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Translation from Cryptic Reading Frames of
DNA Vaccines Generates an Extended
Repertoire of Immunogenic, MHC Class
I-Restricted Epitopes
Reinhold Schirmbeck, Petra Riedl, Nicolas Fissolo, Francois
A. Lemonnier, Antonio Bertoletti and Jörg Reimann
J Immunol 2005; 174:4647-4656; ;
doi: 10.4049/jimmunol.174.8.4647
http://www.jimmunol.org/content/174/8/4647
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Copyright © 2005 by The American Association of
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References
The Journal of Immunology
Translation from Cryptic Reading Frames of DNA Vaccines
Generates an Extended Repertoire of Immunogenic, MHC
Class I-Restricted Epitopes1
Reinhold Schirmbeck,2* Petra Riedl,* Nicolas Fissolo,* Francois A. Lemonnier,†
Antonio Bertoletti,‡ and Jörg Reimann*
he CD8⫹ T cell is an important specific effector cell
against many intracellular pathogens and tumors that specifically recognize antigenic, MHC class I-binding peptides. The consensus is that the primary source of antigenic peptides presented by class I molecules are normal or defective
nascent proteins that arise by translating either conventional (primary) or alternative (cryptic) reading frames (1–3). The latter can
result from the use of alternative reading frames of a coding sequence, or of untranslated regions of DNA, mRNAs, introns, or
intron/exon junctions. Aberrant initiation of mRNA or protein synthesis may result from, e.g., initiation codons in translated or untranslated sequences, frame shifting during translation, defective
splicing of primary transcripts, or overriding of stop signals (3).
These events seem to be errors intrinsic to the complex transcription/translation machinery. T cell-stimulating epitopes generated
from cryptic products translated from alternative open reading
frames (ORFs)3 have been found in (human and mouse) tumors
T
*Institute of Medical Microbiology and Immunology, University of Ulm, Ulm, Germany; †Institut Pasteur, Unité d’Immunité Cellulaire Antivirale, Department d‘Immunologie, Paris, France; and ‡Institute of Hepatology, University College London,
London, United Kingdom
Received for publication August 2, 2004. Accepted for publication January 26, 2005.
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.
1
This work was supported by grants from the Deutsche Forschungsgemeinschaft
(DFG Schi 505/2-2) (to R.S.) and the European Commission (QLK2-CT-2002-00700)
(to J.R., A.B., and R.S.).
2
Address correspondence and reprint requests to Dr. Reinhold Schirmbeck, Institute for
Medical Microbiology and Immunology, University of Ulm, Albert Einstein Allee 11,
D-89081 Ulm, Germany. E-mail address: [email protected]
3
Abbreviations used in this paper: ORF, open reading frame; HBsAg, hepatitis B
surface Ag; HBV, hepatitis B virus; HCMV, human CMV; hsp, heat shock protein;
Copyright © 2005 by The American Association of Immunologists, Inc.
and in virus infections (4 –10). It is unknown whether delivering
Ag by DNA vaccines (11–13) primes T cell responses to immunogenic epitopes generated from alternative ORFs.
Genetic (nucleic acid or DNA) vaccination is a potent tool to
elicit CD8⫹ T cell responses (11, 12). Expression vectors used in
DNA vaccines contain a transcription unit composed of a heterologous promoter/enhancer sequence, the Ag-encoding sequence,
and termination sequences. The constructs are designed such that
the Ag is expressed from the primary open reading frame with the
first ATG motif of the Ag-encoding sequence located downstream
of the promoter. We have reported different strategies to construct
vectors that can deliver an extended spectrum of immunogenic
information. These involved the following approaches: 1) different
Ags were coexpressed from a single transcription unit by incorporating internal ribosomal entry sequences between two (or even
more) genes cloned into a bi-(multi)cistronic vector to allow their
coexpression under control of a single promoter (14, 15). 2) Bidirectional human CMV (HCMV)-derived promoter elements allow
coexpression of two Ags by generating two separate expression
units up- and downstream of the promoter sequence (16). 3) Two
overlapping reading frames of a single sequence that encode two
defined viral Ags (under a single heterologous promoter control)
allow the delivery of more antigenic information in a compact
form by an expression plasmid of limited size (17). In the present
study, we analyzed whether CD8⫹ T cell responses can be generated from cryptic products translated from other than the primary
ORF encoded by DNA vaccines (3).
The 3.2-kb genome of the hepatitis B virus (HBV), one of the
smallest known viral genomes, contains four partially overlapping
ORFs encoding the core, polymerase (Pol), surface (HBsAg), and
LS, large HBsAg; MS, middle HBsAg; Pol, HBV polymerase; S, small HBsAg;
T-Ag, large SV40 tumor Ag; tg, transgenic.
0022-1767/05/$02.00
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To test whether simple expression units used in DNA vaccines can generate immunogenic, MHC class I-binding epitopes by
translating other than the primary open reading frame (ORF), we constructed a vector (pCI/SX) that encodes the small hepatitis
B surface Ag in the primary ORF, and a C-terminal fragment (residue 344 – 832) of the polymerase (Pol) in an alternative
(out-of-frame) reading frame. pCI/SX efficiently primed multispecific, HLA-A2-restricted CD8ⴙ T cell responses to epitopes of
hepatitis B surface Ag and of Pol (Pol3, Pol803– 811). Pol3-containing products generated from pCI/SX were detected only by T cell
assays, but not by biochemical assays. Priming Pol-specific T cell responses to epitopes generated from alternative ORFs depended
on promoter sequences that drive transcription in the DNA vaccine (human CMV-derived promoter sequences being more efficient
than SV40-derived promoter sequences). Human CMV promoter-driven Pol constructs encoding different Pol fragments in primary or alternative reading frames elicited comparable levels of Pol3-specific T cell responses. We confirmed efficient T cell
priming to epitopes from alternative ORFs by constructing DNA vaccines that encode an SV40-derived cT1–272 protein fused either
in frame or out of frame with an immunogenic OVA fragment (OVA18 –385). Similar OVA-specific CD8ⴙ T cell responses were
primed by both alternative vaccine constructs. Hence, DNA vaccine-stimulated T cell responses to epitopes generated from
alternative ORFs seem to be a regular event, although its biological role and risks are largely unexplored. The Journal of
Immunology, 2005, 174: 4647– 4656.
4648
CD8⫹ T CELL RESPONSES TO CRYPTIC TRANSLATION PRODUCTS
amplified by PCR from the pCI/SX, using a forward primer with a NheI
site (AAAGCTAGCGACGGAAATTGCACCTGTAT) and a reverse
primer (CGAGAGAGTCCCAAGCGA). The product was cloned into
NheI/BamHI cut pCI/SX vector. This vector encodes the S144–226 and
the Pol487–832 in an overlapping sequence. pCI/SXnonsense C: An HBV
pol fragment was amplified by PCR from the pCI/SX, using a forward
primer with a NheI site (AAAGCTAGCCAAGTGTTTGCTGACG
CAA) and a reverse primer (CGAGAGAGTCCCAAGCGA). The product was cloned into pCI/SX vector using NheI/BamHI. This vector encodes the Pol685–832 sequence.
OVA-encoding plasmids: pCI/OVA. An OVA construct was generated
that started at the methionine at OVA position 8 (for detail, see Swiss-Prot:
OVAL_CHICK). The OVA8–385-encoding sequence was amplified using a
forward primer with a XhoI site (GGCTCGAGGCAGCAAGCATGGA
ATTTTG) and a reverse primer with a NotI site (GGGCGGCCGCTTA
AGGGGAAACACATCTGCCAA). The product was cloned into pCI vector using XhoI/NotI to generate the pCI/OVA. pCI/cT-OVA1: An OVA18–
385-encoding fragment was amplified by PCR from the pCI/OVA plasmid,
using a forward primer with a KpnI site (AAAGGTACCATTCAAG
GAGCTCAAAGTCCACC) and a reverse primer with a NotI site
(AAAGCGGCCGCTTAAGGGGAAACACATCTGCC). Compared with
the wild-type OVA sequence, the forward primer contained one additional
A nucleotide. The product was cloned into pCI/cT1–272 vector to generate
the pCI/cT-OVA1 construct. This construct encodes a fusion protein
consisting of the cT1–272 fragment and, mediated by the frame shift of the
additional A nucleotide in the forward primer, 10 residues not related to
OVA or large SV40 tumor Ag (T-Ag) (IQGAQSPPCQ), followed by a stop
signal (cT ⫹ 10). Due to a start signal in the alternative reading frame, this
construct additionally encodes an N-terminal 40-residue product not related to OVA or T-Ag (MILIQKKQRKLNKCPGSLVPFKELKVH
HANENIFYCPIAI), followed by the OVA35–385 fragment (OVA*). pCI/
cT-OVA2: The OVA18–385-coding fragment was amplified by PCR from
the pCI/OVA plasmid using a forward primer with a KpnI site (AAAGG
TACCATTCAAGGGCTCAAAGTCCACC) and a reverse primer with a
NotI site (AAAGCGGCCGCTTAAGGGGAAACACATCTGCC). The
product was cloned into pCI/cT1–272 vector to generate the pCI/cT-OVA2
DNA. This construct expresses the cT1–272/OVA18–385 fusion protein plus
3-aa spacer (IQG) not related to T-Ag or OVA (cT-OVA).
Hepatitis B core Ag-encoding plasmid. The pCI/C DNA vaccine has been
described (30).
Materials and Methods
Chicken hepatoma LMH cells (CRL-2117; American Type Culture Collection; Promochem) or human epithelial kidney HEK293 cells (CRL1573; American Type Culture Collection; Promochem) were transiently
transfected with plasmid DNA using the Ca2PO4 method, as described
previously (28, 29). Where indicated, cells were labeled with [35S]methionine, extracted with lysis buffer, and immunoprecipitated using the antiPol mAb 9F9 (31), rabbit anti HBsAg serum, or anti-SV40 T-Ag mAb 419
and protein A-Sepharose. The precipitates were processed for SDS-PAGE,
followed by fluorography. Alternatively, cellular extracts from unlabeled
cells were directly processed for SDS-PAGE and analyzed by Western
blotting using the anti-Pol mAb 9F9, polyclonal rabbit anti-SV40 T-Ag
serum, or polyclonal anti-OVA serum (C-6534; Sigma-Aldrich), followed
by 35S-labeled protein A (28, 29).
Mice
C57BL/6J (B6) mice (H-2b) and HLA-A2 transgenic (A2-tg) HHB mice
were bred and kept under standard pathogen-free conditions in the animal
colony of Ulm University. Male and female mice were used at 12–16 wk
of age.
Vector constructs
HBV Pol-encoding plasmids: pCI/Pol. The HBV Pol was amplified by
PCR from the plasmid pTKTHBV2(a generous gift of A. Meyer, Martinsried, Munich, Germany), using a forward primer with a KpnI site (AAAG
GTACCATGCCCCTATCCTATCAACACTTCCG) and a reverse primer
with a SalI site (AAAGTCGACTCACGGTGGTCTCCATGCGAC). The
product was cloned into pCI vector (catalogue number E1731; Promega)
using KpnI/SalI. pCI/cT-Pol585–832: The Pol585–832 -encoding fragment was
amplified by PCR from the plasmid pTKTHBV2, using a forward primer
with a KpnI site (AAAGGTACCATGGGTTATGTCATTGGATGTTA
TGGG) and a reverse primer with a SalI site (AAAGTCGACTCACGGT
GGTCTCCATGCGAC). The product was cloned in frame behind the
cT272-encoding sequence of pCI/cT272 vector (28) using KpnI/SalI.
pCI/SX and its derivates. The XhoI/BglII fragment of HBV encoding the
small HBsAg up to the HBV poly(A) sequence was cloned into the XhoI/
BamHI-digested pCI vector to generate the pCI/SX plasmid. pCI/SX⌬int:
the intron sequence was deleted from the pCI/SX vector by AflII restriction
and religation to create pCI/SX⌬int. pCI/SX⌬P: the pCI/SX vector was digested with BglII/XhoI, blunted with Klenow, and religated to create the
promoterless pCI/SX⌬P. pCI/S: pCI/SX plasmid was digested with XhoI/
StuI, and the HBsAg-encoding fragment was cloned into XhoI/SmaI-digested pCI. pSI/SX: the HCMV promoter of pCI/SX vector was exchanged with the SV40 early promoter sequences derived from pSI
vector (catalogue number E1712; Promega) to generate the plasmid
pSI/SX. pCI/SXnonsense A: The pCI/SX vector was digested using NheI/
XbaI and religated. This vector encodes the S42–226 and the Pol386–832 in
an overlapping sequence. pCI/SXnonsense B: A HBV Pol fragment was
Transient expression assays
DNA vaccination
We injected 100 ␮g of DNA (50 ␮l of PBS containing 1 ␮g/␮l plasmid
DNA) into each tibialis anterior muscle, as described (32).
Peptides
Synthetic, HPLC-purified peptides used in the described experiments were
obtained from Jerini BioTools.
Splenic specific CD8⫹ T cell frequencies
Spleen cells (1.5 ⫻ 107/ml) were incubated for 1 h in serum-free BioWhittaker-Ultra Culture medium (catalogue number BE12-725F; Cambrex Bio
Science) with the indicated amounts of the specific or the control peptides.
Thereafter, 5 ␮g/ml brefeldin A was added, and the cultures were incubated for further 4 h. Cells were harvested and surface stained with PEconjugated anti-CD8 mAb (catalogue number 01045B; BD Pharmingen).
Surface-stained cells were fixed with 2% paraformaldehyde in PBS before
intracellular staining for IFN-␥. Fixed cells were resuspended in permeabilization buffer (HBSS, 0.5% BSA, 0.5% saponin, and 0.05% sodium
azide) and incubated with FITC-conjugated anti-IFN-␥ mAb (catalogue
number 55441; BD Pharmingen) for 30 min at room temperature and
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trans activator (X) proteins (Fig. 1A) (18, 19). The HBsAg-encoding sequence overlaps the sequence encoding Pol (Fig. 1B) (18). In
the present study, we analyzed an HCMV-driven DNA vaccine
that encodes the small HBsAg in the primary ORF and an overlapping C-terminal Pol344 – 832 fragment in an alternative (out-offrame) ORF. No endogenous HBV-specific promoter sequences
(19, 20) are located in this HBV sequence. Multispecific MHC
class I-restricted epitopes to Pol and HBsAg have been characterized (21–27). The Pol344 – 832 fragment encoded in the alternative
ORF contained at least three different A2-restricted epitopes (Figs.
1 and 2) and, thus, it can be used as a defined model Ag for cryptic
transcription/translation products expressed from alternative reading frames. Immunization experiments showed that the pCI/SX
vaccine efficiently coprimed CD8⫹ T cells specific for A2-restricted epitopes processed from HBsAg and from cryptic Polspecific translation products. We showed that: 1) the HCMV promoter plays a critical role in generating cryptic translational
products from alternative Pol ORF that give rise to immunogenic
Pol803– 811 epitope, and 2) HCMV promoter-driven Pol constructs,
encoding different Pol fragments in the alternative or primary reading frame, elicited comparable levels of Pol3-specific T cell
responses.
In the second part of the study, we generated different constructs
producing an immunogenic epitope of OVA. The OVA18 –385 fragment (containing the well-characterized Kb-restricted SIINFEKL
epitope) was cloned C terminally (in frame or out of frame) behind
the SV40 cT1–272 protein (28, 29). The OVA-specific CD8⫹ T cell
responses primed by constructs producing the epitope from either
an out-of-frame or an in-frame translation product were compared.
B6 (H-2b) mice vaccinated with these constructs developed similar
OVA-specific CD8⫹ T cell responses. Hence, CD8⫹ T cell responses can be primed equally well by immunogenic products generated from either the primary or an alternative ORF of a DNA
vaccine.
The Journal of Immunology
washed in permeabilization buffer. Stained cells were resuspended in PBS/
0.3% w/v BSA supplemented with 0.1% w/v sodium azide. We determined
the frequencies of IFN-␥⫹ CD8⫹ T cells by flow cytometry analyses. The
mean numbers of IFN-␥⫹ CD8⫹ T cells/105 CD8⫹ T cells are shown.
Results
Expression of viral Ags from alternative reading frames of a
DNA vaccine
FIGURE 1. The overlapping reading frames of Pol
and HBsAg. A, Map of the HBV genome. B, The
HBsAg-encoding sequence is encoded in an alternative
reading frame of the Pol-encoding sequence. The relative amino acid positions of the Pol and small HBsAg
and their epitopes are indicated. C, Maps of the Pol
(pCI/Pol; pCI/cT-Pol585– 832)- and HBsAg-encoding
(pCI/SX) vectors. D, Chicken hepatoma LMH cells
were transiently transfected with pCI/Pol, pCI/SX, pCI/
cT-Pol585– 832, or control pCI DNA. Cells were labeled
with [35S]methionine and immunoprecipitated either
for HBsAg (using a polyclonal rabbit anti-HBsAg serum) or Pol (using the anti-Pol mAb 9F9). cT-Pol585–
832 fusion protein was immunoprecipitated with the
anti-T-Ag mAb PAB419. For Western blot analyses,
transfected cells were lysed, and extracts were directly
applied to SDS-gel electrophoresis. Blots were treated
with anti-Pol mAb 9F9, rabbit anti-mouse Ab, and
35
S-labeled protein A. The positions of the (glycosylated and nonglycosylated) forms of HBsAg (LS, MS,
S), hsp73, and cT-Pol 585– 832 fusion protein are
indicated.
An immunogenic Pol epitope is generated from a product
translated from the alternative reading frame of the HBsAgencoding expression plasmid pCI/SX
A2-tg mice were injected with the pCI/SX DNA vaccine. Spleen
cells obtained from these mice 12–14 days postvaccination were
restimulated ex vivo with titrated amounts of the Pol- or HBsAgderived peptides (Fig. 2B). The numbers of CD8⫹ T cells specifically inducible to IFN-␥ expression by each of the peptides were
determined by flow cytometry. The pCI/SX DNA vaccine efficiently primed CD8⫹ T cell responses to the A2-restricted epitopes
S1 and S2 of HBsAg and also to the Pol3 epitope of Pol (Fig. 3).
This vaccine did not prime CD8⫹ T cell responses to the Pol1 and
Pol2 epitope of Pol (Fig. 3). The pCI/Pol DNA vaccine (which
coexpresses HBsAg and Pol from alternative reading frames) efficiently primed CD8⫹ T cell responses to the Pol1 and Pol3, but
prime responses inefficiently to the Pol2 epitope and the HBsAg
epitopes S1 and S2 (Fig. 3). CD8⫹ T cell responses to Pol3 were
always higher than those to Pol1 (Fig. 3). Interestingly, the magnitude of the Pol3-specific T cell responses primed by either pCI/
Pol (Pol3 epitope processed from the primary ORF) or pCI/SX
(Pol3 epitope processed from the alternative ORF) was comparable (Fig. 3). Stimulation of splenic T cells ex vivo with titrated
amounts of the different MHC-I-binding peptides of Pol reached
saturating conditions when ⬎2.5 ⫻ 10⫺7 M respective peptides
were used (Fig. 3). Under these saturating conditions, CD8⫹
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The pCI/SX vector contains sequences encoding the small HBsAg
(S) and its 3⬘ untranslated sequence up to the HBV poly(A) sequence. It also contains in an overlapping, alternative ORF the
sequence encoding the C-terminal Pol344 – 832 fragment (Fig. 1, B
and C). The nucleic acid sequence as well as the amino acid sequences of the alternative HBsAg- and Pol-specific ORFs for the
pCI/SX DNA vaccine as well as the A2-restricted epitopes are
shown in Fig. 2. The pCI/Pol vector encodes the 832 residue Pol
protein, and the large HBsAg (LS) in an alternative reading frame
(Fig. 1, B and C) (17). Cells transfected with pCI/SX plasmid DNA
efficiently expressed (glycosylated and nonglycosylated) small
HBsAg (S) (Fig. 1D). Cells transfected with the pCI/Pol vector
expressed lower levels of the (glycosylated and nonglycosylated)
large pre-S1/pre-S2/S (LS), middle pre-S2/S (MS), and small (S)
HBsAg (Fig. 1D) (17). We did not detect expression of Pol or its
fragments in cells transfected with either pCI/SX or pCI/Pol plasmid DNA using anti-Pol mAb 9F9 that binds an extreme C-terminal linear epitope with high affinity (Fig. 1D) (31).
4649
4650
CD8⫹ T CELL RESPONSES TO CRYPTIC TRANSLATION PRODUCTS
FIGURE 2. A, Nucleic acid and amino acid sequences of pCI/SX-encoding HBsAg in the primary
ORF and Pol344 – 832 in an alternative ORF. B, The
HLA-A2-restricted epitopes of Pol, core, and HBsAg.
Stable and unstable expression of Pol (fragments) gives rise to
Pol3 peptides with comparable immunogenicity for CD8⫹ T
cells
T cell-stimulating epitopes generated from products translated
from alternative ORFs have been identified in different experimental systems (4 –10), but we report in this work the first example that
it also operates in DNA vaccination. Sensitive CD8⫹ T cell priming/presentation assays have been used to detect expression of
such cryptic protein products. Confirming this finding, we found
strong T cell responses to the Pol3 epitope generated from products of alternative reading frames. But, it proved difficult to detect
cryptic translation products in biochemical assays. We could not
detect Pol fragments in lysates of metabolically labeled, pCI/SXtransfected cells using immunoprecipitation and/or Western blotting with the Pol-specific mAb 9F9 (Fig. 1D). Also, Pol expression
was not detected in pCI/Pol-transfected cells using the same protocols (Fig. 1D) (17). These data show that Pol protein expression
is difficult to detect. We cannot distinguish between low Pol expression and a high turnover rate of Pol. Hence, specific T cell
priming by DNA vaccination is more a sensitive readout than most
currently available biochemical tools to detect products from inframe or out-of-frame transcription/translation.
We next asked whether stable overexpression of a Pol fragment
enhances Pol3-specific T cell priming. The Pol585– 832 fragment
encodes only the Pol3 epitope (but not the Pol1 or Pol2 epitope)
(Figs. 1, B and C, and 2A). The heat shock protein 73 (hsp73)facilitated expression system (28) was used to overexpress this Pol
fragment. Its sequence was fused in frame behind the hsp73-capturing, DnaJ-homologous cT1–272 protein domain derived from a cytosolic SV40 T-Ag variant. This generated the pCI/cT-Pol585– 832
DNA vaccine (Fig. 1C). The hsp73-bound cT-Pol585– 832 fusion protein was efficiently expressed in transfected cells and accumulated to
high levels (Fig. 1D). A2-tg mice vaccinated with this DNA vaccine
developed a Pol3-specific CD8⫹ T cell response. The magnitude of
this response was comparable to that primed by DNA vaccines expressing Pol either in frame (pCI/Pol) or out of frame (pCI/SX) (Fig.
3). Thus, DNA vaccines with strikingly different levels of Pol expression prime Pol3-specific T cell responses with similar efficacy. The
magnitude of Pol expression thus does not correlate with efficient
generation of immunogenic epitopes. Rapid Pol degradation may
make its detection of expression from pCI/Pol or pCI/SX difficult, but
may facilitate the generation of immunogenic peptides.
Different HBsAg-encoding expression constructs support CD8⫹
T cell priming to the Pol3 epitope
We constructed variants of plasmid pCI/SX, tested their HBsAg
expression, and measured their relative efficacy to prime A2-restricted T cell responses. We introduced the following deletions
into the pCI/SX construct (Fig. 4A): deletion of C-terminal sequences encoding the Pol570 – 832 sequence (construct 2: pCI/S);
elimination of the intron between the promoter and the start codon
of HBsAg (construct 3: pCI/SX⌬int); or elimination of the promoter (construct 5: pCI/SX⌬P). Furthermore, we exchanged
HCMV-derived promoter sequences with SV40 promoter sequences (construct 4: pSI/SX). These constructs were tested for
HBsAg expression (Fig. 4B). Efficient HBsAg expression was apparent from pCI/SX (lane 1) and pCI/S (lane 2). Expression of
HBsAg was lower when the intron was deleted (pCI/SX⌬int; lane
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IFN-␥⫹ T cell frequencies were comparable between the different
groups. Control experiments confirmed the specificity of the responses to Pol and HBsAg. These responses did not cross-react to
the C1 epitope of the control core Ag of HBV (Fig. 3). Similarly,
vaccination with pCI/C (encoding the HBV core protein) induced
a C1-specific CD8⫹ T cell response that did not cross-react with
the Pol or HBsAg epitopes (Fig. 3). Thus, our data demonstrate
that the Pol3 epitope can be efficiently processed from peptides
generated from translation products of an alternative ORF.
The Journal of Immunology
4651
FIGURE 3. HLA-A2-restricted and Pol- and HBsAgspecific CD8⫹ T cell responses in tg mice. A2-tg mice
were vaccinated i.m. with pCI/SX, pCI/Pol, pCI/cTPol585– 832, or control pCI/C plasmid DNA. Spleen cells
obtained 12–14 days postvaccination were restimulated
ex vivo for 4 h with titrated amounts of antigenic peptides. The cells were surface stained for CD8 and intracellularly stained for IFN-␥. Mean numbers of
IFN-␥⫹ CD8⫹ T cells/105 CD8⫹ splenic T cells (⫾SD)
of three mice from a representative (of three independent) experiment(s) are shown.
Expression constructs encoding Pol fragments in alternative and
primary reading frames support Pol3-specific CD8⫹ T cell
priming
Vaccinating mice with the pCI/SX DNA vaccine (encoding the
Pol344 – 832 fragment in an alternative ORF) primed CD8⫹ T cells
specific for the Pol3, but not the Pol1 epitope (Fig. 3). A switch to
the alternative ORF that generates the immunogenic Pol products
may thus operate downstream of the position of the Pol1 epitope
(Fig. 2A). To test whether immunogenic Pol3 peptides are translated from different cryptic or primary reading frames dependent
on specific Pol sequences, we constructed pCI/SX-based nonsense
vectors. The pCI/SXnonsense A vector was created by deleting the
NheI/XbaI fragment (encoding the S-specific start codon) from the
pCI/SX plasmid. The remaining sequence encodes the Pol386 – 832
and S42–226 fragments (Fig. 5A). Assuming that the first ATG mo-
tif is used to start translation, this construct may produce a S75–226
fragment from the primary ORF and a Pol433– 832 fragment from an
alternative ORF (Fig. 5A). Transient transfection followed by Polspecific immunoprecipitation and/or Western blot analyses did not
detect Pol or S fragments (data not shown), but a similar 1–74
deletion construct of HBsAg has been detected (33).
A2-tg mice were vaccinated with the pCI/SX or pCI/SXnonsense
⫹
A constructs. CD8
T cell priming to the S2, Pol1, and Pol3
epitopes (present on both constructs) was determined (Fig. 5).
Pol3-specific (but not Pol1-specific) CD8⫹ T cell responses were
efficiently elicited in mice vaccinated with both constructs. The
pCI/SXnonsense A DNA coinduced S2-specific CD8⫹ T cell responses
(Fig. 5B). Thus, truncated coding sequences support generation of
immunogenic epitopes from both reading frames.
We further constructed the pCI/SXnonsense B vector containing
the overlapping coding regions Pol487– 832 and S144 –226, and the
pCI/SXnonsense C vector containing only the Pol685– 832-coding regions (Fig. 5A). These constructs did not express detectable levels
of Pol or S (data not shown). Assuming that the first ATG motif is
used to start translation, these constructs may produce the Pol506 – 832
or Pol699 – 832 proteins from the primary ORF (Fig. 5A). A2-tg mice
vaccinated with these constructs generated high CD8⫹ T cell responses to Pol3 (Fig. 5B). Priming of Pol3-specific CD8⫹ T cells is
comparable when translation products were generated from Pol fragments expressed from alternative ORFs (pCI/SX; pCI/SXnonsense A) or
primary ORFs (pCI/SXnonsense B; pCI/SXnonsense C) (Fig. 5). The vector pCI/SXnonsense B encodes the S144 –226 sequence, but there is no
ATG in this ORF preceding the S2 epitope (Fig. 5A). Hence, no S2specific responses were primed with this construct.
Induction of OVA-specific CD8⫹ T cell responses
We extended the study to an unrelated Ag system using OVA. We
compared generation of an immunogenic Kb-restricted OVA
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3), or when the promoter sequences were changed (pSI/SX; lane
4), confirming previous results (32). No HBsAg expression was
detected from the pCI/SX⌬P plasmid (lane 5). Pol-specific translation products were not detected in transfected cells (data not
shown).
In A2-tg mice vaccinated with these constructs, CD8⫹ T cell
priming to the S2 and Pol3 epitopes was determined (Fig. 4C).
S2-specific CD8⫹ T cell responses were efficiently elicited in mice
vaccinated with the pCI/SX, pCI/S, pCI/SX⌬int, or pSI/SX DNA
vaccines. The pCI/SX⌬P did not elicit S2-specific CD8⫹ T cell
responses. CD8⫹ T cell responses to Pol3 were efficiently induced
in mice by vaccination with pCI/SX or pCI/SX⌬int, but not with
pCI/S, pCI/SX⌬P, or pSI/SX. HBsAg expression from HCMVderived, but not SV40-derived promoter sequences thus supported
generation of the immunogenic Pol3 epitope from a translation
product of an alternative ORF. Intron sequences were not necessary for Pol3 priming.
4652
CD8⫹ T CELL RESPONSES TO CRYPTIC TRANSLATION PRODUCTS
epitope from primary and an alternative translation product. We
designed a pCI/cT-OVA1 expression plasmid that encodes the
hsp73-binding cT1–272 fragment and a 10-residue spacer IQG
AQSPPCQ in the primary ORF (cT ⫹ 10; Fig. 6, A and B). This
construct also encodes (in an alternative open reading frame) a
fusion protein containing N terminally a 40-residue fragment
MILIQKKQRKLNKCPGSLVPFKELKVHHANENIFYCPIAI
initiated from an upstream ATG start signal and the OVA35–385
(OVA*; Fig. 6, A and B). In the control pCI/cT-OVA2 construct,
we inserted the OVA18 –385 sequence C terminally in frame to the
cT1–272 fragment to generate the cT-OVA18 –385 fusion protein
(cT-OVA; Fig. 6, A and B). We further cloned a OVA8 –385
sequence into the pCI vector to generate the pCI/OVA construct
(OVA; Fig. 6A). These constructs allowed the comparative evaluation of the immunogenicity of the Kb-restricted OVA257–264
epitope SIINFEKL expressed from either the alternative (pCI/cTOVA1) or primary (pCI/cT-OVA2, pCI/OVA) OVA-encoding
ORFs. OVA-specific Western blotting revealed efficient expression of the OVA protein or the cT-OVA fusion protein by cells
transfected with pCI/OVA or pCI/cT-OVA2 plasmid DNA (Fig.
6C). Cells transiently transfected with pCI/cT-OVA1 plasmid
DNA efficiently expressed the N-terminal cT ⫹ 10 protein, but not
the OVA* fragment, as expected (Fig. 6C).
B6 mice were vaccinated with pCI/cT-OVA1, pCI/cT-OVA2,
pCI/OVA, or control pCI plasmid DNA (100 ␮g/mouse i.m.). The
OVA- and T-Ag-specific CD8⫹ T cell responses were read out 12
days postvaccination. CD8⫹ T cell responses of B6 mice specific
for the Kb-restricted OVA257–264 epitope were comparable when
the epitope was generated by translation products generated from
primary ORFs (pCI/cT-OVA2, pCI/OVA) or an alternative ORF
(pCI/cT-OVA1) (Fig. 6D). pCI/cT-OVA1 and pCI/cT-OVA2
DNA vaccines primed similar CD8⫹ T cell responses to the SV40
T-Ag, i.e., the Db-restricted T206 –215 epitope SAINNYAQKL (Fig.
6D) (34). These data confirm the efficient generation of CD8⫹ T
cell-stimulating epitopes by HCMV-driven translation products of
alternative reading frames.
Discussion
We used in vivo priming by a single injection of a DNA vaccine
to assess (in a quantitative and specific CD8⫹ T cell readout) the
relative efficacy with which immunogenic epitopes are generated
from in-frame (primary ORF) vs out-of-frame (alternative ORF)
translation products using different Ag systems. We demonstrate
that CD8⫹ T cell responses specific for unrelated epitopes processed from cryptic translation products are efficiently primed by
simple DNA vaccines. These data support the notion that expressing Ag in the primary ORF of a DNA vaccine may lead to copriming of immunogenic, MHC class I-binding epitopes generated
from cryptic translation products of alternative reading frames.
The HBsAg-encoding pCI/SX DNA vaccine efficiently primed
CD8⫹ T cell responses to the A2-restricted epitopes S1 and S2 of
HBsAg, and to the Pol3 epitope of Pol. Thus, T cell responses were
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FIGURE 4. Variant HBsAg-encoding DNA vaccines prime Pol-specific T cell responses. A, Maps of
the DNA vaccines used: pCI/SX (1), pCI/S (2), pCI/
SX⌬int (3), pSI/SX (4), and pCI/SX⌬P (5). B, Cells transiently transfected with these plasmid DNAs were
grown for 2 days, labeled with [35S]methionine, lysed,
immunoprecipitated by an anti-HBsAg serum, and analyzed for HBsAg expression by SDS-PAGE. The position of the (glycosylated and nonglycosylated) small
HBsAg (S) is indicated. C, A2-tg mice were vaccinated
i.m. with the DNA constructs 1–5. Spleen cells obtained 12 days postvaccination were restimulated ex
vivo for 4 h with titrated amounts of the antigenic S2,
Pol3, or (control) C1 peptides. Cells were surface
stained for CD8 and intracellularly stained for IFN-␥.
Mean numbers of IFN-␥⫹ CD8⫹ T cells/105 CD8⫹
splenic T cells (⫾SD) of three mice from a representative experiment are shown.
The Journal of Immunology
4653
efficiently primed by immunogenic translation products of inframe and out-of-frame reading frames of a DNA vaccine. The
Pol1 and Pol2 epitopes also encoded in this alternative reading
frame were not primed by the pCI/SX vaccine (Fig. 3). The pCI/
Pol DNA vaccine (expected to express the complete Pol) efficiently primed CD8⫹ T cell responses to the Pol3 and Pol1
epitopes. CD8⫹ T cell responses to Pol3 were always higher than
those to Pol1 (Fig. 3). Binding affinities of the antigenic Pol and
HBsAg peptides to HLA-A2 have been published and are in a
similar range (10 –38 nM; Fig. 2B). It is considered that an affinity
threshold of ⬍500 nM is required for induction of CD8⫹ T cell
responses (21). Thus, differences in the binding affinities of the
antigenic peptides to their restricting element are unlikely to underlie the observed immunogenicity of Pol epitopes, focusing on
the question as to how these epitopes are generated.
Conventional expression vectors used in DNA vaccines contain
a transcription unit encoding the Ag sequence cloned downstream
from a strong promoter/enhancer sequence that drives expression
of the immunogenic protein from a primary reading frame. Promoter sequences used in DNA vaccines are often from viruses,
e.g., CMV, SV40, or retroviruses (12). Our data suggest that the
type of promoter used to express an Ag in a DNA vaccine has an
influence on the efficacy of generating epitopes from alternative
reading frames. HCMV-driven pCI/SX, but not SV40-driven
pSI/SX DNA vaccines induced Pol3-specific T cell responses.
Hence, HCMV-derived promoter sequences are apparently more
efficient than SV40-derived promoter sequences in supporting generation of cryptic translation products from which immunogenic
epitopes can be processed, but the molecular events underlying this
finding are unclear. We have shown that DNA vaccines with
SV40-derived promoter sequences produce lower levels of Ag
from the primary reading frame than vaccines in which Ag expression is driven by HCMV-derived promoter sequences (Fig.
4B) (32). Thus, the efficacy of a promoter may play a role in the
decreased generation of cryptic Pol-specific translation products.
Alternatively, different promoter sequences may have different fidelities with which they use a primary reading frame.
We used different approaches to detect Pol-derived translation
products from alternative reading frames using the anti-Pol mAb
9F9 directed against the extreme C terminus of Pol close to the
location of the Pol3 epitope. We tried to detect newly synthesized
Pol or Pol fragments in lysates of 35S-labeled transfectants or their
steady state levels by Western blotting. We were only successful in
detecting hsp73-associated cT-Pol585– 832 fusion Ag, but not in detecting the Pol protein nor any cryptic Pol fragments (Fig. 1D).
Thus, Pol expression does not seem to correlate with efficient generation of immunogenic epitopes because apparently very different
levels of Pol expression from DNA vaccines primed T cell responses
in mice of comparable efficacy (Fig. 3). Rapid Pol degradation (that
may make detection of its expression from pCI/Pol or the pCI/SX
difficult) may facilitate the generation of immunogenic peptides from
this protein, a finding compatible with the notion that degradation of
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FIGURE 5. DNA constructs that encode different HBsAg and Pol fragments in overlapping reading frames. A,
Maps of the DNA constructs pCI/SX (1), pCI/SXnonsense A
(2), pCI/SXnonsense B (3), and pCI/SXnonsense C (4). The
vector-encoded amino acids and the respective ATG
motifs of HBsAg and Pol are indicated. Furthermore,
the primary (p) and alternative (a) reading frames (defined by the first ATG start signal downstream of the
promoter) are indicated. B, HLA A2-tg mice were vaccinated i.m. with DNA of the constructs 1– 4. Spleen
cells obtained 12 days postvaccination were restimulated ex vivo for 4 h with titrated amounts of the antigenic S2, Pol1, Pol3, or (control) C1 peptides. Cells
were surface stained for CD8 and intracellularly stained
for IFN-␥. Mean numbers of IFN-␥⫹ CD8⫹ T cells/105
CD8⫹ splenic T cells (⫾SD) of three representative
mice are shown.
4654
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FIGURE 6. CD8⫹ T cell responses to OVA expressed by DNA vaccines in a primary or an alternative
reading frame. A and B, Maps and sequences of the
pCI/cT-OVA1, pCI/cT-OVA2, and pCI/OVA constructs. The pCI/cT-OVA1 construct encodes two proteins: the cT ⫹ 10 protein that encodes the cT1–272 protein plus 10 (C-terminal) residues not related to OVA or
T-Ag (IQGAQSPPCQ) and, in an alternative ORF, the
OVA* protein that encodes 40 N-terminal residues
(MILIQKKQRKLNKCPGSLVPFKELKVHHANE
NIFYCPIAI) and the OVA35–385. The pCI/cT-OVA2
construct encodes the cT1–272-OVA18 –385 (cT-OVA)
fusion protein in the primary reading frame. pCI/OVA
encodes a OVA8 –385 (OVA) protein in the primary
ORF. C, Expression of OVA-encoding constructs. Human epithelial kidney HEK293 cells were transiently
transfected with DNA from the pCI/cT-OVA1, pCI/cTOVA2, pCI/OVA, or (control) pCI constructs. Nonlabeled extracts of transfected cells were processed for
OVA- or T-Ag-specific Western blotting (using polyclonal anti-OVA or anti-T-Ag sera and 35S-labeled
protein A). The position of OVA, cT-OVA, and cT ⫹
10 is indicated. D, B6 mice were vaccinated i.m. with
the pCI/cT-OVA1, pCI/cT-OVA2, pCI/OVA, or noncoding pCI DNA vaccines. Spleen cells obtained 12–14
days postvaccination were restimulated for 4 h ex vivo
with the antigenic, Kb-binding OVA257–264, Db-binding
T206 –215, or (control) HBsAg peptide. Cells were surface stained for CD8 and intracellularly stained for
IFN-␥. Mean numbers of IFN-␥⫹ CD8⫹ T cells/105
CD8⫹ splenic T cells (⫾SD) of three mice from a
representative (of three independent) experiment(s) are
shown.
CD8⫹ T CELL RESPONSES TO CRYPTIC TRANSLATION PRODUCTS
The Journal of Immunology
Acknowledgments
We are grateful for the excellent technical assistance of Tanja Güntert and
Katrin Ölberger. We thank Drs. Hansjörg Hauser (Gesellschaft fur Biotechnologishe Forschung, Braunschweig, Germany) and R. Carlson and
J. Wands (Molecular Hepatology Laboratory, Massachusetts General Hospital Cancer Centre, Boston, MA) for helpful advice and reagents.
Disclosures
The authors have no financial conflict of interest.
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nascent protein (but not the stability of mature proteins) is critical in
generating MHC class I-binding epitopes (3).
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Pol fragments from alternative reading frames of the same sequence. In the pCI/SXnonsense A construct, two reading frames apparently generated protein fragments that could not be detected by
biochemical assays (Fig. 5). However, this nonsense construct induced CD8⫹ T cell responses to Pol and HBsAg. HCMV-derived
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that can elicit CD8⫹ T cell responses in vivo (Fig. 5).
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phenomenon seems to have general importance. We demonstrated
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possibility to concomitantly deliver self in an immunogenic form.
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