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 Virology 347 (2006) 127 – 139
www.elsevier.com/locate/yviro

SARS coronavirus nucleocapsid immunodominant T-cell epitope

cluster is common to both exogenous recombinant and endogenous
DNA-encoded immunogens
Vandana Gupta a, Tani M. Tabiin a, Kai Sun a, Ananth Chandrasekaran a, Azlinda Anwar a,
Kun Yang b, Priya Chikhlikar b, Jerome Salmon b, Vladimir Brusic c,d, Ernesto T.A. Marques b,e,f,
Srinivasan N. Kellathur a,b, Thomas J. August a,b,*
a
Division of Biomedical Sciences, Johns Hopkins in Singapore, 31 Biopolis Way, #02-01 The Nanos, Singapore 138669, Singapore
b
Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205, USA
c
Institute for Infocomm Research, 21 Heng Mui Keng Terrace, Singapore 119613, Singapore
d
School of Land and Food Sciences and the Institute for Molecular Bioscience, University of Queensland, Brisbane 4072, Australia
e
Department of Medicine, Division of Infectious Diseases, The Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21218, USA
f
Virology and Experimental Therapy Laboratory, Aggeu Magalhaes Research Center, Recife, PE 50670-420, Brazil

Received 11 August 2005; returned to author for revision 22 September 2005; accepted 22 November 2005
Available online 4 January 2006

Abstract

Correspondence between the T-cell epitope responses of vaccine immunogens and those of pathogen antigens is critical to vaccine efficacy. In the
present study, we analyzed the spectrum of immune responses of mice to three different forms of the SARS coronavirus nucleocapsid (N): (1)
exogenous recombinant protein (N-GST) with Freund’s adjuvant; (2) DNA encoding unmodified N as an endogenous cytoplasmic protein (pN); and
(3) DNA encoding N as a LAMP-1 chimera targeted to the lysosomal MHC II compartment (p-LAMP-N). Lysosomal trafficking of the LAMP/N
chimera in transfected cells was documented by both confocal and immunoelectron microscopy. The responses of the immunized mice differed
markedly. The strongest T-cell IFN-g and CTL responses were to the LAMP-N chimera followed by the pN immunogen. In contrast, N-GST elicited
strong T cell IL-4 but minimal IFN-g responses and a much greater antibody response. Despite these differences, however, the immunodominant T-
cell ELISpot responses to each of the three immunogens were elicited by the same N peptides, with the greatest responses being generated by a cluster
of five overlapping peptides, N76 – 114, each of which contained nonameric H2d binding domains with high binding scores for both class I and, except
for N76 – 93, class II alleles. These results demonstrate that processing and presentation of N, whether exogenously or endogenously derived, resulted
in common immunodominant epitopes, supporting the usefulness of modified antigen delivery and trafficking forms and, in particular, LAMP
chimeras as vaccine candidates. Nevertheless, the profiles of T-cell responses were distinctly different. The pronounced Th-2 and humoral response
to N protein plus adjuvant are in contrast to the balanced IFN-g and IL-4 responses and strong memory CTL responses to the LAMP-N chimera.
D 2005 Elsevier Inc. All rights reserved.

Keywords: SARS coronavirus; Nucleocapsid protein; N DNA construct; LAMP-N DNA construct; Recombinant N; Rodent; T cells; Antigen peptide epitopes;
ELISpot

Introduction
Abbreviations: SARS, severe acute respiratory syndrome; SARS CoV,
SARS-associated coronavirus; N, nucleocapsid protein; N-GST, N protein
fused to glutathione S-transferase; N-His, N protein fused to histidine; LAMP1, Vaccines designed to prevent viral infection must promote
lysosome associated membrane protein-1; MIIC, MHC class II containing the adaptive expansion of T cells that are associated with B-
compartments; p-hLAMP-N, p43-human LAMP1 SARS CoV N construct; p- and T-cell long-term memory of epitopes identical to or cross-
N, p43-SARS CoV N construct. reactive with those of the natural pathogen (Welsh et al., 2004;
* Corresponding author. Department of Pharmacology and Molecular
Sciences, The Johns Hopkins University School of Medicine, 725 N. Wolfe
Crotty and Ahmed, 2004). The processing and presentation of
Street, Baltimore, MD 21205, USA. Fax: +1 410 502 3066. antigen epitopes of vaccine immunogens must therefore mimic
E-mail address: taugust@bs.jhmi.edu (T.J. August). that of pathogens. The conventional understanding has been
0042-6822/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.virol.2005.11.042
128 V. Gupta et al. / Virology 347 (2006) 127 – 139

that epitopes presented by MHC class I are derived from the antigen sequences to the trafficking/targeting signals of the
endogenous cellular proteins that have been cleaved by lysosome-associated membrane protein-1 (LAMP-1) (Chen et
proteasomal and post-proteasomal peptidases (Villadangos et al., 1985). LAMP molecules are known to traffic to lysosomes
al., 1999; Yewdell and Bennink, 2001; Kloetzel 2004) and that and to specialized multilaminar vesicular compartments of
the epitope loading of MHC class II molecules generally immature APCs, termed MIIC, where MHC II antigen
involves exogenous proteins that are taken into antigen- processing and the formation of antigenic peptide –MHC II
presenting cells (APCs) by specialized endocytic receptors complexes occur (Kleijmeer et al., 1997; Geuze 1998; Murk et
and processed by proteases of the endosomal/lysosomal al., 2004). Studies of several antigens (including HIV-1 Env
compartments (Moreno et al., 1991; Bryant and Ploegh, 2004). and Gag, HPV E7, cytomegalovirus pp65, carcinoembryonic
It has been widely reported that various antigen-processing antigen, dengue preM/E and yellow fever Env, and the catalytic
pathways may affect immune epitope formation either posi- subunit of the telomerase reverse transcriptase (hTERT))
tively or negatively and may influence the repertoire of T-cell expressed from viral vectors and naked DNA or RNA have
epitope-specific immune responses either quantitatively or shown that LAMP-targeted antigens generated greater antigen-
qualitatively (Pamer and Cresswell, 1998; Kessler et al., specific lymphoproliferative activity, antibody titers, and
2002; Lautwein et al., 2004; Kloetzel, 2004). Such specificity cytotoxic T-lymphocyte activities than do DNA constructs
in antigen processing would suggest that the form and delivery encoding wild-type antigens (Rowell et al., 1995; Wu et al.,
of an immunogen can be critical to the generation of 1995; Ruff et al., 1997; Nair et al., 1998; Raviprakash et al.,
biologically effective antigen epitopes and thus to vaccine 2001; Bonini et al., 2001; Su et al., 2002; Lu et al., 2003;
efficacy. There is, however, abundant evidence for the Marques et al., 2003; Chikhlikar et al., 2004; Barros de Arruda
presentation of exogenous antigens by MHC class I and of et al., 2004; Anwar et al., 2005; Su et al., 2002). Most recently,
endogenous proteins by MHC class II through a variety of in a clinical study, patients who received dendritic cells
alternative antigen trafficking and processing mechanisms, transfected with RNA encoding an hTERT/LAMP chimera
including specialized processing pathways (Reimann and developed higher frequencies of hTERT-specific T-cell
Schirmbeck, 1999; Norbury et al., 2001; Bonifaz et al., 2002; responses than did subjects receiving dendritic cells transfected
Imai et al., 2004), cross priming and presentation (Brode and with the unmodified hTERT template (Su et al., 2005).
Macary, 2004; Tewari et al., 2005), alternative endolysosomal Despite this abundance of evidence of increased immune
pathways (Brooks et al., 1991; Lich et al., 2000; Chen and responses to LAMP chimeras of antigen proteins, the biolog-
Jondal, 2004), vesicular translocation (Reis e Sousa and ical consequences of the enhanced immune responses in
Germain, 1995; Ackerman and Cresswell, 2004), and phago- relation to protective immunity have not been defined, and
cytic or autophagic mechanisms for antigen delivery to both the there remains a critical question of the identity of T-cell
MHC class I and II compartments (Houde et al., 2003; epitopes of LAMP-targeted antigens as compared to those of
Guermonprez et al., 2003; Nimmerjahn et al., 2003; Rocha natural pathogens. LAMP trafficking is not a normal mecha-
and Tanchot, 2004; Paludan et al., 2005). nism for antigen access to the MHC class II compartment of
Nevertheless, little is known about the consequences of antigen-presenting cells, and the epitope specificity of antigen
these alternative antigen trafficking and processing pathways processing and presentation in vivo in comparison to other
for the production of pathogen-specific antigen epitopes. Thus, antigen delivery systems remains to be demonstrated.
an examination of T-cell epitopes is a critical early step in the In the present study, we have used the severe acute
development of a new generation of vaccines, including DNA respiratory syndrome coronavirus (SARS CoV) nucleocapsid
vaccines, chimeras with other protein sequences, or epitope- (N) as a model antigen to address this issue of immune
based antigens, all of which involve antigens as non-viral response and T-cell epitope presentation as a function of
structures that may access processing pathways that differ from antigen formulation. The N protein of 422 amino acids is
those followed by the native pathogens. These issues regarding abundantly expressed and highly immunogenic (Lau et al.,
the pathways used for antigen delivery and processing are 2004; Huang et al., 2004a). The primary function of this
particularly relevant to MHC class II epitopes. Presentation of structural protein in virus assembly is to bind to SARS CoV
antigen epitopes and activation of CD4 T cells by MHC class II RNA during the formation of the ribonucleoprotein complex
molecules are critical steps in generating memory B cell and (Huang et al., 2004b). Previous studies have shown that both
CD8+ T-cell phenotypes (Wang and Livingstone, 2003; Sun et the N protein (Lin et al., 2003; Zhao et al., 2005) and DNA
al., 2004), and failure to elicit immune memory is one of the encoded N (Kim et al., 2004; Zhu et al., 2004; Yang et al.,
recognized problems of conventional genetic vaccines 2004) elicit antibody and cytotoxic T-cell responses in mice. In
(Maecker et al., 1998). This defect may reflect an inadequate this analysis of the effect of immunogen formulation and
level of antigen expression or restricted access to the MHC II delivery on immune responses in vivo, we have utilized three
processing and presentation compartments by DNA-encoded forms of N delivery: (1) recombinant N-GST protein combined
endogenous antigens that lack a dedicated trafficking pathway with Freund’s adjuvant as a classical exogenous antigen under
(Martins et al., 1995; Maecker et al., 1998; Oehen et al., 2000; conditions of profound activation of the innate immune
Rush et al., 2002). response; (2) N encoded in a DNA plasmid as an unmodified
In response to this problem, we have characterized DNA endogenous cytoplasmic/nuclear antigen; and (3) a DNA
vaccine constructs synthesized in the form of chimeras that link construct with N inserted into the luminal domain of the entire
 V. Gupta et al. / Virology 347 (2006) 127 – 139 129

LAMP-1 molecule as an unnatural endogenous antigen to make the p-hLAMP-N vector (Fig. 1A). Western blot
targeted to the luminal compartment of endosomal/lysosomal analysis of N in cell lysates and culture supernatants of
vesicles (Guarnieri et al., 1993). transfected COS-7 cells showed the presence of the ¨42 kDa N
protein at comparable levels in cell lysates and culture
Results supernatants of p-N and p-hLAMP-N-transfected COS-7 cells
and of various forms of the higher molecular weight LAMP-N
Expression of N in cells transfected with p-N and p-LAMP-N chimera in the cell lysate and culture supernatant of p-hLAMP-
DNA plasmids N-transfected cells (Fig. 1B). The N-like protein in the cell
lysate of p-hLAMP-N-transfected cells is attributed to the
Validation of N expression by p-N and p-LAMP-N DNA cleavage of N from the protease-resistant LAMP at some stage
constructs was carried out by analyzing cells transfected in in the cellular trafficking of the molecule. Intact or near-intact
vitro. cDNA encoding the N protein was prepared by RT-PCR HIV-1 Gag protein was also observed with cells transfected
with N-specific primers and SARS RNA purified from a with the corresponding LAMP/Gag DNA vaccine construct
Singapore clinical isolate (GenBank accession no. AY307165). (Marques et al., 2003). Several forms of the larger LAMP-N
The corresponding 1269-bp N sequence was inserted into the molecule were also present in both the cell lysate and culture
p43 vector (Kessler et al., 1996) to form the wild-type p-N supernatants of the transfected cells. The intact LAMP-N is
DNA expression vector and into the p43-hLAMP-1 construct found at about 150 kDa, and the other bands are attributed to
in a position 5V-proximal to the LAMP transmembrane domain various degradation products. However, we are cautious about

Fig. 1. Construction and expression of DNA plasmids encoding SARS CoV N and hLAMP-N. Transgenic N and hLAMP-N chimera are expressed at comparable
levels in cell lysate and culture supernatant of transfected cells. (A) Schematic representation of the plasmid constructs. Arrows indicate the schematic position of the
various sequences: SARS CoV native nucleocapsid (SARS CoV N), hLAMP1 luminal domain (luminal domain), the hLAMP1 transmembrane and cytoplasmic
domains (TM/cyto), and the adeno-associated virus inverted terminal repeat sequences (AAV ITR). p-hLAMP: a control plasmid encoding the full human LAMP1
luminal, transmembrane (TM) and cytoplasmic (cyto) domains in the p43 expression vector. p-N: the unmodified SARS CoV N sequence in the p43 plasmid. p-
hLAMP-N: SARS CoV N sequences inserted into the full hLAMP1 sequence proximal to the hLAMP TM domain. Boxed areas represent the open reading frames as
indicated. The ovals indicate the AAV-ITR sequences of the p43 plasmid. (B) N and hLAMP-N protein expression in transfected monkey kidney COS-7 cells.
Western blot analysis of p-hLAMP (control), p-N and p-hLAMP-N-transfected cells with anti-N-GST polyclonal serum showed comparable amounts of N and
hLAMP-N protein in both the cell lysate and culture supernatant. The positions of the protein markers in kDa are shown on the right.
130 V. Gupta et al. / Virology 347 (2006) 127 – 139

drawing conclusions from the in vitro experiments as such F). MHC class II and LAMP-N in transfected melanoma cells
experiments may have little relationship to the expression of were both densely present throughout the cytoplasm and were
the immunogen in the cells transfected in vivo. markedly colocalized (Figs. 2M – O). Unmodified N was
densely expressed throughout the cytoplasm of the transfected
Confocal and immune electron microscopy cells, again in apparently vesicular structures, but with little or
no colocalization with either the endogenous LAMP-1 (Figs.
The presumed cell trafficking pathway for the newly 2G –I) or MHC class II (Figs. 2J– L) in B cells or melanoma
synthesized N, which lacks an endoplasmic reticulum translo- cells (Figs. 2P –R).
cation (signal) sequence, is to the cell cytoplasm. In contrast, Greater definition of the localization of N in transfected
the LAMP-N chimera is expected to traffic to endosomal/ cells was obtained by analyzing of immunolabeled ultrathin
lysosomal vesicles because of the LAMP vesicular trafficking cryosections of mouse LB 27.4 B cells. The endocytic
sequences. We used confocal microscopy to validate these compartments of these B cells are morphologically similar to
trafficking destinations for the immunoreactive unmodified N those of other reported mouse B-cell lines (6H5.DM and
antigens and N of the LAMP-N chimera expressed in A20.Ab), with well-defined multivesicular and multilaminar
transfected mouse LB 27.4 B cells and human melanoma MIICs and end organelles (Geuze, 1998). Double immunogold
Mel-Juso cells, comparing their cellular distribution to that of labeling of the transfected B cells confirmed that N of the
the endogenously expressed LAMP-1 and MHC class II LAMP-N chimera was present with both endogenous LAMP
proteins (Fig. 2). The colocalization of endogenous LAMP and MHC class II molecules in the MIIC multilaminar vesicles
and MHC class II proteins in vesicular compartments of APCs (Figs. 3A, B). In contrast, unmodified N was predominantly
has been extensively documented (Kleijmeer et al., 1996; localized in the cytoplasm or in vacuolated structures with no
Geuze, 1998; Murk et al., 2004). Trafficking of the LAMP-N evident colocalization with LAMP or MHC class II (Figs. 3C –
chimera protein to the LAMP and MHC II compartments of the E). Remarkably, lower power magnification showed that some
transfected cells was validated by the colocalization of the of the cytoplasmic vesicles contained large amounts of N.
LAMP-N chimera (labeled by anti-N antibody) with endoge- These vesicles may correspond to the strongly labeled vesicular
nous LAMP-1 and MHC class II in both mouse B (Figs. 2A – structures shown by immunofluorescent microscopy, and the
F) and human melanoma (Figs. 2M– O) cells. Endogenous presence of N in these structures suggests a possible route for
MHC class II of the B cells was distributed both at the the secretion of the protein (Fig. 3E).
periphery of the cell and in intracellular foci, in accordance
with the known maturation trafficking of vesicular MHC class Mouse primary immune responses
II molecules to the cell surface. Colocalization of LAMP-N
with MHC class II of B cells was restricted to the intracellular N antigen in three forms, representing differences in antigen
foci in the endosomal – lysosomal compartment and not with delivery, adjuvants, and peptide processing pathways, were
the mature MHC class II at the plasma membrane (Figs. 2D – administered according to the following protocols: (1) N as an

Fig. 2. Colocalization (yellow) of confocal images of immunolabeled transgenic N (red) with LAMP1 (green) and MHC II (green) in p-hLAMP-N-transfected
murine B cells (A – F) and with MHC II in human Mel-Juso cells (M – O). Colocalization rarely seen in p-N-transfected cells (G – L, P – R).
 V. Gupta et al. / Virology 347 (2006) 127 – 139 131

Fig. 3. Immunoelectron microscopy of murine B cells transfected with p-LAMP-N and p-N. N of p-hLAMP-N-transfected cells localized in typical MIIC
multilaminar vesicles with endogenous LAMP (A) and MHC II (B). N of p-N-transfected cells not found with endogenous LAMP1 (C) or MHC II (D) but present as
aggregates in cytoplasmic vesicles devoid of LAMP1 and MHC class II, sometimes in large numbers (E, low power). MIIC, MHC class II containing compartment;
PM, plasma membrane; L, electron dense lysosomes. Scale bar was adjusted to 200 nm.

exogenous N-GST protein was administered three times, on strongest IFN-g responders were cells from mice immunized
day one and 3 and 6 weeks later, initially with CFA and then with p-hLAMP-N, with comparable but weaker responses by
with IFA. (2) N as an unmodified endogenous, DNA-encoded, the p-N-immunized mice, and minimal responses by cells of
cytoplasmic protein, and (3) N as an artificial chimera encoded the N-GST-immunized mice (Table 1; Fig. 4). The major
in the luminal domain of LAMP and targeted to cellular grouping of IFN-g responses was elicited by peptides of amino
lysosomal and MHC II compartments were administered five acid sequence N76 – 114, encompassing 5 of the overlapping
times, on day 1, and 3, 6, 9, and 14 weeks later, without added peptides. More recently, these results have also been confirmed
adjuvant but subject to the molecular adjuvant effects of the in a new experimental protocol currently being conducted that
DNA CpG motifs and the targeting of the LAMP-N chimera to also show that all of the peptides that encompass N76 – 114 elicit
the MHC II compartment. DNA encoding p-hLAMP was used both CD4- and CD8-specific ELISpot responses (data not
as the negative control. shown). Other dominant responses were to peptides N130 – 149
T-cell responses were assayed by IFN-g ELISpot and and N241 – 258 and the overlapping peptides N352 – 366 and
ELISA assays using spleen cells collected 8 days after the N353 – 370. All significant epitope-specific responses of the pN or
final DNA immunization. For these assays, the T cells were N-GST-immunized mice were shared by the p-hLAMP-N-
stimulated with N-His protein as a positive control or with a immunized mice. The weak IFN-g responses of the N-GST
library of overlapping N peptides, 15 to 20 amino acids in protein immunized mice were observed only with peptides that
length. The results were confirmed in three experiments and in elicited the strongest responses with splenocytes of the DNA-
multiple assays with three independent sets of peptides from plasmid-immunized mice (Figs. 4A – C). This comparatively
different sources. The IFN-g responses were also assayed with weak IFN-g response of N-GST-immunized mice was in marked
an ELISA protocol, with similar results (data not shown). The contrast to the strong IL-4 responses as shown below and to the
132 V. Gupta et al. / Virology 347 (2006) 127 – 139

Table 1
T-cell IFN-g ELISpot positive N-peptide sequences
N peptides IFN-g SFC/106 splenocytes (mean T SD)
Number N-aa Sequence p-hLAMP-N p-N N-GST
11 76 – 93 NTNSGPDDQIGYYRRATR 57 T 2 3T2 6T1
12 80 – 99 GPDDQIGYYRRATRRVRGGD 429 T 8 266 T 18 46 T 12
13 84 – 101 QIGYYRRATRRVRGGDGK 377 T 11 209 T 6 55 T 6
14 92 – 109 TRRVRGGDGKMKELSPRW 309 T 13 212 T 7 24 T 5
15 100 – 114 GKMKELSPRWYFYYL 75 T 12 55 T 7 9T2
21 130 – 149 GIVWVATEGALNTPKDHIGT 106 T 4 61 T 8 5T3
35 241 – 258 QQQGQTVTKKSAAEASKK 131 T 20 23 T 3 2T1
51 352 – 366 ILLNKHIDAYKTFPP 137 T 10 34 T 8 38 T 4
52 353 – 370 LLNKHIDAYKTFPPTEPK 118 T 11 50 T 8 46 T 11
All ELISpot responses were to a group of 9 dominant peptides, shown by peptide number, amino acid region in N, sequence, and number of IFN-g-positive cells of
mice immunized with the three immunogens. A cluster (N76 – 114) of 5 peptides of 15 to 20 aa, overlapping by 10 to 16, contains 3 of the strongest dominant epitopes.

exceptional strong antibody responses. The N-specific antibody titer of about 1:25,000 on days 15 and 22 following activation
titers to N-GST immunization on day 42 (after the first boost) of the N-GST-immunized mice as compared to the responses of
was 1:25,000 and at day 63 (after the second boost) was about 1:7000 by the p-hLAMP-N-immunized mice. There was
1:67,500, as compared to titers increasing to a maximum of relatively little response (1:1000) by mice initially immunized
1:3200 on days 84 and 105 in response to immunization with the with p-N.
p-N and p-hLAMP-N DNA constructs.
Correlation of ELISpot-positive peptides and computational
Memory immune responses predictions of N-specific H2d binding motifs

Memory immune responses (total IgG antibody titers, CTL Peptide epitope activation of T cells relies on receptor
activity, and ELISpot IFN-g and IL-4 responses) were (TCR) recognition of complexes of antigen peptide sequences
examined by use of a separate experimental protocol with bound to the nine amino acids of the binding clefts of MHC
groups of mice treated with the same three N antigen class I and II molecules (Benacerraf, 1978). It is postulated that
preparations but receiving only three inoculations over 6 the kinetic stability of the MHC– peptide complexes is a key
weeks. After an interval of 36 weeks, the memory immune parameter that dictates immunodominance (Lazarski et al.,
response was activated by a single injection at week 42 of the 2005). In the further analysis of the T-cell epitope specificity of
p-N construct as a surrogate of virus infection. Antibody and T- N, the predicted H2d class I (H2-Kd, -Ld, -Dd) and class II (I-
cell ELISpot and CTL responses were measured immediately Ed, I-Ad) binding motifs of the 60 SARS N overlapping
before p-N injection at 42 weeks and three times at week peptides were examined by a murine immunoinformatics
intervals thereafter (Figs. 5 and 6). The T-cell assays were system based on quantitative matrices, PREDBALB/c (Zhang
conducted with the selected peptides that had elicited immu- et al., 2005). Each of the 414 nine-amino-acid sequences of N
nodominant responses in pilot experiments for IFN-g, N80 – 99 was analyzed for binding to the core sequence of the 5 H2d
and N241 – 258; IL-4, N80 – 99 and N130 – 149; and CTL, N80 – 99 alleles. All of the nine IFN-g ELISpot-positive peptides scored
and N353 – 370. The residual T-cell IFN-g and IL-4 responses of for a high probability of binding to class I and/or class II alleles
mice sacrificed on day 1 before p-N memory activation were (Table 2), and, in ongoing experiments, all of a new set of
very low and were significant only in mice initially immunized peptides encompassing N76 – 114 elicit both CD4- and CD8-
with p-hLAMP-N (Fig. 5). Following the recall p-N injection, specific responses (data not shown).
increased IFN-g and IL-4 responses were observed on day 8,
reaching an apparent maximum on days 15 to 22. The strongest Discussion
memory T-cell responses in all assays were those of mice
initially immunized with p-hLAMP-N, with strong IFN-g and In the present study, we have demonstrated that the
IL-4 responses to both peptides. N-GST plus Freund’s adjuvant dominant T-cell immune responses of mice immunized with
elicited strong IL-4 responses but minimal IFN-g responses, as the SARS CoV N protein plus adjuvant or with DNA encoding
seen for the primary response. The responses of mice initially two different cellular trafficking forms of N are directed to the
immunized with p-N were uniformly lower than those seen for same peptide epitopes of all three immunogens. This finding
the LAMP chimera and the IL-4 responses to N-GST. points to the existence of versatile antigen-processing mechan-
However, the IFN-g responses were greater for p-N than for isms that are not defined by the initial site of delivery of the
N-GST. Memory CTL responses were strongest in p-hLAMP- protein, whether exogenous or endogenous, or the state of the
N-immunized mice, with a much weaker response to p-N DNA innate responses to the immunogens. The data are consistent
immunization and little if any after immunization with N-GST with multiple in vivo routes for antigen access to proteolytic
with adjuvant (Fig. 6). Memory antibody responses followed processing compartments that produce the same or comparable
the pattern of the primary response, with a much stronger IgG MHC I and II peptide epitopes. This versatility would not
 V. Gupta et al. / Virology 347 (2006) 127 – 139 133

plus CFA. Others have reported similar findings that the

same CTL-defined epitope peptide is generated by an
exogenous protein and by plasmid DNA-encoded antigens
(Schirmbeck et al., 1998) and that the efficiency of MHC
class I cross-presentation of peptides derived from exogenous
antigens is comparable to that for presentation by the
classical MHC class II pathway (Storni and Bachmann,
2004). Additionally, Ria et al. (2004) have compared the
response to hen egg-white lysozyme (HEL) and its reduced
and carboxymethylated form (RCM-HEL) and have found
that RCM-HEL induces an in vivo T-cell response that is
focused on the same immunodominant determinant that
characterizes the response to native HEL but that the
RCM-HEL response is skewed toward the Th1 pathway.
No difference in the efficiency of processing was observed
between HEL and RCM-HEL. We conclude that the makeup
of the T-cell epitopes of an antigen is primarily determined
by the amino acid composition of the protein in the context
of the peptide binding sites of MHC molecules, and not by
the mechanism of delivery of the protein. These findings
support the usefulness of novel immunogens, such as antigen
chimeras and DNA vaccines encoding antigens with modified
cell trafficking signals, as vaccine candidates, despite their
differences from normal pathogen proteins in terms of
antigen structure, delivery, and processing mechanisms.
The differences in the results we obtained for the three
antigen preparations were largely related to the character and
magnitude of the immune responses. Mice immunized with
exogenous N protein plus Freund’s adjuvant responded with a
greatly enhanced antibody response and a high ratio of T-cell IL-
4 to IFN-g responses to the antigen peptides. In contrast, the
same peptides elicited a balanced IFN-g and IL-4 response and
lower antibody responses in mice immunized with DNA
vaccines. Freund’s adjuvant has been associated with the
preferential induction of T-cell Th2 responses (Shibaki and
Katz, 2002; Yip et al., 1999; Billiau and Matthys, 2001), and
different T-cell cytokine responses to the same epitope (Varga et
Fig. 4. Primary T-cell IFN-g ELISpot responses of (A) p-hLAMP-N, (B) p-N,
and (C) N-GST-immunized mice. As described in Materials and methods, mice
al., 2000) have been attributed to variable expression patterns of
were immunized five times with the DNA immunogens and three times with N- single cells and populations that reflect transient rather than
GST plus adjuvant, and the mice were sacrificed for ELISpot assay of antigen- heritable differences in the expression profile (Kelso and
specific splenocyte IFN-g secretion 8 days after the final immunization. The Groves, 1997; Kelso et al., 2002). Another difference between
splenocytes were stimulated with recombinant N-His protein and with N the vaccines was the comparatively poor memory response to
peptides from panels of overlapping peptides spanning the entire molecule,
with mice immunized by hLAMP plasmid as the negative control. All
the N-GST protein and p-N DNA construct as compared to the
significant T-cell peptide-specific IFN-g responses elicited by each immunogen p-hLAMP-N chimera construct. The limited efficacy of the
were stimulated by the same peptides, with the strongest responses to p- conventional p-N DNA construct is consistent with many
hLAMP-N and the weakest to N-GST. previous studies of our and other laboratories (Martins et al.,
1995; Maecker et al., 1998; Oehen et al., 2000; Rush et al., 2002;
preclude quantitative variation in epitope presentation as a Marques et al., 2003; Barros de Arruda et al., 2004). Although it
result of differences in the levels of antigen, the degree of is likely that there are multiple factors involved in memory T-cell
processing, or access to cellular compartments for antigen differentiation (Masopust et al., 2004), we attribute the enhanced
presentation. For example, in this study, the greater response T-cell and memory responses to p-LAMP-N, at least in part, to
to the LAMP-N chimera could be related to the quantita- the increased efficiency of trafficking of the LAMP-N chimera
tively greater delivery of N to the MHC class II compart- to the MHC II compartments of APCs.
ment of APCs. There can also be major differences in the The localization of the dominant T-cell responses to a cluster
repertoire or functionality of T cells responding to any given of overlapping peptides epitopes within N76 – 114 is similar to
epitope, such as the differences in the IFN-g and IL-4 findings from other ongoing studies of HIV-1 Gag and yellow
responses of mice immunized with DNA or with N-GST fever virus envelope proteins where the major T-cell responses
134 V. Gupta et al. / Virology 347 (2006) 127 – 139

Fig. 5. Kinetics of memory IFN-g and IL-4 ELISpot responses of N-immunized mice. Mice were immunized three times over 6 weeks with p-hLAMP
(control 0), p-N (n), p-hLAMP-N (r), N-GST (). At week 42, after an interval of 8 months, the memory immune response was activated by a single
injection of the p-N construct. Mice were sacrificed 1 day before the final p-N injection and three times at weekly intervals thereafter (days 1, 8, 15, and 22).
All assays were conducted in triplicate with cells from each of three mice from each experimental group and at each time point. ELISpot IFN-g memory was
measured in response to N80 – 99 (A) and N241 – 258 (B); and IL-4 in responses to N80 – 99 (C) and N130 – 149 (D). The p-hLAMP-N immunogen elicited the
strongest IFN-g and IL-4 ELISpot memory T-cell responses. N-GST immunogen elicited strong IL-4, but little IFN-g response.

also involve clustered epitopes (unpublished observations). and, as such, may facilitate the development of epitope-based
Regions of overlapping T-cell epitopes are also reported in vaccines if a single or few hot spots elicit all of the required T-
studies of HIV-1 proteins (Shankar et al., 1996; Surman et al., cell functions. The basis for such hot spots is unknown but is
2001; Brown et al., 2003; Berzofsky et al., 1991), the outer likely to be related in some measure to the binding affinities of
membrane protein of Chlamydia trachomatis (Kim and the peptide sequences to MHC molecules as well as to other
DeMars, 2001), and in predictions of HLA T-cell epitopes functional parameters that affect MHC –peptide binding to T-
(Srinivasan et al., 2004; Zhang et al., 2005). Clustered T-cell cell receptors during T-cell activation (Chen et al., 2005).
epitopes appear to be a common, perhaps ubiquitous occurrence Analysis of the nonameric MHC binding motifs of SARS N by

Fig. 6. Kinetics of memory CTL responses of N-immunized mice. Mice were immunized and investigated under the same protocol as described in Fig. 5 with p-
hLAMP (control 0), p-N (n), p-hLAMP-N (r), and N-GST (). CTL activity was assayed in responses to N80 – 99 (A) and N353 – 370 (B). The p-hLAMP-N
immunogen elicited the strongest memory CTL responses with relatively little response of mice immunized with N-GST protein or DNA encoding unmodified N.
 V. Gupta et al. / Virology 347 (2006) 127 – 139 135

Table 2
Correlation of ELISpot-positive peptides and computational predictions of N-specific H2d binding motifs
IFN-g N peptides PREDBALB/c H2d alleles
H2-Kd H2-Ld H2-Dd I-Ad I-Ed
76NTNSGPDDQIGYYRRATR93 – – SGPDDQIGY – –
80GPDDQIGYYRRATRRVRGGD99 YYRRATRRV GPDDQIGYY IGYYRRATR GYYRRATRR GYYRRATRR
GYYRRATRR
84QIGYYRRATRRVRGGDGK101 YYRRATRRV – IGYYRRATR GYYRRATRR RRVRGGDGK
GYYRRATRR
92TRRVRGGDGKMKELSPRW109 – – GGDGKMKEL – RRVRGGDGK
VRGGDGKMK
GKMKELSPR
100GKMKELSPRWYFYYL114 ELSPRWYFY LSPRWYFYY LSPRWYFYY – GKMKELSPR
SPRWYFYYL LSPRWYFYY
130GIVWVATEGALNTPKDHIGT149 ALNTPKDHI VWVATEGAL – EGALNTPKD WVATEGALN
TEGALNTPK
241QQQGQTVTKKSAAEASKK258 – – – QQGQTVTKK –
GQTVTKKSA
VTKKSAAEA
TKKSAAEAS
352ILLNKHIDAYKTFPP366 LLNKHIDAY – – NKHIDAYKT LLNKHIDAY
NKHIDAYKT
353LLNKHIDAYKTFPPTEPK370 LLNKHIDAY – – NKHIDAYKT LLNKHIDAY
NKHIDAYKT
The N-specific H2d binding motifs were analyzed by the PREDBALB/c system. Each of the IFN-g ELISpot peptides contained multiple nine amino acids binding
motifs that were within the top 5% of all binding scores to the five H2d alleles. All of the IFN-g ELISpot peptides except N241 – 258 scored were predicted to contain
class I binding motifs. Similarly, class II binding motifs were predicted in all except N76 – 93 and N130 – 149.

a newly developed PREDBALB/c computational system for SARS CoV N DNA plasmids
predicting the probability of binding of each nine-amino-acid
sequence of an antigen to the H2d class I (H2-Kd, -Ld, -Dd) The SARS CoV nucleocapsid (N) cDNA was prepared from
and class II (I-Ed, I-Ad) binding motifs (Zhang et al., 2005) a Singapore clinical isolate and purified total RNA. The 1269 bp
showed that each of the SARS N peptides that elicited N gene sequence was obtained by PCR with primers 5V-
ELISpot-positive IFN-g T-cell responses contained high- CGGCTAGCATGTCTGATAATGGACCCCAATC-3V and 5V-
probability binding motifs for MHC class I and/or II alleles. GCGGTACCTTATGCCTGAGTTGAATCAGCAG-3V, having
There may also be protein structural effects in the selection of NheI and KpnI sites. The PCR product was cloned into the p43
epitope hot spots. The three-dimensional structure of the N expression vector containing AAV-ITR sequences flanking the
amino-terminal domain, N45 – 181, of a truncated model lacking expression elements and a CMV promoter (Kessler et al., 1996)
N1 – 44 and N182 – 422 has been determined by NMR spectros- to make the wild-type plasmid p-N. This N-sequence (without
copy (Huang et al., 2004b). Two of the ELISpot-positive the stop codon) was amplified, and XholI and EcoRI sites were
sequences, N76 – 114 and N130 – 149, were present on the exposed introduced by using primers 5V-CGCTCGAGATGTCTGA-
surface of this structure. The N76 – 114 T-cell hot spot sequence TAATGGACCCCAATC-3V and 5V-CGGAATTCTGCCT-
also contains the N76 – 101 sequence reported to comprise a B GAGTTGAATCAGCAGAA-3V. The p-hLAMP-N chimera
cell epitope (Huang et al., 2004a). An association between T- vector was constructed by substitution of the HIV-gag sequence
and B-cell epitopes has also been found for the HIV envelope; into the p-hLAMP-gag vector (Chikhlikar et al., 2004). Due to
these are seen to be present in exposed loops or strands when the presence of internal NheI or XhoI sites in the N sequence,
mapped onto the crystal structure of the protein (Brown et al., the p-N and p-hLAMP-N plasmids were constructed by
2003; Del Porto et al., 2002; Shirai et al., 1999). sequential cloning of the two fragments of the N gene.

Materials and methods E. coli and baculovirus N-expression systems

Cell lines SARS-CoV N was amplified from the RT-PCR sequence

using forward primer 5V-ACGTCAGAATTCATGTCTGA-
Mouse B lymphoma and monkey kidney cell lines (LB27.4 TAATGGACCCCAA with an EcoRI site and reverse primer
and COS-7) were obtained from ATCC (Rockville, MD). 5V-AGTTTAGCGGCCGCTGCCTGAGTTGAATCAGCAG
Human melanoma cell line (Mel-Juso) was obtained from with a NotI site. The cDNA sequence was cloned into the
German Collection of Microorganisms and Cell Cultures EcoRI and NotI sites of the pGEX6P-1 (Invitrogen, a kind gift
(Braunschweig Germany). Insect cell lines SF9 and High5 from Dr. Roland of the Institute of Molecular and Cell Biology,
were obtained from Invitrogen. All the cell lines were Singapore) and pFastBac HT-A (Invitrogen, Carlsbad, CA)
maintained according to the supplier’s protocols. vectors. All clones were verified by sequencing.
136 V. Gupta et al. / Virology 347 (2006) 127 – 139

Expression of the SARS CoV N and LAMP-N proteins (prepared in our laboratory). Localization of the viral proteins
in the MHC class II-containing compartments of Mel-Juso cells
Expression of the N protein from the vaccine plasmids p-N was analyzed with anti-HLA-DR, DP, and DQ antibodies (BD
and p-LAMP-N in the COS-7 cell line was analyzed by Pharmingen) and rabbit anti-N antibody (IMG-549; Imgenex).
Western blot analysis. Cells were transfected with the SARS
plasmids using Polyfect Transfection reagent (Qiagen GmBH). Mouse immunization
Cell culture and polyacrylamide gel electrophoresis were
carried out as described (Chikhlikar et al., 2004). The N Three immunization protocols were carried out: (1) three
protein was detected by incubation with polyclonal serum from groups (p-hLAMP control, p-N, and p-hLAMP-N immuno-
mice immunized with N-GST protein followed by horseradish- gens) of 5 female (6 to 8 weeks old) BALB/c mice per group
peroxidase-conjugated goat anti-mouse IgG monoclonal anti- were immunized subcutaneously at the base of the tail with 50
body (Pierce Biotechnology). Binding of secondary antibody Ag of specified endotoxin-free DNA plasmid diluted in PBS on
was visualized with the SuperSignal West Pico Chemilumi- days 1, 22, 43, 64, and 100. Blood samples were collected by
nescent Kit or DAB (Pierce Biotechnology). phlebotomy from the tail vein on days 0, 21, 42, 63, 84, 99, and
108, and the animals were sacrificed on day 108, 8 days after
Recombinant N protein expression, purification, and Western the last immunization. (2) Another group was immunized
blot analysis intraperitoneally with 10 Ag of N-GST protein emulsified with
CFA followed by two booster immunizations 3 weeks apart
Recombinant N protein was generated by use of two DNA with N-GST protein emulsified with IFA. Blood samples were
plasmid expression systems, N-GST in E. coli for use as an collected as outlined above on days 0, 21, and 42, and the
antigen for mouse immunization and N-His in a baculovirus animals were sacrificed on day 50, 8 days after the last
system as an antigen for ELISA and ELISpot assays. immunization. Protocols 1 and 2 were carried out twice with
Recombinant N protein was expressed as a GST fusion in five mice per group each time. (3) An additional protocol was
Top10 E. coli cells (Invitrogen) after IPTG induction. The carried out for analysis of memory responses: four groups of 16
protein was purified by affinity chromatography with glutathi- mice each (p-hLAMP control, p-N, p-hLAMP-N, and N-GST
one S – Sepharose beads (Amersham Pharmacia Biotech AB), immunogens) were immunized as outlined above, but with
and the preparation was validated by Western blot analysis only three injections over 6 weeks. At week 42, after an
using mouse monoclonal anti-GST (Santa Cruz Biotechnology) interval of 36 weeks, the memory immune response was
and rabbit polyclonal anti-N (Imgenex) antibodies. The Bac to activated by a single injection of the p-N construct with p-
Bac baculovirus expression system version C (Invitrogen) was hLAMP as the negative control. Three mice of each group were
used for N protein expression in insect cells. pFastBacHT-A-N sacrificed 1 day before the final p-N injection and at weekly
was constructed by cloning the EcoRI- and NotI-digested intervals for 3 weeks thereafter. These experiments were
SARS CoV N fragment into the pFastBacHT-A vector. N performed as approved by the Johns Hopkins University
protein was purified from lysates of infected High5 cells using Animal Care and Use Committee under Protocol Number
Ni-NTA agarose beads (Invitrogen), and the protein prepara- MO04M178.
tion was verified by Western blot analysis with mouse
monoclonal Penta-His-antibody (Qiagen, GmBH) and anti-N ELISA for N-specific antibody analysis
(Imgenex) antibodies. Purification of the culture products of
each expression system yielded fractions enriched for the 73- Anti-SARS CoV N IgG antibodies in the serum from each
kDa N-GST and 55-kDa N-His proteins. mouse were assayed by ELISA for seroconversion at a dilution
of 1:300, and the total IgG titers of 3-fold serial dilutions,
Immunofluorescent confocal and immunoelectron microscopy starting from 1:100, were determined by standard quantitative
ELISA. In brief, ELISA plates (96-well, MaxiSorb F96; Nunc
The cellular localization of the expressed viral proteins in Inc.) were coated overnight at 4 -C with 1 Ag/ml recombinant
transfected mouse B lymphoma cells was studied by confocal N-His protein in PBS followed by blocking with ELISA buffer
and immunoelectron microscopy and confirmed by confocal (PBS containing 2.5% milk and 0.05% Tween 20). After
microscopy of transfected human Mel-Juso cells. Cells were washing, appropriately diluted mouse serum was added and
transfected with the SARS CoV plasmids (p-hLAMP-N and p- incubated for 2 h at room temperature. Total IgG was detected
N) using Polyfect (Qiagen, GmBH). The expression of the with the Mouse Extravidin alkaline phosphatase staining kit
recombinant viral proteins was visualized by the use of rabbit (Sigma).
polyclonal anti-N (Imgenex) or mouse monoclonal anti-human
LAMP1 antibody H4A3 (obtained from supernatant of ELISpot analysis of antigen-activated T cells
hybridoma cultures prepared in our laboratory). Localization
of the transgenic proteins in MHC-class-II-containing compart- The response of antigen-specific T cells from immunized
ments and lysosomes of B cells was determined by the use of mice was measured by the use of IFN-g and IL-4 ELISpot sets
anti-mouse MHC class II antibody M5/114.15.2 (BD Bios- (BD Pharmingen) according to the manufacturer’s protocol.
ciences) and anti-mouse LAMP1 (mLAMP1) ID4B antibody Splenocytes were stimulated with 5 Ag/ml of recombinant N-
 V. Gupta et al. / Virology 347 (2006) 127 – 139 137

His (baculovirus-produced) or 10 Ag/ml of the overlapping of the BALB/c proteome. The support of the NIH AIDS
SARS CoV synthetic peptides from three sources (NIH AIDS Research and Reference Reagent Program in providing the
Research and Reference Reagent Program, Rockville, MD; SARS N peptides is gratefully acknowledged.
SynPep Corp, Dublin, CA; the Johns Hopkins Biochemistry
Core Facility). Negative control stimulation produced less than References
five spots per well in >90% of experiments. The average
number of spots in the negative control wells was 1.67 T 1.87. Ackerman, A.L., Cresswell, P., 2004. Cellular mechanisms governing cross-
Each experiment was repeated at least twice on different groups presentation of exogenous antigens. Nat. Immunol. 5 (7), 678 – 684.
of mice from two immunizations. Anwar, A., Chandrasekaran, A., Lee Ng, M., Marques, E., August, J.T., 2005.
West Nile premembrane-envelope genetic vaccine encoded as a chimera
containing the transmembrane and cytoplasmic domains of a lysosome
Cytotoxic T-lymphocyte assay associated membrane protein: increased cellular concentration of the
transgene product, targeting to the MHC II compartment and enhanced
CTL assays were performed using the Cytotox 96 non- neutralizing antibody response. J. Virol. 332 (1), 66 – 77.
radioactive cytotoxicity assay kit (Promega Corporation, USA). Barros de Arruda, L., Chikhlikar, P.R., August, J.T., Marques, E.T., 2004. DNA
vaccine encoding human immunodeficiency virus-1 Gag, targeted to the
The pooled splenocytes (from two mice in the N-GST group major histocompatibility complex II compartment by lysosomal-associated
and three mice in each of the other groups) obtained at each membrane protein, elicits enhanced long-term memory response. Immunol
time point (days 1, 8, 15, and 22) after memory recall with pN 112 (1), 126 – 133.
were stimulated in vitro with each of the two peptides, N80 – 99 Benacerraf, B., 1978. A hypothesis to relate the specificity of T lymphocytes and
and N353 – 370, at a final concentration of 10 Ag/ml for 4 days. the activity of I region-specific Ir genes in macrophages and B lymphocytes.
J. Immunol. 120 (6), 1809 – 1812.
SP2/O target cells were pulsed overnight separately with 10 Ag/ Berzofsky, J.A., Pendleton, C.D., Clerici, M., Ahlers, J., Lucey, D.R., Putney,
ml of each of the two peptides, washed two times, resuspended S.D., Shearer, G.M., 1991. Construction of peptides encompassing multi-
in RPMI supplemented with 2% FCS, and seeded onto a 96- determinant clusters of human immunodeficiency virus envelope to induce
well U-bottom plate (1 104 cells/well in 50 Al of culture in vitro T cell responses in mice and humans of multiple MHC types. J. Clin.
Invest. 88 (3), 876 – 884.
medium) with an equal volume of pooled, in-vitro-stimulated
Billiau, A., Matthys, P., 2001. Modes of action of Freund’s adjuvants in
splenocytes from recalled mice (effector cells) at different experimental models of autoimmune diseases. J. Leukocyte Biol. 70 (6),
effector/target ratios, 20:1, 40:1, and 80:1. All incubations were 849 – 860.
done in quadruplicate. The negative control contained an equal Bonifaz, L., Bonnyay, D., Mahnke, K., Rivera, M., Nussenzweig, M.C.,
concentration of unrelated peptide, and controls for effector cell Steinman, R.M., 2002. Efficient targeting of protein antigen to the dendritic
spontaneous release, target spontaneous release, and target cell receptor DEC-205 in the steady state leads to antigen presentation on
major histocompatibility complex class I products and peripheral CD8+ T
maximum release were included in the assays. All the values cell tolerance. J. Exp. Med. 196 (12), 1627 – 1638.
were computed as the mean T standard deviations of the four Bonini, C., Lee, S.P., Riddell, S.R., Greenberg, P.D., 2001. Targeting antigen in
replicate assays after subtracting the average of the values mature dendritic cells for simultaneous stimulation of CD4+ and CD8+ T
obtained for the negative controls. The use of overlapping cells. J. Immunol. 166 (8), 5250 – 5257.
peptides for antiviral CD8 and CD4 T-cell responses has been Brode, S., Macary, P.A., 2004. Cross-presentation: dendritic cells and
macrophages bite off more than they can chew! Immunology 112 (3),
described (Maecker et al., 2001; Draenert et al., 2003). 345 – 351.
Brooks, A., Hartley, S., Kjer-Nielsen, L., Perera, J., Goodnow, C.C., Basten,
Prediction of T-cell binding motifs A., McCluskey, J., 1991. Class II-restricted presentation of an endoge-
nously derived immunodominant T-cell determinant of hen egg lyso-
zyme. Proc. Natl. Acad. Sci. U.S.A. 88 (8), 3290 – 3294.
BALB/c mouse H2d T-cell binding motifs of the 60
Brown, S.A., Stambas, J., Zhan, X., Slobod, K.S., Coleclough, C., Zirkel, A.,
overlapping N peptides were predicted by use of the Surman, S., White, S.W., Doherty, P.C., Hurwitz, J.L., 2003. Clustering of
PREDBALB/c class I (H2-Kd, -Ld, -Dd) and class II (I-Ed, -Ad) Th cell epitopes on exposed regions of HIV envelope despite defects in
models (Zhang et al., 2005). The PREDBALB/C system scores antibody activity. J. Immunol. 171 (8), 4140 – 4148.
the binding index of all immunogen nine-amino-acid sequences Bryant, P., Ploegh, H., 2004. Class II MHC peptide loading by the
to H2d molecules, based on quantitative matrices. professionals. Curr. Opin. Immunol. 16 (1), 96 – 102.
Chen, L., Jondal, M., 2004. Endolysosomal processing of exogenous antigen
into major histocompatibility complex class I-binding peptides. Scand. J.
Acknowledgments Immunol. 59 (6), 545 – 552.
Chen, J.W., Murphy, T.L., Willingham, M.C., Pastan, I., August, J.T., 1985.
This research has been funded in part by the National Identification of two lysosomal membrane glycoproteins. J. Cell Biol. 10
Institute of Allergy and Infectious Diseases, National Institutes (1), 85 – 95.
Chen, J.L., Stewart-Jones, G., Bossi, G., Lissin, N.M., Wooldridge, L., Choi,
of Health, Department of Health and Human Services, contract E.M., Held, G., Dunbar, P.R., Esnouf, R.M., Sami, M., Boulter, J.M.,
no. HHSN266200400085C, and by contracts from the Agency Rizkallah, P., Renner, C., Sewell, A., van der Merwe, P.A., Jakobsen, B.K.,
for Science, Technology and Research, Singapore to the Griffiths, G., Jones, E.Y., Cerundolo, V., 2005. Structural and kinetic basis
Division of Biomedical Sciences, Johns Hopkins in Singapore, for heightened immunogenicity of T cell vaccines. J. Exp. Med. 201 (8),
and Institute for Infocomm Research, Singapore. We thank A/P 1243 – 1255.
Chikhlikar, P., Barros de Arruda, L., Agrawal, S., Byrne, B., Guggino, W.,
Ng Mah Lee and A/P Charanjit Kaur (National University of August, J.T., Marques, E.T., 2004. Inverted terminal repeat sequences of
Singapore) for critical analysis of the IEM images and J.L. Koh adeno-associated virus enhance the antibody and CD8 (+) responses to a
(Institute for Infocomm Research) for computational analysis HIV-1 p55Gag/LAMP DNA vaccine chimera. Virology 323 (2), 220 – 232.
138 V. Gupta et al. / Virology 347 (2006) 127 – 139

Crotty, S., Ahmed, R., 2004. Immunological memory in humans. Semin. Lau, S.K., Woo, P.C., Wong, B.H., Tsoi, H.W., Woo, G.K., Poon, R.W., Chan,
Immunol. 16 (3), 197 – 203. K.H., Wei, W.I., Peiris, J.S., Yuen, K.Y., 2004. Detection of severe acute
Del Porto, P., Puntoriero, G., Scotta, C., Nicosia, A., Piccolella, E., 2002. High respiratory syndrome (SARS) coronavirus nucleocapsid protein in SARS
prevalence of hypervariable region 1-specific and-cross-reactive CD4 (+) T patients by enzyme-linked immunosorbent assay. J. Clin. Microbiol. 42 (7),
cells in HCV-infected individuals responsive to IFN-alpha treatment. 2884 – 2889.
Virology 269 (2), 313 – 324. Lautwein, A., Kraus, M., Reich, M., Burster, T., Brandenburg, J., Overkleeft,
Draenert, R., Altfeld, M., Brander, C., Basgoz, N., Corcoran, C., Wurcel, A.G., H.S., Schwarz, G., Kammer, W., Weber, E., Kalbacher, H., Nordheim, A.,
Stone, D.R., Kalams, S.A., Trocha, A., Addo, M.M., Goulder, P.J., Walker, Driessen, C., 2004. Human B lymphoblastoid cells contain distinct patterns
B.D., 2003. Comparison of overlapping peptide sets for detection of of cathepsin activity in endocytic compartments and regulate MHC class II
antiviral CD8 and CD4 T cell responses. J. Immunol. Methods 275 (12), transport in a cathepsin S-independent manner. J. Leukocyte Biol. 75 (5),
19 – 29. 844 – 855.
Geuze, H.J., 1998. The role of endosomes and lysosomes in MHC class II Lazarski, C.A., Chaves, F.A., Jenks, S.A., Wu, S., Richards, K.A., Weaver,
functioning. Immunol. Today 19 (6), 282 – 287. J.M., Sant, A.J., 2005. The kinetic stability of MHC Class II: peptide
Guarnieri, F.G., Arterburn, L.M., Penno, M.B., Cha, Y., August, J.T., 1993. The complexes is a key parameter that dictates immunodominance. Immunity
motif Tyr – X – X-hydrophobic residue mediates lysosomal membrane 23 (1), 29 – 40.
targeting of lysosome-associated membrane protein 1. J. Biol. Chem. 268 Lich, J.D., Elliott, J.F., Blum, J.S., 2000. Cytoplasmic processing is a
(3), 1941 – 1946. prerequisite for presentation of an endogenous antigen by major histocom-
Guermonprez, P., Saveanu, L., Kleijmeer, M., Davoust, J., Van Endert, P., patibility complex class II proteins. J. Exp. Med. 191 (9), 1513 – 1524.
Amigorena, S., 2003. ER-phagosome fusion defines an MHC class I cross- Lin, Y., et al., 2003. Identification of an epitope of SARS-coronavirus
presentation compartment in dendritic cells. Nature 425 (6956), 397 – 402. nucleocapsid protein. Cell Res. 13 (3), 141 – 145.
Houde, M., Bertholet, S., Gagnon, E., Brunet, S., Goyette, G., Laplante, A., Lu, Y., Raviprakash, K., Leao, I.C., Chikhlikar, P.R., Ewing, D., Anwar, A.,
Princiotta, M.F., Thibault, P., Sacks, D., Desjardins, M., 2003. Phagosomes Chougnet, C., Murphy, G., Hayes, C.G., August, T.J., Marques, E.T., 2003.
are competent organelles for antigen cross-presentation. Nature 425 (6956), Dengue 2 PreM-E/LAMP chimera targeted to the MHC class II compart-
402 – 406. ment elicits long-lasting neutralizing antibodies. Vaccine 21 (17 – 18),
Huang, L.R., Chiu, C.M., Yeh, S.H., Huang, W.H., Hsueh, P.R., Yang, W.Z., 2178 – 2189.
Yang, J.Y., Su, I.J., Chang, S.C., Chen, P.J., 2004a. Evaluation of antibody Maecker, H.T., Umetsu, D.T., DeKruyff, R.H., Levy, S., 1998. Cytotoxic T cell
responses against SARS coronaviral nucleocapsid or spike proteins by responses to DNA vaccination: dependence on antigen presentation via
immunoblotting or ELISA. J. Med. Virol. 73 (3), 338 – 346. class II MHC. J. Immunol. 161 (12), 6532 – 6536.
Huang, Q., Yu, L., Petros, A.M., Gunasekera, A., Liu, Z., Xu, N., Hajduk, P., Maecker, H.T., Dunn, H.S., Suni, M.A., Khatamzas, E., Pitcher, C.J., Bunde, T.,
Mack, J., Fesik, S.W., Olejniczak, E.T., 2004b. Structure of the N-terminal Persaud, N., Trigona, W., Fu, T.M., Sinclair, E., Bredt, B.M., McCune,
RNA-binding domain of the SARS CoV nucleocapsid protein. Biochem- J.M., Maino, V.C., Kern, F., Picker, L.J., 2001. Use of overlapping peptide
istry 43 (20), 6059 – 6063. mixtures as antigens for cytokine flow cytometry. J. Immunol. Methods 255
Imai, J., Hasegawa, H., Maruya, M., Koyasu, S., Yahara, I., 2004. Exogenous (1 – 2), 27 – 40.
antigens are processed through the endoplasmic reticulum-associated Marques, E.T., Chikhlikar, P., Barros de Arruda, L., Leao, I.C., Lu, Y., Wong,
degradation (ERAD) in cross-presentation by dendritic cells. Int. Immunol. J., Chen, J.S., Byrne, B., August, J.T., 2003. HIV-1 p55Gag encoded in the
17 (1), 45 – 53. lysosome-associated membrane protein-1 as a DNA plasmid vaccine
Kelso, A., Groves, P., 1997. A single peripheral CD8+ T cell can give rise to chimera is highly expressed, traffics to the major histocompatibility class
progeny expressing type 1 and/or type 2 cytokine genes and can retain its II compartment, and elicits enhanced immune responses. J. Biol. Chem. 278
multipotentiality through many cell divisions. Proc. Natl. Acad. Sci. U.S.A. (39), 37926 – 37936.
94 (15), 8070 – 8075. Martins, L.P., Lau, L.L., Asano, M.S., Ahmed, R., 1995. DNA vaccination
Kelso, A., Costelloe, E.O., Johnson, B.J., Groves, P., Buttigieg, K., Fitzpatrick, against persistent viral infection. Virology 69 (4), 2574 – 2582.
D.R., 2002. The genes for perforin, granzymes A – C and IFN-gamma are Masopust, D., Kaech, S.M., Wherry, E.J., Ahmed, R., 2004. The role of
differentially expressed in single CD8 (+) T cells during primary activation. programming in memory T-cell development. Curr. Opin. Immunol. 16 (2),
Int. Immunol. 14 (6), 605 – 613. 217 – 225.
Kessler, P.D., Podsakoff, G.M., Chen, X., McQuiston, S.A., Colosi, P.C., Moreno, J., Vignali, D.A., Nadimi, F., Fuchs, S., Adorini, L., Hammerling, G.J.,
Matelis, L.A., Kurtzman, G.J., Byrne, B.J., 1996. Gene delivery to skeletal 1991. Processing of an endogenous protein can generate MHC class II-
muscle results in sustained expression and systemic delivery of a restricted T cell determinants distinct from those derived from exogenous
therapeutic protein. Proc. Natl. Acad. Sci. U.S.A. 93 (24), 14082 – 14087. antigen. J. Immunol. 147 (10), 3306 – 3313.
Kessler, B.M., Glas, R., Ploegh, H.L., 2002. MHC class I antigen processing Murk, J.L., Lebbink, M.N., Humbel, B.M., Geerts, W.J., Griffith, J.M.,
regulated by cytosolic proteolysis—short cuts that alter peptide generation. Langenberg, D.M., Verreck, F.A., Verkleij, A.J., Koster, A.J., Geuze, H.J.,
Mol. Immunol. 39 (3 – 4), 171 – 179. Kleijmeer, M.J., 2004. 3-D structure of multilaminar lysosomes in antigen
Kim, S.K., DeMars, R., 2001. Epitope clusters in the major outer presenting cells reveals trapping of MHC II on the internal membranes.
membrane protein of Chlamydia trachomatis. Curr. Opin. Immunol. 13 Traffic 5 (12), 936 – 945.
(4), 429 – 436. Nair, S.K., Boczkowski, D., Morse, M., Cumming, R.I., Lyerly, H.K., Gilboa,
Kim, T.W., Lee, J.H., Hung, C.F., Peng, S., Roden, R., Wang, M.C., Viscidi, R., E., 1998. Induction of primary carcinoembryonic antigen (CEA)-specific
Tsai, Y.C., He, L., Chen, P.J., Boyd, D.A., Wu, T.C., 2004. Generation and cytotoxic T lymphocytes in vitro using human dendritic cells transfected
characterization of DNA vaccines targeting the nucleocapsid protein of with RNA. Nat. Biotechnol. 16 (4), 364 – 369.
severe acute respiratory syndrome coronavirus. J. Virol. 78 (9), 4638 – 4645. Nimmerjahn, F., Milosevic, S., Behrends, U., Jaffee, E.M., Pardoll, D.M.,
Kleijmeer, M.J., Raposo, G., Geuze, H.J., 1996. Characterization of MHC Bornkamm, G.W., Mautner, J., 2003. Major histocompatibility complex
class II compartments by immunoelectron microscopy. Methods 10 (2), class II-restricted presentation of a cytosolic antigen by autophagy. Eur. J.
191 – 207. Immunol. 33 (5), 1250 – 1259.
Kleijmeer, M.J., Morkowski, S., Griffith, J.M., Rudensky, A.Y., Geuze, H.J., Norbury, C.C., Princiotta, M.F., Bacik, I., Brutkiewicz, R.R., Wood, P., Elliott,
1997. Major histocompatibility complex class II compartments in human T., Bennink, J.R., Yewdell, J.W., 2001. Multiple antigen-specific processing
and mouse B lymphoblasts represent conventional endocytic compartments. pathways for activating naive CD8+ T cells in vivo. J. Immunol. 166 (7),
J. Cell Biol. 139 (3), 639 – 649. 4355 – 4362.
Kloetzel, P.M., 2004. Generation of major histocompatibility complex class I Oehen, S., Junt, T., López-Macı́as, C., Kramps, T.A., 2000. Antiviral protection
antigens: functional interplay between proteasomes and TPPII. Nat. after DNA vaccination is short lived and not enhanced by CpG DNA.
Immunol. 5 (7), 661 – 669. Immunology 99 (2), 163 – 169.
 V. Gupta et al. / Virology 347 (2006) 127 – 139 139

Paludan, C., Schmid, D., Landthaler, M., Vockerodt, M., Kube, D., Tuschl, T., Storni, T., Bachmann, M.F., 2004. Loading of MHC class I and II
Munz, C., 2005. Endogenous MHC class II processing of a viral nuclear presentation pathways by exogenous antigens: a quantitative in vivo
antigen after autophagy. Science 307 (5709), 593 – 596. comparison. J. Immunol. 172 (10), 6129 – 6135.
Pamer, E., Cresswell, P., 1998. Mechanisms of MHC class I-restricted antigen Su, Z., Vieweg, J., Weizer, A.Z., Dahm, P., Yancey, D., Turaga, V., Higgins, J.,
processing. Annu. Rev. Immunol. 16, 323 – 358. Boczkowski, D., Gilboa, E., Dannull, J., 2002. Enhanced induction of
Raviprakash, K., Marques, E., Ewing, D., Lu, Y., Phillips, I., Porter, K.R., telomerase-specific CD4 (+) T cells using dendritic cells transfected with
Kochel, T.J., August, T.J., Hayes, C.G., Murphy, G.S., 2001. Synergistic RNA encoding a chimeric gene product. Cancer Res. 62 (17), 5041 – 5048.
neutralizing antibody response to a dengue virus type 2 DNA vaccine by Sun, J.C., Williams, M.A., Bevan, M.J., 2004. CD4+ T cells are required for the
incorporation of lysosome-associated membrane protein sequences and use maintenance, not programming, of memory CD8+ T cells after acute
of plasmid expressing GM-CSF. Virol 290 (1), 74 – 82. infection. Nat. Immunol. 5 (9), 927 – 933.
Reimann, J., Schirmbeck, R., 1999. Alternative pathways for processing Surman, S., Lockey, T.D., Slobod, K.S., Jones, B., Riberdy, J.M., White, S.W.,
exogenous and endogenous antigens that can generate peptides for MHC Doherty, P.C., Hurwitz, J.L., 2001. Localization of CD4+ T cell epitope
class I-restricted presentation. Immunol. Rev. 172, 131 – 152. hotspots to exposed strands of HIV envelope glycoprotein suggests
Reis e Sousa, C., Germain, R.N., 1995. Major histocompatibility complex class structural influences on antigen processing. Proc. Natl. Acad. Sci. U.S.A.
I presentation of peptides derived from soluble exogenous antigen by a 98 (8), 4587 – 4592.
subset of cells engaged in phagocytosis. J. Exp. Med. 182 (3), 841 – 851. Tewari, M.K., Sinnathamby, G., Rajagopal, D., Eisenlohr, L.C., 2005. A
Ria, F., Gallard, A., Gabaglia, C.R., Guery, J.C., Sercarz, E.E., Adorini, L., 2004. cytosolic pathway for MHC class II-restricted antigen processing that is
Selection of similar naive T cell repertoires but induction of distinct T cell proteasome and TAP dependent. Nat. Immunol. 6 (3), 287 – 294.
responses by native and modified antigen. J. Immunol. 172 (6), 3447 – 3453. Varga, S.M., Wissinger, E.L., Braciale, T.J., 2000. The attachment (G)
Rocha, B., Tanchot, C., 2004. Towards a cellular definition of CD8+ T-cell glycoprotein of respiratory syncytial virus contains a single immuno-
memory: the role of CD4+ T-cell help in CD8+ T-cell responses. Curr. Opin. dominant epitope that elicits both Th1 and Th2 CD4+ T cell responses.
Immunol. 16 (3), 259 – 263. J. Immunol. 165 (11), 6487 – 6495.
Rowell, J.F., Ruff, A.L., Guarnieri, F.G., Staveley-O’Carroll, K., Lin, X., Tang, Villadangos, J.A., Bryant, R.A., Deussing, J., Driessen, C., 1999. Proteases
J., August, J.T., Siliciano, R.F., 1995. Lysosome-associated membrane involved in MHC class II antigen presentation. Immunol. Rev. 172,
protein-1-mediated targeting of the HIV-1 envelope protein to an 109 – 120.
endosomal/lysosomal compartment enhances its presentation to MHC class Wang, J.C., Livingstone, A.M., 2003. Cutting edge: CD4+ T cell help can be
II-restricted T cells. J. Immunol. 155 (4), 1818 – 1828. essential for primary CD8+ T cell responses in vivo. J. Immunol. 171 (12),
Ruff, A.L., Guarnieri, F.G., Staveley-O’Carroll, K., Siliciano, R.F., August, 6339 – 6343.
J.T., 1997. The enhanced immune response to the HIV gp160/LAMP Welsh, R.M., Selin, L.K., Szomolanyi-Tsuda, E., 2004. Immunological
chimeric gene product targeted to the lysosome membrane protein memory to viral infections. Annu. Rev. Immunol. 22, 711 – 743.
trafficking pathway. J. Biol. Chem. 272 (13), 8671 – 8678. Wu, T.C., Guarnieri, F.G., Staveley-O’Carroll, K.F., Viscidi, R.P., Levitsky,
Rush, C., Mitchell, T., Garside, P., 2002. Efficient priming of CD4+ and CD8+ T H.I., Hedrick, L., Cho, K.R., August, J.T., Pardoll, D.M., 1995.
cells by DNA vaccination depends on appropriate targeting of sufficient Engineering an intracellular pathway for major histocompatibility complex
levels of immunologically relevant antigen to appropriate processing class II presentation of antigens. Proc. Natl. Acad. Sci. U.S.A. 92 (25),
pathways. J. Immunol. 169 (9), 4951 – 4960. 11671 – 11675.
Schirmbeck, R., Wild, J., Reimann, J., 1998. Similar as well as distinct MHC Yang, Z.Y., Kong, W.P., Huang, Y., Roberts, A., Murphy, B.R., Subbarao, K.,
class I-binding peptides are generated by exogenous and endogenous Nabel, G.J., 2004. A DNA vaccine induces SARS coronavirus neutraliza-
processing of hepatitis B virus surface antigen. Eur. J. Immunol. 28 (12), tion and protective immunity in mice. Nature 428 (6982), 561 – 564.
4149 – 4161. Yewdell, J.W., Bennink, J.R., 2001. Cut and trim: generating MHC class I
Shankar, P., Fabry, J.A., Fong, D.M., Lieberman, J., 1996. Three regions of peptide ligands. Curr. Opin. Immunol. 13 (1), 13 – 18.
HIV-1 gp160 contain clusters of immunodominant CTL epitopes. Immunol. Yip, H.C., Karulin, A.Y., Tary-Lehmann, M., Hesse, M.D., Radeke, H.,
Lett. 52 (1), 23 – 30. Heeger, P.S., Trezza, R.P., Heinzel, F.P., Forsthuber, T., Lehmann, P.V.,
Shibaki, A., Katz, S.I., 2002. Induction of skewed Th1/Th2 T-cell differenti- 1999. Adjuvant-guided type-1 and type-2 immunity: infectious/nonin-
ation via subcutaneous immunization with Freund’s adjuvant. Exp. fectious dichotomy defines the class of response. J. Immunol. 162 (7),
Dermatol. 11 (2), 126 – 134. 3942 – 3949.
Shirai, M., Arichi, T., Chen, M., Masaki, T., Nishioka, M., Ikeda, K., Zhang, G.L., Srinivasan, K.N., Veeramani, A., August, J.T., Brusic, V., 2005.
Takahashi, H., Enomoto, N., Saito, T., Major, M.E., Nakazawa, T., PREDBALB/c: a system for prediction of peptide binding to the H2d
Akatsuka, T., Feinstone, S.M., Berzofsky, J.A., 1999. T cell recognition molecules, a haplotype of the BALB/c mouse. Nucleic Acids Res. 33,
of hypervariable region-1 from hepatitis C virus envelope protein with W180 – W183.
multiple class II MHC molecules in mice and humans: preferential help for Zhao, P., Cao, J., Zhao, L.J., Qin, Z.L., Ke, J.S., Pan, W., Ren, H., Yu, J.G., Qi,
induction of antibodies to the hypervariable region. J. Immunol. 162 (1), Z.T., 2005. Immune responses against SARS-coronavirus nucleocapsid
568 – 576. protein induced by DNA vaccine. Virology 331 (1), 128 – 135.
Srinivasan, K.N., Zhang, G., Khan, A.M., August, J.T., Brusic, V., 2004. Zhu, M.S., Pan, Y., Chen, H.Q., Shen, Y., Wang, X.C., Sun, Y.J., Tao, K.H.,
Prediction of Class I T-cell epitopes: evidence of presence of immunolog- 2004. Induction of SARS-nucleoprotein-specific immune response by use
ical hot spots inside antigens. Bioinformatics 20 (Suppl. 1), I297 – I302. of DNA vaccine. Immunol. Lett. 92 (3), 237 – 243.