Translating innate response into long-lasting antibody response by the intrinsic antigen-adjuvant properties of papaya mosaic virus.

Abstract

Identifying the properties of a molecule involved in the efficient activation of the innate and adaptive immune responses that lead to long-lasting immunity is crucial for vaccine and adjuvant development. Here we show that the papaya mosaic virus (PapMV) is recognized by the immune system as a pathogen-associated molecular pattern (PAMP) and as an antigen in mice (Pamptigen). A single immunization of PapMV without added adjuvant efficiently induced both cellular and specific long-lasting antibody responses. PapMV also efficiently activated innate immune responses, as shown by the induction of lipid raft aggregation, secretion of pro-inflammatory cytokines, up-regulation of co-stimulatory molecules on dendritic cells and macrophages, and long-lasting adjuvant effects upon the specific antibody responses to model antigens. PapMV mixed with Salmonella enterica serovar Typhi (S. typhi) outer membrane protein C increased its protective capacity against challenge with S. typhi, revealing the intrinsic adjuvant properties of PapMV in the induction of immunity. Antigen-presenting cells loaded with PapMV efficiently induced antibody responses in vivo, which may link the innate and adaptive responses observed. PapMV recognition as a Pamptigen might be translated into long-lasting antibody responses and protection observed. These properties could be used in the development of new vaccine platforms.

Full text

Page 1

Translating innate response into long-lasting antibody response
by the intrinsic antigen-adjuvant properties of papaya mosaic virus
Elizabeth Acosta-Ramı ´rez,1,? Rebeca
Pe ´rez-Flores,1,? Nathalie Majeau,2
Rodolfo Pastelin-Palacios,3Cristina
Gil-Cruz,1,? Maricela Ramı ´rez-
Saldan ˜a,1,? Nataly Manjarrez-
Ordun ˜o,4Luisa Cervantes-
Barraga ´n,1,? Leopoldo Santos-
Argumedo,4Leopoldo Flores-
Romo,5Ingeborg Becker,6Armando
Isibasi,1Denis Leclerc2and
Constantino Lo ´pez-Macı ´as1
1Medical Research Unit on Immunochemistry,
Specialties Hospital, National Medical Centre
‘Siglo XXI’. Mexican Institute for Social Secu-
rity (IMSS), Mexico City,2Infectology Research
Centre, Laval University, Quebec, Canada,
3Faculty of Chemistry, National Autonomous
University of Mexico (UNAM), Mexico City,
4Molecular Biomedicine Department, Center
for Advance Studies and Research (CINVE-
STAV) of the National Polythechnic Institute
(IPN), Mexico City,5Department of Cell Biol-
ogy, CINVESTAV-IPN, Mexico City, and
6Department of Experimental Medicine,
Faculty of Medicine, UNAM, Mexico City
doi:10.1111/j.1365-2567.2007.02753.x
Received 6 June 2007; revised 11 October
2007; accepted 12 October 2007.
?PhD program on Biomedical Sciences,
UNAM, Mexico City.
?PhD program on Immunology, National
School of Biological Sciences (ENCB) IPN,
Mexico City.
Correspondence: Dr C. Lo ´pez-Macı ´as,
UIM en Inmunoquı ´mica, Coordinacio ´n
de Investigacio ´n en Salud, PO
Box A047, C.P. 06703, Me ´xico D.F.
Email: constantino@sminmunologia.org;
constantino.lopez@imss.gob.mx
Senior author: Constantino Lo ´pez-Macı ´as
Summary
Identifying the properties of a molecule involved in the efficient activation
of the innate and adaptive immune responses that lead to long-lasting
immunity is crucial for vaccine and adjuvant development. Here we show
that the papaya mosaic virus (PapMV) is recognized by the immune sys-
tem as a pathogen-associated molecular pattern (PAMP) and as an anti-
gen in mice (Pamptigen). A single immunization of PapMV without
added adjuvant efficiently induced both cellular and specific long-lasting
antibody responses. PapMV also efficiently activated innate immune
responses, as shown by the induction of lipid raft aggregation, secretion
of pro-inflammatory cytokines, up-regulation of co-stimulatory molecules
on dendritic cells and macrophages, and long-lasting adjuvant effects
upon the specific antibody responses to model antigens. PapMV mixed
with Salmonella enterica serovar Typhi (S. typhi) outer membrane protein
C increased its protective capacity against challenge with S. typhi, reveal-
ing the intrinsic adjuvant properties of PapMV in the induction of immu-
nity. Antigen-presenting cells loaded with PapMV efficiently induced
antibody responses in vivo, which may link the innate and adaptive
responses observed. PapMV recognition as a Pamptigen might be trans-
lated into long-lasting antibody responses and protection observed. These
properties could be used in the development of new vaccine platforms.
Keywords: adjuvants; antigens; memory; papaya mosaic virus; vaccination
Abbreviations: APC, antigen-presenting cell; BCR, B-cell receptor; BMDC, bone marrow-derived dendritic cells; BMDM, bone marrow-
derived macrophages; CFA, complete Freund’s adjuvant; CFSE, 5(6)-carboxyfluorescein diacetate N-succinimidyl ester; CP, coat protein;
CTB, cholera toxin B subunit; DC, dendritic cell; DMEM, Dubecco’s modified Eagle’s minimal essential medium; DTH, delayed-type
hypersensitivity; ELISA, enzyme-linked immunosorbent assay; FBS, fetal bovine serum; FCS, fetal calf serum; FITC, fluorescein
isothiocyanate; HEL, hen egg- white lysozyme; i.p., intraperitoneal; IFA, incomplete Freund’s adjuvant; IL, interleukin; LD50,50% lethal
dose; LPS, lipopolysaccharide; MHC class II, major histocompatibility complex class II; OmpC, outer membrane protein C; OVA,
ovalbumin; PAMP, pathogen-associated molecular pattern; Pamptigen, pathogen-associated molecular pattern and antigen; PapMV,
papaya mosaic virus; PBS, phosphate-buffered saline; PE, phycoerythrin; PerCP, peridinin chlorophyll protein; PRR, pattern recognition
receptor; s.c., subcutaneous; SD, standard deviation; TCR, T-cell receptor; TLR, Toll-like receptor; TNF-a, tumour necrosis factor-a.
? 2007 The Authors Journal compilation ? 2007 Blackwell Publishing Ltd, Immunology
1
IMMUNOLOGY ORIGINAL ARTICLE

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Introduction
The onset of the immune response against a given patho-
gen depends on the recognition of microbial components
by non-clonal (innate) and clonal (adaptive) receptors
expressed on the appropriate cells. Innate receptors
known as pattern-recognition receptors (PRR) allow the
recognition of microbial products known as pathogen-
associated molecular patterns (PAMP).1In contrast, the
adaptive immune response is characterized by clonally
expressed B-cell receptor (BCR) or T-cell receptor (TCR)
recognition of molecules, which define the antigenicity of
these molecules.2The outcome of the immune response
activated through these receptors is that protection or
immunity (which may last for many years or a lifetime)
is generated against the infecting pathogen3,4and this
phenomenon is the basis of vaccination. However, the
immunological mechanisms mediating the induction of
long-term immunity remain obscure.
High, long-lasting protective
induced mainly after pathogenic infection, or by vaccina-
tion with an attenuated pathogen or with purified anti-
gens mixed with adjuvants.5,6Two key factors achieve
this response. First, antigen immunogenicity depends on
the antigen molecular nature and structure.2Thus, viruses
that infect mammals exhibit a quasicrystalline, highly
organized surface with a regular array of epitopes that
efficiently cross-link epitope-specific membrane immuno-
globulins on B cells, inducing a T-cell-independent anti-
body response.7
Second, the presence of adjuvants
increases the magnitude and duration of the immune
response by activating the innate immune system.8The
adjuvant effect of most molecules depends on activation
of the innate immune response through PRR.9This opens
the possibility that pattern recognition through the innate
immune system could be responsible for shaping the
long-lasting protective response induced by pathogens or
vaccines.
Plant viruses are effective tools for antigen expression.10
Unlike conventional live attenuated or recombinant vac-
cines, plant viruses are considered non-pathogenic for
mammals10
and fulfilthe
described for mammal-infecting viruses. Thus, they repre-
sent an excellent non-infectious model for using to study
immunogenicity and adjuvanticity. Our experimental
model is the papaya mosaic virus (PapMV), a potexvirus
that infects Carica papaya (papaya) leaves.11,12The Pap-
MV virion is a flexible rod, 500 nm long and 15 nm in
diameter, composed of 1400 subunits of the viral coat
protein (CP) assembled around the genomic positive
single-stranded RNA.13In this study, we show that a
single immunization with PapMV in the absence of added
adjuvant efficiently induced both cellular and specific
long-lasting antibody responses. In addition, PapMV
efficiently activated innate immune responses and, when
antibody titresare
immunogenicproperties
used as an adjuvant for model antigens or for an experi-
mental vaccine, promoted a long-lasting specific antibody
response and increased the protective capacity of the
experimental vaccine. We propose that this strong
immunogenicity shown by PapMV is the consequence of
its recognition both as a PAMP and as an antigen [patho-
gen-associated molecular pattern and antigen (Pampti-
gen)] by the immune system, thus translating the innate
immune recognition into long-lasting antibody responses
and protection. These properties could be used in the
development of new vaccine platforms to support the
induction of long-lasting immunity.
Materials and methods
Bacterial strains
The wild-type S. typhi strain was obtained from the
American Type Culture Collection 9993 (ATCC, Mana-
ssas, VA). Isogenic Salmonella mutant strain STYF302
(DompF,Kanamycin resistance
OmpC, has been described previously.14,15
(KmR)), expressing
Antigens
PapMV was purified as described previously.16Lipopoly-
saccharide (LPS)-free ovalbumin (OVA) grade VI was
from Sigma-Aldrich (St Louis, MO, USA). Hen egg-white
lysozyme (HEL) was from Research Organics Inc. (Cleve-
land, OH, USA). LPS was from Escherichia coli O111:B4
(Sigma-Aldrich). Outer membrane protein C (OmpC)
was purified from S. typhi STYC302 (DompF, KmR), as
described previously.15
Mice
BALB/c (Harlan, Mexico City, Mexico) and C3H/HeJ17
mice, 6–8 weeks of age, were housed in the animal facilities
of the Experimental Medicine Department, Faculty of
Medicine, UNAM, and were cared for in conformity with
good laboratory practice guidelines. These studies were
reviewed and approved by the IMSS National Scientific
Research Committee (project no. 2005-785-016).
Immunizations
Groups of mice were immunized intraperitoneally (i.p.)
on day 0 with 30 lg of PapMV diluted in sterile isotonic
saline solution (saline) to a total volume of 0?5 ml. To
study the effects of adjuvant, groups of mice were immu-
nized i.p. on day 0 with 2 mg of OVA or HEL alone, or
with 30 lg of PapMV and 2 mg of OVA or HEL in com-
plete Freund’s adjuvant (CFA) 1:1 (v/v) or with 5 lg of
LPS from E. coli O111:B4 (Sigma-Aldrich). To study the
adjuvant effect of PapMV in the anti-OmpC response,
2
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mice were immunized i.p. on day 0 with 10 lg of OmpC
alone or with 30 lg of PapMV and 10 lg of OmpC in
incomplete Freund’s adjuvant (IFA) 1:1 (v/v). On day 15,
mice received 10 lg of OmpC i.p. without adjuvant.
Control mice were injected with saline or with 30 lg of
PapMV only. Blood samples were collected at various
time-points, as indicated in each figure. Individual serum
samples were stored at )20? until analysis. The number
of mice used in the experiment is indicated in the figure
legends.
Determination of antibody titres by enzyme-linked
immunosorbent assay
High-binding 96-well polystyrene plates (Corning, New
York, NY, USA) were coated with 1 lg/ml of PapMV,
10 lg/ml of OmpC, 100 lg/ml of HEL or 150 lg/ml of
OVA in 0?1 M carbonate–bicarbonate buffer (pH 9?5).
Enzyme-linked immunosorbent assay (ELISA) was per-
formed as described previously.15Antibody titres are
given as –log2 dilution · 40 or as the inverse of serum
dilution. A positive titre was defined as three standard
deviations (3 SD) above the mean value of the negative
control.
Delayed-type hypersensitivity test
BALB/c mice were immunized subcutaneously (s.c.) in
the footpad with 30 lg of wild-type PapMV, 30 lg of
ultraviolet light-inactivated PapMV, buffer (Tris–HCl, pH
6?8) or sterile pyrogen-free saline solution. Seven days
after immunization, mice were challenged by s.c. injection
of 3 lg of PapMV into the right ear. Ear thickness was
measured before the challenge and 24 hr after injection
using a digital micrometer (Mituyoto, Tokyo, Japan).
Generation of bone marrow-derived macrophages
Bone
obtained from the femurs of BALB/c mice (6–8 weeks of
age) and cultured in Dulbecco’s modified Eagle’s minimal
essential medium (DMEM) with bone marrow medium
[30% L cell-conditioned medium, 20% heat-inactivated
fetal bovine serum (FBS) and 50% DMEM)], as described
previously.18
marrow-derived macrophages(BMDM)were
Generation of bone marrow-derived dendritic cells
Bone marrow-derived dendritic cells (BMDC) were gener-
ated by 6 days of culture of BALB/c bone marrow cells
with granulocyte–macrophage colony-stimulating factor
(GM-CSF)-containing supernatant from the cell line
X63-GM-CSF (kindly provided by Dr Antonius Rolink,
University of Basel, Basel, Switzerland). Dendritic cells
(DC) were further purified using Optiprep density-gradi-
ent centrifugation (Sigma-Aldrich Co., Basel, Switzer-
land). CD11c+B220)cells were more than 95% pure
when analysed by flow cytometry.
Peritoneal macrophage purification
Thioglycolate-elicited macrophages were collected from
the peritoneal cavity of BALB/c mice, cultured at
1 · 106cells per well in DMEM (containing 10% FCS,
L-glutamine 2 mM, penicillin 100 U/ml, streptomycin
100 lg/ml) overnight at 37?. Non-adherent cells were
removed by washing with cold phosphate-buffered saline
(PBS).
Lipid raft aggregation
Ten-thousand BMDM were cultured on sterile circular
slides and stimulated for 30 min with 1 lg/ml of
PapMV in 100 ll of DMEM. After the stimulus was
removed, the slides were washed twice with PBS con-
taining 2% FBS and once with PBS alone. The cells were
fixed using 3% p-formaldehyde and incubated at 4? for
20 min. Cells were washed as described above. Cholera
toxin B subunit (CTB) coupled to fluorescein isothiocya-
nate (FITC) (CTB–FITC, 1:500; Sigma-Aldrich) was
added and the slides were incubated in the dark at
room temperature.Non-specific
blocked with goat serum (1:100 dilution) for 1 hr at
room temperature. After incubation, the slides were
washed three times with PBS and then rat anti-PapMV
(1:100 dilution) was added. The slides were incubated
overnight at 4?. As an isotype control, mouse anti-
PapMV (1:100) was used. The slides were washed three
times with PBS and incubated for 1 hr at room temper-
ature with tetramethylrhodamine isothiocyanate-tagged
anti-rat IgG (1:200). The slides were washed three times
with PBS, mounted and assessed using a confocal micro-
scope (Zeiss Axiovert 100 M microscope with
expert mode software; Carl Zeiss, Aalen, Germany).
binding siteswere
LSM510
BMDM, peritoneal macrophage and BMDC stimulation,
and determination of cytokine production
One-million BMDM, peritoneal macrophages or BMDC
were stimulated with 1 lg/ml of PapMV or with 100 ng/ml
of LPS from E. coli O111:B4 (Sigma-Aldrich). Unstim-
ulated cells were used as a control. At the time-points
indicated in the graphs, the culture supernatant was har-
vested and stored at )70? until analysis. Tumour necrosis
factor-a (TNF-a) and interleukin (IL)-6 were quantified
by ELISA (all OptEIA sets; BD Pharmingen, San Diego,
CA, USA), following the manufacturer’s instructions. The
concentration of final reaction product was determined at
450 nm in an ELISA plate reader (Dynex Technologies
MRII, Chantilly, VA) with BIOLINX 2.22 software (Dynex
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Strong immunogenicity and adjuvanticity of papaya mosaic virus

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Technologies). The detection limit was 15?6 pg/ml for
both cytokines. Mouse interferon-a (IFN-a) concentra-
tion in cell culture supernatants was measured using
ELISA (PBL Biomedical Laboratories, NJ, USA) according
to the manufacturer’s instructions.
In vivo DC, macrophage and B-lymphocyte activation
assay
BALB/c mice (7 weeks of age) were immunized i.p. with
30 lg of PapMV and 50 lg of Poly I:C suspended
respectively in 500 ll of saline. Saline (500 ll) was used
as a control, and 24 hr after immunization lymph nodes
and spleens were obtained. Cell suspensions were pre-
pared from these organs, and flow cytometry staining
was performed using phycoerythrin (PE)–CD11c, allo-
phycocyanin–CD11b, B220–peridinin chlorophyll protein
(PerCP), FITC–CD80, FITC–CD86, FITC–CD40, FITC–
CD69 and FITC–major histocompatibility complex class
II (MHC class II) antibodies (all purchased from BD
Pharmingen).
Transference of BMDM
BMDM were pulsed with 30 lg/ml of PapMV in 5 ml of
DMEM containing 10% FBS and incubated for 3 hr at
37?. The supernatant was removed, the cells were washed
four times with PBS, and, before harvesting, the last cell
wash was collected. Totals of 104, 105and 106harvested
cells were immediately transferred i.v. into BALB/c mice
in a final volume of 300 ll. Culture supernatant, the last
cell wash supernatant, 105unstimulated BMDM and
30 lg of PapMV were suspended in a final volume of
300 ll and transferred i.v. into the control mice. The
numbers of mice used are indicated in the figure legends.
Protection assay
BALB/c mice were immunized i.p. on day 0 with 10 lg of
OmpC or 10 lg of OmpC that had been incubated previ-
ously for 1 hr with 30 lg of PapMV at 4?. A boost on
day 15 was performed using 10 lg of OmpC only. Con-
trol mice were injected with saline or 30 lg PapMV
alone. The challenge was performed on day 21 with 100
and 500 lethal dose 50% (LD50) of 5% mucin-suspended
S. typhi, and the survival rate was registered for 10 days
after the challenge, as described previously.19The num-
bers of mice used are indicated in the figure legends.
B-cell activation and proliferation
One-million splenic B cells per well were incubated
with PapMV (1 lg/ml) and harvested after 18 hr to
assess the activation markers, and were harvested again
after 80 hr to assess cell differentiation into plasma
cells. LPS (10 lg/ml) from E. coli O111:B4 (Sigma-
Aldrich) was used as a positive control. Flow cytometry
staining was performed with CD44–FITC, MHC class
II–PE, CD86–FITC, CD22–PE, CD138–PE and B220–all-
ophycocyanin antibodies (BD Pharmingen). One-million
cells were incubated for 15 min with a mixture of fluo-
rescein-, PE- and allophycocyanin-conjugated antibodies.
Cell proliferation was tested using fifty-million splenic
cells suspended in 1 ml of PBS containing 1 ll of
a 1 mM solution of 5(6)-carboxyfluorescein diacetate
N-succinimidyl ester (CFSE) (Molecular Probes, Eugene,
OR, USA). Cells were stimulated as described above.
Data were acquired using a FACSCalibur (Becton Dick-
inson, San Jose, CA, USA) and analysed using CELLQUEST
software (Becton Dickinson).
Results
PapMV induces a long-lasting antibody immune
response
B-cell activation is facilitated by antigenic organization
and molecular repetitiveness (more than 20 subunits), as
shown in a paracrystalline array.20PapMV virions contain
about 1400 copies of a single CP arranged in a helical
form.21We immunized BALB/c mice i.p. with 30 lg of
PapMV and measured PapMV-specific antibody titres for
1 year. During the primary antibody response on day 8,
we observed a 1024-fold increase in IgM titre and a 256-
fold increase in IgG titre (Fig. 1a). In the absence of
boosting, the IgM response decreased to the basal level on
day 20 and remained there until day 365. In contrast, the
IgG titre reached its maximum, a 1024-fold increase,
around day 21 and remained at this level until the end of
the experiment 1 year later. PapMV induced production
of the four subclasses of IgG. The antibody titres of the
IgG subclasses were observed first between days 4 and 12
and all reached their maximum values around day 20,
where they remained until day 365 (Fig. 1a). Similar
results were obtained when 50 lg of PapMV was used for
immunization (data not shown). In addition, i.v. and s.c.
immunization with PapMV induced similar antibody
kinetics (data not shown). This antibody response was
observed for 1 year after a single immunization without
adjuvants.
PapMV did not induce naı ¨ve B-lymphocyte prolifera-
tion in vitro (Fig. 2b); however, it induced minor up-
regulation of activation markers such as CD44, CD86,
CD22 and MHC class II (Fig. 2a). Additionally, PapMV
induced in vivo up-regulation of CD69 and CD40 on B
cells (Fig. 3). PapMV did not induce B-cell differentia-
tion into plasma cells in vitro (Fig. 2c). The experiments
were repeated by adding IL-4 to the PapMV stimulus or
by using 30 or 50 lg/ml of PapMV (data not shown),
confirming that PapMV did not activate B cells from
4
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E. Acosta-Ramı ´rez et al.

Page 5

naı ¨ve mice and did not prepare B cells for activation by
IL-4. We conclude that PapMV is not a thymus-inde-
pendent type 1 antigen. However, it induces some up-
regulation of activation marker expression levels in B
cells.
To determine whether the antibody response observed
was influenced by the presence of undetected traces of
LPS or polysaccharides,22,23which could have contami-
nated the PapMV preparation during purification, C3H/
HeJ mice [an LPS hyporesponsive Toll-like receptor 4
(TLR-4) mutant strain] were immunized with 30 lg of
PapMV. IgM and IgG titre kinetics were similar to those
observed in BALB/c mice (data not shown). Thus, unde-
tected contamination with TLR-4 ligands that could be
present in the PapMV did not contribute to the antibody
response observed. These results show that the PapMV is
a strong immunogenic particle able to induce a sustained
and specific long-lasting antibody response comprising all
IgG subclasses.
PapMV-induced T-cell responses
The presence of all IgG subclasses revealed that T cells
must help in the response induced by PapMV. A delayed-
type hypersensitivity (DTH) test was performed to test
thecapacityofPapMV
responses. Groups of five BALB/c mice were immunized
s.c. with 30 lg of intact or UV-inactivated PapMV.
UV-inactivated PapMV was used to control the influence
of virus viability in the response observed, even though
the plant virus replication machinery is not compatible
with mammalian systems and plant viruses are non-
pathogenic in mammals.10PapMV and UV-inactivated
PapMV induced a strong DTH response compared with
toinducesystemicT-cell
0 8 16 24 50 100 150 200 300 400 32
0 8 16 24 50 100 150 200 300 400
32
0816 24 50 100150200300 40032
0
2
4
6
8
10
12
14
0
2
4
6
8
10
12
14
0
2
4
6
8
10
12
14
Days after immunization
Antibody titre (–log2 × 40)
(a)
0
2
4
6
8
01
2
1
41
0
2
4
6
8
0 1
2 1
4 1
PapMV
Saline
IgG2b IgG3
IgG2a
IgG IgM
08 162432
0
2
4
6
8
01
21
41
50 100150 200 300 4000
8
16 24 32 50 100 150 200 300 400
16 24 32 50 100 150 200 300 400
Saline
Buffer
UV
PapMV
PapMV
DTH (μm)
**
**
(b)
140
120
100
80
60
40
20
0
08
Figure 1. Papaya mosaic virus (PapMV) induces T-cell and long-lasting antibody immune responses. (a) Titres of anti-PapMV IgM and IgG,
and of IgG subclasses IgG1, IgG2a, IgG2b and IgG3, were measured in female BALB/c mice immunized intraperitoneally (i.p.) with 30 lg of
PapMV or with saline. Blood samples were collected at the time-points depicted. Results are expressed as the mean ± standard deviation (SD) of
five mice per group. Data correspond to one representative result from five independent experiments. (b) T-cell responses were assessed by the
delayed-type hypersensitivity (DTH) test. Groups of five female BALB/c mice were immunized subcutaneously (s.c.) in the footpad with 30 lg of
wild-type PapMV or with 30 lg of ultraviolet light (UV) light-inactivated PapMV. Buffer (Tris–HCl, pH 6?8) alone or saline was used as a con-
trol. On day 7, the mice were challenged with 3 lg of PapMV administered s.c. in the ear. Ear thickness was measured using a micrometer before
and 24 hr after injection. Means were analysed by the Student’s t-test. Significant differences are indicated by asterisks: **P < 0?001. One repre-
sentative result from two independent experiments is shown.
? 2007 The Authors Journal compilation ? 2007 Blackwell Publishing Ltd, Immunology
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Strong immunogenicity and adjuvanticity of papaya mosaic virus

Page 6

the control (Fig. 1b). Thus, PapMV induced a strong
T-cell response in addition to the sustained long-lasting
antibody response. Taken together, these results show that
PapMV is a highly immunogenic antigen for both the
humoral and cellular immune responses.
PapMV induces the aggregation of lipid rafts
on BMDM
During the molecular recognition process on antigen-
presenting cells (APC), a cluster of surface molecules
present in lipid raft aggregates, allowing the induction of a
signalling cascade to induce cell activation.24To determine
whether PapMV induces lipid raft aggregation, murine
BMDM were stimulated with 1 lg/ml of PapMV for
30 min. The cells were then stained with cholera toxin
subunit B coupled with FITC to target the lipid raft-
resident glycolipid, GM1. PapMV stimulated the BMDM,
which showed an increased aggregation of lipid rafts.
Moreover, on these cells, PapMV co-localized with these
aggregated lipid rafts (Fig. 4a panel ii). By contrast, lipid
raft aggregation was not observed in unstimulated cells
(Fig. 4a panel i). Aggregation was manifested as the accu-
mulation of lipid rafts in localized regions of the stimulated
cells, whereas FITC staining in unstimulated cells was
homogenous around the cell periphery. These results
suggest that PapMV is recognized at the surface of BMDM.
PapMV induces pro-inflammatory cytokine secretion
in APC
To determine whether the lipid raft aggregation induced
by PapMV is associated with cell activation, we measured
cytokine secretion after PapMV stimulation. BMDM were
stimulated with 0?01, 0?1 (data not shown) or 1 lg/ml of
PapMV, and the secretion of TNF-a and IL-6 was mea-
sured in the culture supernatants. As a control, 100 ng/ml
of E. coli UHO111 LPS was used. Only stimulation with
1 lg/ml of PapMV induced IL-6 secretion in BMDM
12 hr after the initial stimulation. The maximum amount
of IL-6 was detected at 50 hr, after which time IL-6 con-
centrations intheculture
(Fig. 4c). TNF-a secretion was detected in culture super-
natants 4 hr after stimulation with 1 lg/ml of PapMV,
and an increased amount of TNF-a was present in cell
supernatants during the 24 hr of the experiment (Fig. 4c).
Thus, in addition to lipid raft aggregation, PapMV
induces the secretion of pro-inflammatory cytokines in
BMDM. As DC are an important link between innate and
adaptive immune responses, we evaluated the production
of IL-6 and TNF-a by BMDC pulsed with PapMV. Both
cytokines were observed in BMDC culture supernatants
18 and 24 hr after PapMV stimulation (Fig. 4d). Because
type I IFN is suggested to contribute to the generation of
a virus-specific adaptive immune response, we measured
supernatantsdecreased
CFSE low cells (%)
Medium 2·27
0·56
15·65
PapMV 2·87
LPS 75·15
(b)
CFSE
Max (%)
Max (%)
Max (%)
Max (%)
Max (%)
(a)
MHC II CD22
CD44 CD86
100
80
60
40
20
0
100
100
80
60
40
20
0
100
100
80
60
40
20
0
100
100
80
60
40
20
0
100
100
80
60
40
20
0
100
(c)
LPS
PapMV
B220
CD138
101
102
103
101
102
103
101
102
103
100
101
101
102
102
103
103
104
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
101
102
103
Figure 2. Papaya mosaic virus (PapMV) did not induce in vitro naı ¨ve B-lymphocyte proliferation; however, it induced a small shift in the
expression of activation markers on these cells. Flow cytometric analysis of B cells was performed after stimulation with PapMV. (a) Induction of
B-cell activation markers. (b) B-cell proliferation. Naı ¨ve B cells were pulsed after 18 hr with 100 lg/ml of lipopolysaccharide (LPS) (grey solid
line) or with 1 lg/ml of PapMV (black solid line); non-pulsed cells are shown by shading. (c) To assess B-cell differentiation into plasma cells
induced by PapMV, cells were stimulated with the virus for 80 hr and stained for B220 and CD138. One representative result from three experi-
ments is shown. CFSE, 5(6)-carboxyfluorescein diacetate N-succinimidyl ester.
6
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Page 7

IFN-a production by macrophages and DC after PapMV
stimulation. Peritoneal macrophages and BMDC were
pulsed with PapMV, and, after 24 hr of stimulation, IFN-a
was measured in the cell supernatants. Peritoneal macro-
phages did not produce IFN-a after PapMV stimulation
(Fig. 4c), whereas PapMV-stimulated BMDC produced
this cytokine more efficiently than did BMDC stimulated
with LPS (Fig. 4d).
BMDM bearing PapMV participate in the induction
of the specific antibody response
APC can directly deliver antigen to B cells for the induc-
tion of the antibody response.25,26To address the contri-
bution of APC in the induction of the antibody responses
against PapMV, BMDM were cultured with 30 lg/ml of
PapMV for 3 hr. After incubation, cells were injected i.v.
into BALB/c mice at one of three doses (104, 105or 106
cells). As the control for the free virus, the cell culture
supernatant from the PapMV-stimulated cells and the last
wash supernatant of these cells were independently
injected i.v. in mice. As a positive control, mice were
injected i.v. with 30 lg of PapMV, and 105non-pulsed
BMDM were transferred as a control (Fig. 4b). Neither
104nor 105PapMV-pulsed cells induced a specific IgG
titre. By contrast, transfer of 106PapMV-stimulated cells
induced a specific antibody titre on day 12 after transfer.
The titre increased 16-fold around day 20 and remained
at this level until the last day tested (day 30; Fig. 4b).
Neither the last cell wash supernatant nor the 105unstim-
ulated BMDM induced a PapMV-specific antibody titre,
showing that transferred cells did not contain free virus
and that the antibody response induced was specific.
Moreover, both the culture supernatant from PapMV-
stimulated BMDM, and PapMV injected i.v. induced spe-
cific antibody titres (Fig. 4b).
These results show that BMDM were able to provide
PapMV to B cells efficiently, inducing a specific antibody
Lymph nodes Spleen
CD11c+
CD11b–
CD11c–
CD11b+
CD11c–
B220+
CD11c+
CD11b–
CD11c–
CD11b+
CD11c–
B220+
CD80
100
0
128
Events
101
102
FITC-A
103
104
100
101
102
103
104
100
101
102
103
104
100
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102
103
104
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FITC-A
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FITC-A
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FITC-A
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100
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102
FITC-A
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104
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FITC-A
103
104
100
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FITC-A
103
104
100
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FITC-A
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104
0
32
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0
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0
256
Events
FITC-A
0
512
Events
0
223
Events
0
64
Events
FITC-A
0
64
Events
0
756
Events
0
512
Events
0
64
Events
FITC-A
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64
Events
FITC-A
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64
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FITC-A
0
256
Events
0
223
Events
FITC-A
0
256
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128
CD40
0
Events
CD86
0
56
Events
FITC-A
0
32
Events
FITC-A
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32
Events
FITC-A
0
32
Events
FITC-A
0
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Events
0
256
Events
FITC-A
0
1024
Events
FITC-A
0
512
Events
FITC-A
0
64
Events
FITC-A
CD69
0
128
Events
FITC-A
MHC II
0
64
Events
FITC-A
0
32
Events
FITC-A
Figure 3. Papaya mosaic virus (PapMV) induces co-stimulatory molecule up-regulation in dendritic cells (DC), macrophages and B cells in vivo.
BALB/c mice were immunized intraperitoneally (i.p.) with 30 lg of saline-diluted PapMV (grey line) or with 50 lg of Poly I:C (black line), and
control mice were injected with saline (filled histogram). Twenty-four hours after immunization, lymph nodes and spleen cells were collected
and stained. Flow cytometry was performed to analyse cell populations defined as: CD11c+CD11b)(DCs), CD11c)CD11b+(macrophages) and
CD11c)B220+(B cells). A representative result of two experiments is shown. FITC, fluorescein isothiocyanate; MHC II, major histocompatibility
complex class II.
? 2007 The Authors Journal compilation ? 2007 Blackwell Publishing Ltd, Immunology
7
Strong immunogenicity and adjuvanticity of papaya mosaic virus

Page 8

immune response. This mechanism could also be involved
in translating the innate immune response induced by
PapMV into a specific long-lasting antibody response.
PapMV induces the up-regulation of activation
markers on DC, macrophages and B cells in vivo
If PapMV is indeed recognized by the immune system as
a PAMP, it should drive the maturation of immature DC
or activate APC, as do other PRR agonists. To character-
ize the capacity of PapMV to stimulate APC in vivo, we
isolated lymph nodes and spleens of mice immunized
with PapMV and analysed the up-regulation of co-stimu-
latory and activation molecules (CD80, CD86, CD40,
CD69 and MHC class II). Mice were immunized i.p. with
30 lg of PapMV, 50 lg of Poly I:C (both in saline) or
saline alone (control). Twenty-four hours after immuniza-
tion, lymph nodes and spleen cells were harvested. As
shown in Fig. 3, PapMV induced the up-regulation of
CD80andCD69 in
(CD11c+CD11b)) and CD40 and CD69 in spleen DC.
For macrophages (CD11c)CD11b+), up-regulation of
CD80 and CD69 was observed in LN, whereas CD40
and CD69 up-regulation was observed in the spleen.
CD69 was up-regulated in LN B lymphocytes (CD11c)
B220+), and CD40 and CD69 were up-regulated in sple-
nic B cells. The TLR-3 agonist, Poly I:C, induced the up-
regulation of the markers tested, although the profile
observed was markedly different from that induced by
PapMV (Fig. 3).
lymphnode(LN)DC
(b)
0510 1520253035
0
1
2
3
4
5
6
7
8
9
10
PapMV
106 BMDM + PapMV
105 BMDM + PapMV
104 BMDM + PapMV
Cell wash
105 BMDM
Culture supernatant
IgG
Days after transference
Antibody titre (–log2 × 40)
(a)
(i)
CTB-
FITC
CTB-
FITC
PapMV
PapMVMerge
Merge
(ii)
(d)
Hours after stimulation
04812 16 20 24 28
0
20
40
60
80
100
Hours after stimulation
(c)
0 4 812 16 2024 28
0
200
400
600
800
1000
PapMV 1·0 μg/ml
LPS 100 ng/ml
40 50 60 70 80
IL-6 (pg/ml)
IL-6 (pg/ml)
Macrophages
Dendritic cells
1824
0
1000
2000
3000
20 000
22 500
25 000
1824
0
100
200
300
2000
12 000
1824
0
5
10
15
20
25
IFN-α (pg/ml)
TNF-α (pg/ml)
IFN-α (pg/ml)
TNF-α (pg/ml)
18 24
0
25
50
75
100
Medium
PapMV
LPS
Medium
PapMV
LPS
Figure 4. Papaya mosaic virus (PapMV) efficiently activates antigen-presenting cells (APC) and these cells participate in the induction of the
specific antibody response. (a) Aggregation of lipid rafts induced by PapMV. (i) Unstimulated cells. (ii) Cells pulsed with 1 lg/ml of PapMV.
Original magnification ·100. (b) Bone marrow-derived macrophages (BMDM) pulsed with PapMV efficiently induced specific IgG titres. BMDM
were stimulated with 30 lg/ml of PapMV and transferred intravenously (i.v.) to BALB/c mice. Non-pulsed cells, culture supernatant, final-wash
supernatant and 30 lg of PapMV were injected i.v. as controls. The PapMV-specific IgG titres in mice sera were measured by enzyme-linked
immunosorbent assay (ELISA) at the time-points depicted in the graph. Results are expressed as the mean ± standard deviation (SD) of six mice
per group. Data correspond to one representative result from three independent experiments. (c,d) Production of inflammatory cytokines in
macrophages and dendritic cells induced by 1 lg/ml of PapMV or by 100 ng/ml of lipopolysaccharide (LPS) from Escherichia coli UHO111 (as a
positive control). Results are expressed as the mean ± SD of four wells per time chased. Data correspond to one representative result from three
independent experiments. CTB, cholera toxin B subunit; FITC, fluorescein isothiocyanate; IFN-a, interferon-a; IL-6, interleukin-6; TNF, tumour
necrosis factor.
8
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E. Acosta-Ramı ´rez et al.

Page 9

PapMV strengthens the antibody response
to model antigens
Adjuvants are substances capable of augmenting the anti-
body response or the cellular immune response against
poor immunogenic antigens.27Adjuvant effects are medi-
ated by the activation of DC and macrophages through
PRR such as TLR.28
As mentioned above, PapMV
induced the activation and maturation of APC, suggesting
that it has intrinsic adjuvant properties. To determine
whether PapMV is an adjuvant that can promote a long-
lasting antibody response to other antigens, BALB/c mice
were immunized i.p. on day 0 with 2 mg of OVA or
HEL, either alone or together with the following adju-
vants: PapMV, CFA or LPS. The IgG titre specific for
OVA or HEL was measured by ELISA at the time-points
indicated (Fig. 5a). The adjuvant effect of PapMV was
observed on the total IgG response to OVA and HEL in
immunized animals. An adjuvant effect induced by Pap-
MV was observed for HEL on day 30 after immunization,
when the antibody titre increased eightfold compared
with the antibody titre induced by HEL alone. This differ-
ence in antibody titre was maintained until day 120, but
not to day 400 (Fig. 5a). Although LPS induced an adju-
vant effect only in the first 30 days after immunization,
CFA showed the strongest adjuvant effect from day 8 to
the end of the experiment on day 400 after immunization
(Fig. 5a). For immunization with OVA, the adjuvant
effect on total IgGtitres was observed only until day 120
after the first immunization, after which the antibody
titre decreased with time, but was still fourfold higher
than for OVA on day 400 (Fig. 5a). Further analysis was
performed on day 20 to identify which IgG subclasses
were induced by OVA and by OVA co-immunized with
adjuvants (where PapMV did not show an adjuvant effect
on the total IgG response). PapMV, LPS and CFA
induced OVA-specific IgG2a and IgG2b titres, whereas
OVA alone induced only IgG1-specific antibody titres
(Fig. 5a). No adjuvant effect for IgG1 was observed when
OVA was co-administered with any of the adjuvants used.
Thus, PapMV, LPS and CFA induce an adjuvant effect on
the IgG subclass responses to OVA. Moreover, PapMV
exhibits adjuvant properties that induce a long-lasting
increase in specific antibody titres to model antigens.
Taken together, these data suggest that PapMV has intrin-
sic adjuvant properties that may have mediated the trans-
lation of the innate response into an antigen-specific
long-lasting antibody response.27
Co-administration of PapMV and S. typhi OmpC
porin increases the OmpC protective capacity against
S. typhi challenge
An adjuvant is also used to promote the antigen protec-
tive capacity.27To test whether the adjuvant properties of
PapMV could increase the protection of a vaccine, we co-
administered PapMV with an experimental vaccine to
typhoid fever, composed of S. typhi OmpC porin, devel-
oped in our laboratory.15,29Compared to mice immu-
nized with the OmpC porin, mice immunized with
OmpC mixed with PapMV showed a survival rate 30%
higher after challenge with 100 and 500 LD50of S. typhi
(Fig. 6b). No protection was observed in mice immunized
only with PapMV on days 0 and 15 and challenged on
day 21 (Fig. 6b) or in mice immunized with PapMV and
challenged 24 hr later (data not shown). These data reject
IgG
010 20 30
Days after immunization
0
2
4
6
8
10
12
14
PapMV + HEL
HEL
LPS + HEL
CFA + HEL
ISS
100200300400500
IgG
010 20
Days after immunization
30
0
2
4
6
8
10
12
14
OVA
LPS + OVA
ISS
100 200 300 400 500
CFA + OVA
PapMV + OVA
Antibody titre (–log2 × 40)
Antibody titre (–log2
× 40)
Antibody titre
(–log2
× 40)
(a)
(b)
IgG2b
0
Saline
2
4
6
8
10
12
IgG2a
0
2
4
6
8
10
12
IgG1
Saline
PapMV
OVA
PapMV + OVA
LPS + OVA
CFA + OVA
Saline
PapMV
OVA
PapMV + OVA
LPS + OVA
CFA + OVA
PapMV
OVA
PapMV + OVA
LPS + OVA
CFA + OVA
0
2
4
6
8
10
12
14
16
18
20
Figure 5. Papaya mosaic virus (PapMV) strengthens the antibody response to model antigens. (a) Groups of three female BALB/c mice were
immunized with 2 mg of ovalbumin (OVA) or hen egg-white lysozyme (HEL), either alone or with the following adjuvants: 30 lg of PapMV,
complete Freund’s adjuvant (CFA) (1:1, v/v) or 5 lg of lipopolysaccharide (LPS) from Escherichia coli O111:B4. Control mice were injected with
saline only (isotonic saline solution (ISS)). Serum was obtained from the immunized mice at the time-points depicted. A representative result
from two experiments is shown. (b) The PapMV adjuvant effect is characterized by the induction of higher OVA-specific IgG2a and IgG2b titres.
Analysis of IgG subclasses present on day 20 in serum from mice immunized with OVA, PapMV–OVA, LPS–OVA and CFA–OVA.
? 2007 The Authors Journal compilation ? 2007 Blackwell Publishing Ltd, Immunology
9
Strong immunogenicity and adjuvanticity of papaya mosaic virus

Page 10

the idea that the protection observed is the result of
enhanced inflammation induced by PapMV. To test if
increased protection correlates with an increase in the
antibody titre specific for OmpC, we measured the
PapMV adjuvant effect on the OmpC-specific antibody
titres. PapMV co-immunization with OmpC induced an
increase in the anti-OmpC IgG1, IgG2a, IgG2b and IgG3
titres (Fig. 6a).These results further corroborate that the
adjuvant properties of PapMV potentiate both innate and
adaptive immune responses elicited by OmpC to achieve
protection against S. typhi challenge.
Discussion
The characterization and study of molecules able to
induce long-lasting immune responses, and the mecha-
nisms involved in their recognition, are important for
understanding the generation of long-term immunity.15,30
We found, in this study, that PapMV is a strong immuno-
genic antigen able to induce a long-lasting antibody
response with a single dose and without added adjuvants
(Fig. 1a). We also observed that PapMV induces a T-cell
response (Fig. 1b). Because PapMV is not pathogenic in
mammals,10the mammalian immune system has had
no evolutionary selective pressure to generate immunity
against PapMV. Therefore, the highly immunogenic activ-
ity of this virus suggests that the molecular patterns rec-
ognized by PRR on cells from the innate immune system
and by B and T cells – rather than pathogenicity of the
virus – might be the driving force in the recognition of
PapMV. The relationship between the distribution of epi-
topes within a molecule and the capacity to activate B
lymphocytes independently of T cells has been described
previously,31suggesting that highly repetitive and orga-
nized antigens promote the establishment of a long-
lasting B-cell response. Similarly to many vertebrate
pathogenic viruses, PapMV repetitive-pattern epitopes
may cross-link specific immunoglobulins efficiently on
B-cell surfaces. The specificity of PapMV recognition by B
cells is supported by the observation that PapMV is not a
0 2
Days after challenge
4 6 8 10
0
20
40
60
80
100
OmpC
OmpC
OmpC + PapMV
OmpC + PapMV
PapMV
ISS
Survival (%)
(b)
Antibody titre (1/dilution)
(a)
350 000
IgG1
0
50 000
IgG2a
OmpC
OmpC + PapMV
OmpC + IFA
PapMV
ISS
OmpC
OmpC + PapMV
OmpC + IFA
PapMV
ISS
0
50 000
100 000
100 000
150 000
150 000
200 000
200 000
250 000
250 000
300 000
300 000
350 000
0
50 000
100 000
150 000
200 000
250 000
300 000
350 000
IgG2b
IgG3
0
5000
10 000
15 000
20 000
25 000
30 000
35 000
40 000
45 000
Figure 6. Papaya mosaic virus (PapMV) promotes the increase of antibody titres and potentiates the protection induced by an experimental vac-
cine. (a) Groups of five female BALB/c mice were immunized intraperitoneally (i.p.) on day 0 with 10 lg of outer membrane protein C (OmpC)
alone or together with 30 lg of PapMV or Freund’s incomplete adjuvant (IFA) (1:1, v/v). On day 15, all mice were boosted with 10 lg of OmpC
only. Control mice were injected with saline only (isotonic saline solution (ISS)). Antibody titres were measured by enzyme-linked immunosor-
bent assay (ELISA) on day 21 after the first immunization. (b) PapMV increased the protective capacity of OmpC porin. Groups of 10 female
BALB/c mice were immunized i.p. with 10 lg of OmpC either alone or with 30 lg of PapMV. The booster was given on day 15 using Omp C
alone. Control mice were injected with saline (ISS) or PapMV. The challenge of experimental groups was performed on day 21 with a 100 (filled
symbols) or a 500 (open symbols) 50% lethal dose (LD50) of Salmonella enterica serovar Typhi (S. typhi), and the survival rate was recorded for
10 days after the challenge. Control groups were challenged with 20 LD50of S. typhi. A representative result of three experiments is shown.
10
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E. Acosta-Ramı ´rez et al.

Page 11

type 1 thymus-independent antigen (Fig. 2). However, the
small up-regulation of B-cell activation markers observed
both in vitro and in vivo suggest that PapMV could also
be recognized by non-BCR receptors on B cells. The con-
tribution of non-BCR receptors, such as TLRs, expressed
on B cells, to the humoral immune response is a topic of
debate.32,33TLR stimulation has been proposed as a third
signal required for the activation of human naı ¨ve B cells34
or as a signal to maintain B-cell memory compartments.35
Thus, PapMV could stimulate B cells to favour processes
such as antigen presentation, co-stimulation or as survival
signal for some B cells.
Recognition of repetitive, organized molecular patterns
is also important to activate the innate immune response.
This has been studied for mannose-binding lectin,36for a
polymeric bacterial protein that signals through TLR-437
and for highly ordered flagellin, a TLR-5 agonist, for
which immunogenicity is dependent on its level of poly-
merization.38Accordingly, PapMV particles efficiently acti-
vated innate immune system cells, as shown in Figs 3 and
4. Although we did not identify the PRR involved in the
innate immune activation, this response indicated that
PapMV is recognized by the immune system similarly to
PAMP. Because PAMP are important components of
many adjuvants, we tested the adjuvant properties of
PapMV. PapMV promoted long-lasting antibody responses
to HEL and to OVA (Fig. 5a) and has been found to
potentiate the protective capacity of an experimental vac-
cine19,29(Fig. 6). Previously, we have reported a lack of
adjuvanticity of the monomeric form of PapMV CP, indi-
cating the critical function of multimerization in the
induction of an antibody response to the expressed anti-
gen.39These data confirm that PapMV activates immune
responses similarly to PAMP.
Antigen presentation by APCs to T cells has shown the
importance of the innate immune system to activate the
adaptive immune system. APCs loaded with PapMV effi-
ciently induced antibody responses (Fig. 4b), as previ-
ously observed for a mammalian infecting virus.25These
data, and the effects elicited by PapMV on APCs (Figs 3
and 4), suggest that PapMV-stimulated APCs provide the
antigen and the cytokine environment needed to promote
an efficient T-cell-dependent antibody response, and this
might be the mechanism involved in translating intrinsic
adjuvant signals delivered by PapMV into the long-lasting
antibody response observed. We propose that PapMV is
sensed by the immune system both as PAMP and as anti-
gen (Pamptigen), simultaneously activating innate and
adaptive immune responses, which would favour the
induction of a memory compartment. Pamptigens could
also bind both BCRs and PRRs, such as TLRs, on specific
B cells, leading to the differentiation of antibody-secreting
cells or to the generation of memory B cells. A Pamptigen
could reduce the antigen threshold required to activate
the immune response. Therefore, small amounts of the
persistent antigen could maintain a high antibody titre.
Most non-infectious antigens, such as OVA or HEL, or
PAMP (such as LPS), do not induce long-lasting antibody
titres on their own. Our results suggest that the innate
immune response makes an important contribution to
the development or to the maintenance of long-lasting
antibody responses.
The strong immunogenicity and intrinsic adjuvant
properties of PapMV translate into a specific long-lasting
antibody response to both PapMV and to model co-
immunized antigens. As PapMV can be produced using
inexpensive procedures and is stable at room temperature,
it offers biotechnology useful advantages to develop new
adjuvant or vaccine platforms39to induce long-lasting
immunity.
Acknowledgements
This study was funded by the Mexican National Science
and Technology Council (CONACyT) Projects SEP-
CONACyT 33137-M and 45261-M and by the Fund
for Research Development (FOFOI) from IMSS Projects
2003-020, 2004-052 and 2006-022 awarded to C. Lo ´pez-
Macı ´as. R. Pastelin-Palacios acknowledge funding from
Programa de Apoyo a Proyectos de Investigacio ´n e Inno-
vacio ´n Tecnolo ´gica (PAPIIT), UNAM, Project: IN224907
andSEP-CONACyT43911-M.
acknowledge scholarships received from CONACyT and
IMSS. We acknowledge the excellent work on animal care
performed by Mr Ricardo Vargas Orozco and Daniel Sa ´n-
chez Almaraz, DVM. We thank Dr Edmundo Calva from
the Biotechnology Institute, UNAM, for providing strain
STYF302.
ThePhDstudents
Conflict of interest
The authors have no financial conflict of interest.
N. Majeau, D. Leclerc and C. Lo ´pez-Macı ´as are co-
inventors on a patent pending for the use of PapMV as
an adjuvant.
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