM-infected insect cells, and DC
subsets from mesenteric lymph nodes were analyzed with the indicated
markers by flow cytometry. The histograms are representative of two mice.
The dashed lines indicate staining from unvaccinated mice, and the solid
lines indicate the maximal staining during the time course (MHC class II
at 2 h, CD80 at 24 h, CD86 and CD70 at 16 h). b, CFSE-labeled Tg T cells
were incubated ex vivo with CD11c?splenocytes pulsed with either 39 or
?-gal peptide (left) or from mice vaccinated with 39-LdTM-infected or
-uninfected insect cells (right). CFSE dilution was determined by flow
cytometry. Percentages shown are the percentages of CFSE-diluted 39-
incubated T cells minus the ?-gal-incubated T cells. Data shown are rep-
resentative of two independent experiments.
Insect cell-derived peptides are presented to T cells by host
mice from CT26 tumor formation and delays formation of tumors. a, Mice
were vaccinated with 5 ? 106peptide-LdTM-infected insect cells 14 and 7
days before s.c. injection of 5 ? 104CT26 tumor cells. Tumors were
measured every 2 days. Mice were sacrificed when the tumor reached 100
mm2. Groups were compared using the log rank test (??, p ? 0.0001,
39-LdTM compared with ?-gal-LdTM; ???, p ? 0.0247, 15-LdTM com-
pared with 39-LdTM). b, Mice were vaccinated with 5 ? 106peptide-
LdTM-infected insect cells 2, 5, 8, 11, and 14 days after s.c. injection of
5 ? 104CT26. Mice were sacrificed when the tumor reached 100 mm2.
Tumors were measured every 2 days, and tumor sizes were compared from
each group on individual days with unpaired two-tailed Student’s t test
(?, p ? 0.0001 for unvaccinated vs 39-LdTM and p ? 0.0044 for ?-gal-
LdTM vs 39-LdTM).
Vaccination with 39-LdTM-infected insect cells protects
194INFECTED INSECT CELLS ELICIT PROTECTIVE ANTITUMOR IMMUNITY
We next vaccinated mice with insect cells infected with BV-
encoding WMF-LdTM to analyze the tumor-specific T cell re-
sponses and to determine whether antitumor activity is afforded by
this high-affinity mimotope. Vaccination with 39-LdTM- and
WMF-LdTM-infected insect cells elicited tumor Ag-specific T
cells, as determined by tetramer staining (Fig. 7c). Although the
affinity of the WMF peptide in the peptide-MHC/TCR interaction
is higher than the intermediate-affinity 39 peptide, fewer AH1-
specific T cells were detected in the blood after vaccination with
WMF-LdTM-infected insect cells. In addition, vaccination with
39-LdTM-infected insect cells protected the majority of mice from
subsequent CT26 tumor challenge, whereas vaccination with
?-gal-LdTM-, AH1-LdTM-, or WMF-LdTM-infected insect cells
failed to protect (Fig. 7d). These results are consistent with those
in Fig. 6 and our previous results showing that peptides that form
an intermediate-affinity peptide-MHC/TCR complex with substi-
tutions in the MHC-contact residues, such as the 39 peptide, pro-
tect mice from tumor challenge (27), whereas those that form a low
(AH1)- or high (peptide 15 or WMF)-affinity peptide-MHC/TCR
complex do not.
We have shown that vaccination with BV-infected insect cells ex-
pressing peptide-MHC complexes generates functional Ag-spe-
cific CD8?T cell responses. This vaccine, which protects against
tumor challenge and delays growth of tumors, is convenient in that
it does not require additional adjuvants to those provided by the
BV and insect cells; it eliminates the need, expense, and potential
artifacts of synthesizing peptides identified in the BV peptide li-
brary before evaluation; and it does not require purification of
proteins or BV before injection. Vaccination with infected insect
cells expressing other recombinant proteins also results in specific
immune cell responses. For example, we have used this vaccina-
tion strategy to develop TCR-specific Abs (data not shown). Like
with the peptide-MHC vaccine, vaccination with infected insect
cells expressing recombinant proteins eliminates the need for pro-
This vaccine strategy is unique and cannot be practically
achieved using other strategies because it provides a method to
analyze peptides identified in BV peptide-MHC libraries that are
otherwise technically difficult to evaluate, such as the WMF pep-
tide (Fig. 7). Amino acids such as cysteine, methionine, and tryp-
tophan are often avoided in peptide libraries due to disulfide bond-
ing, insolubility, and sensitivity to oxidation (27, 42). Small
molecular changes in amino acid residues of the peptide, such as
oxidation or alkyl-chain modifications, can alter epitopes and thus
elicit different T cell responses (43, 44). Furthermore, vaccination
with synthetic peptides does not always stimulate T cells that rec-
ognize endogenously processed peptides, possibly due to oxidation
or cysteinylation of amino acid residues during peptide synthesis
or handling (45–47). Although oxidation does not affect the T cell
response to all Ags, in the BV-infected insect cell vaccine strategy,
the peptide is produced, processed, and cross-presented in a re-
duced intracellular environment similar to natural tumor Ags.
In vitro characterization of peptides derived from libraries re-
quires coexpression of MHC molecules and peptides. Insect cells
infected with rBV-encoding peptide, H-2Ld, and ?2m molecules
bind H-2Ld-specific Abs and the AH1-specific CT-TCR, suggest-
ing that the protein structure is similar to that of mammalian cells.
Furthermore, the avidity of the peptide-MHC/TCR complexes cor-
relates with the affinity of the soluble mimotope-MHC/TCR com-
plexes (Fig. 1) (27). In addition to binding assays for character-
ization of peptides, other in vitro studies using infected insect cells
examine T cell function, such as cytokine production (21, 22). In
vivo, insect cells expressing peptide 39 restricted by H-2Ldelicit
a population of T cells that binds Ag-loaded tetramer (Fig. 2, a and
b), kills Ag-loaded target cells (Fig. 2, c and d), and protects 67%
of mice from subsequent tumor challenge (Fig. 6a). These proof-
of-concept experiments indicate that the T cells elicited by infected
insect cells recognize native peptide on tumor cells.
peptides identified from the BV peptide library. a, Insect cells infected with
the indicated BV were analyzed as in Fig. 1a. The MFI of the CT-TCR at
a specific MHC expression level were 150, 98.8, 22.7, and 2.2 for WMF-
LdTM, 39-LdTM, AH1-LdTM, and ?-gal-LdTM, respectively. These data
are representative of three independent experiments. b, T cells expressing
the CT-TCR were incubated with infected insect cells expressing ICAM
and B7 costimulatory molecules and AH1-LdTM, 39-LdTM, ?-gal-LdTM,
or WMF-LdTM in vitro at a 5:1 ratio. Proliferation was determined by
incorporation of [3H]thymidine and is shown as mean cpm ? SD, n ? 3.
c, Mice were injected with the indicated vaccines, as in Fig. 2b, and the
frequency of CD8?AH1-Ldtet?splenocytes was determined by flow cy-
tometry 14 days after the initial injection. The data shown are representa-
tive plots, and the numbers indicate the average percentage of CD8?
AH1-Ldtet?T cells minus background ?-gal-LdTM tetramer staining (39-
LdTM, n ? 2; WMF-LdTM, n ? 4; unvaccinated, n ? 2). d, Tumor pro-
tection assays were performed as in Fig. 6a. Groups were compared using
the log rank test (?, p ? 0.0044 comparing 39-LdTM with ?-gal-LdTM).
The BV-infected insect cell vaccine can be used to evaluate
195 The Journal of Immunology
To analyze the mechanism of priming by this vaccine, we de-
veloped a new Tg mouse that expresses the ?- and ?-chains of a
TCR specific for the AH1/H-2LdAg. This TCR was derived from
a BALB/c mouse vaccinated with irradiated CT26 tumor cells ex-
pressing GM-CSF (28), i.e., a T cell clone that had escaped neg-
ative selection in the thymus. Because T cells from this mouse
recognize a tumor/self Ag, we are including them in our analyses
to determine the requirements of peptide vaccines that break tol-
erance. Not all tumor-specific T cells generated in this mouse ex-
press CD8 molecules, suggesting that down-regulation of the CD8
molecule is a consequence of tolerance, as reported by others (48,
49). Alternatively, aberrant expression of the TCR during T cell
development may disrupt the expression of the CD8 molecule. The
Ag-specific response of the CD3?CD8?T cells is less robust rel-
ative to the CD3?CD8?cells, suggesting that the coreceptor con-
tributes to the binding avidity of the TCR complex. Consistent
with this possibility, more coreceptor-negative T cells bind 39-Ld
tet than AH1-Ldtet (data not shown).
Although the BV constructions require the MHC molecules for
peptide studies in vitro, it is not required to elicit specific T cells
in vivo. Like other effective antitumor CD8?T cell responses (41,
50), the results we show in this study are consistent with tumor-
specific T cells elicited by cross priming. 1) Direct recognition of
Ag on the surface of infected insect cells is not required to elicit T
cells when the vaccine encodes peptide and MHC (Fig. 4). Insect
cells infected with BV-encoding peptide-Ldcomplexes that are not
expressed on the cell surface elicit a similar frequency of AH1-Ld
tet?T cells as insect cells encoding surface peptide-Ldcomplexes
(Fig. 4a). 2) When the vaccine encodes peptide, but no MHC mol-
ecules (peptide-?2m), similar responses are elicited (Fig. 4). In this
experiment, the only MHC available to present peptide is from the
host cells, not the vaccine. 3) This vaccine induces maturation of
CD11c?DCs from the draining lymph nodes (mesenteric) and
spleen as determined by increased expression of costimulatory and
maturation markers CD80, CD86, MHCII, and CD70 (Fig. 5a).
The expression of CD70, a maturation marker whose expression
correlates with optimal expansion of CD8?T cells following vac-
cination with both CD40 ligands and TLR agonists (51), increased
on DCs after vaccination. 4) CD11c?cells from vaccinated mice
stimulate Tg T cells to proliferate in an Ag-specific manner ex
vivo. No additional Ag or adjuvant is added in these experiments
(Fig. 5b). 5) When the vaccine expresses costimulatory molecules
(ICAM and B7), the T cell response to the vaccine is unchanged
(data not shown). 6) Finally, it is unlikely that peptide-MHC mol-
ecules are produced in BV-infected DCs because the polyhedron
promoter driving transcription of the Ags is active only in insect
cells (9–11). This feature of BV makes them safe to work with.
Alternatively, extracellular Ag processing and presentation by DCs
stimulate CD4?T cells (52). A similar mechanism in which MHC
I-restricted peptides are loaded onto DCs is possible. However,
because this vaccine delivers only an estimated 200 ng of peptide
and 10 ?g of free peptide is required for similar responses (27),
this mechanism most likely accounts for a small fraction of the T
cell response. Thus, like other vaccine delivery systems (53), the
injected insect cells do not present Ags directly, but activate T cells
by transferring the Ag to host professional APCs, resulting in ef-
fective priming of tumor-specific T cell responses. In summary,
use of these BV peptide-MHC constructions provides a stream-
lined system for evaluation of newly discovered peptides.
We thank Drs. Denise Golgher, Haruo Tsuchiya, and Su-Yi Tseng for
assistance with the production of the CT-TCR Tg mice. We also thank
Jennifer McWilliams for constructing the CT-TCR plasmid, Heather
Knowles for assistance with the immunoprecipitation, and Dr. Phillip
Sanchez for help with the DC activation experiments.
The authors have no financial conflict of interest.
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197The Journal of Immunology