Immune response to a potyvirus with exposed amino groups available for chemical conjugation.

Abstract

The amino terminus of the tobacco etch virus (TEV) capsid protein is located on the external surface of infectious TEV particles, as proposed by previous studies and an in silico model. The epsilon amino groups on the exposed lysine residues are available for chemical conjugation to any given protein, and can thus act as antigen carriers. The availability of amino groups on the surfaces of TEV particles was determined and the immune response to TEV evaluated.
Using a biotin-tagged molecule that reacts specifically with amino groups, we found that the TEV capsid protein has amino groups on its surface available for coupling to other molecules via crosslinkers. Intraperitoneal TEV was administered to female BALB/c mice, and both their humoral and cellular responses measured. Different IgG isotypes, particularly IgG2a, directed against TEV were induced. In a cell proliferation assay, only spleen cells from vaccinated mice that were stimulated in vitro with TEV showed significant proliferation of CD3+/CD4+ and CD3+/CD8+ subpopulations and secreted significant amounts of interferon γ.
TEV has surface amino groups that are available for chemical coupling. TEV induces both humoral and cellular responses when administered alone intraperitoneally to mice. Therefore, TEV should be evaluated as a vaccine adjuvant when chemically coupled to antigens of choice.

Full text

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RESEARCHOpen Access
Immune response to a potyvirus with exposed
amino groups available for chemical conjugation
Carlos Alberto Manuel-Cabrera1, Ana Márquez-Aguirre1, Hernández-Gutiérrez Rodolfo1, Pablo César Ortiz-Lazareno2,
Gabriela Chavez-Calvillo3,4, Mauricio Carrillo-Tripp4, Laura Silva-Rosales3and Abel Gutiérrez-Ortega1*
Abstract
Background: The amino terminus of the tobacco etch virus (TEV) capsid protein is located on the external surface
of infectious TEV particles, as proposed by previous studies and an in silico model. The epsilon amino groups on
the exposed lysine residues are available for chemical conjugation to any given protein, and can thus act as
antigen carriers. The availability of amino groups on the surfaces of TEV particles was determined and the immune
response to TEV evaluated.
Results: Using a biotin-tagged molecule that reacts specifically with amino groups, we found that the TEV capsid
protein has amino groups on its surface available for coupling to other molecules via crosslinkers. Intraperitoneal
TEV was administered to female BALB/c mice, and both their humoral and cellular responses measured. Different
IgG isotypes, particularly IgG2a, directed against TEV were induced. In a cell proliferation assay, only spleen cells
from vaccinated mice that were stimulated in vitro with TEV showed significant proliferation of CD3+/CD4+and
CD3+/CD8+subpopulations and secreted significant amounts of interferon g.
Conclusions: TEV has surface amino groups that are available for chemical coupling. TEV induces both humoral
and cellular responses when administered alone intraperitoneally to mice. Therefore, TEV should be evaluated as a
vaccine adjuvant when chemically coupled to antigens of choice.
Keywords: Tobacco etch virus, capsid protein, amino groups, chemical conjugation, immune response
Background
Tobacco etch virus (TEV) belongs to the genus Poty-
virus, the largest and economically most important
genus of the recognized plant virus groups and families
[1]. The genomes of the potyviruses are single positive-
stranded RNAs, surrounded by approximately 2,000 sub-
units of the coat protein (CP) [2]. A previous study has
demonstrated that the CP amino and carboxy termini of
several potyviruses are located on the surface of the
infectious particle and bear the most immunogenic epi-
topes [3]. Based on biochemical and immunological evi-
dence, two other studies have suggested that the first 29
amino acids of the TEV capsid protein are hydrophilic
and are located at or near the particle’s surface [4,5].
Generally, viruses induce good immune responses,
which are dependent on their surface structures. These
structures consist of one or a few proteins and are highly
organized and repetitive in nature. This repetitiveness
could be recognized by the immune system as a patho-
gen-associated geometric pattern similar to pathogen-
associated molecular patterns [6]. Viruses are good
immunogens because they facilitate the crosslinking of B-
cell receptors, enhancing the host antibody response
[7,8]. Viruses are also efficiently internalized, processed,
and presented by antigen-presenting cells [9]. These fea-
tures make viruses good candidates for the presentation
of foreign antigens on their surfaces. By exploiting these
features, several plant viruses have been used as antigen-
presenting platforms for the development of subunit vac-
cines directed against a variety of human and animal
pathogens. This is normally achieved by inserting DNA
sequences in-frame with the CP-encoding gene. The
viruses used for this purpose include the tobacco mosaic
* Correspondence: aortega@ciatej.net.mx
1Unidad de Biotecnología Médica y Farmacéutica, Centro de Investigación y
Asistencia en Tecnología y Diseño del Estado de Jalisco, Normalistas 800,
Colinas de la Normal, Guadalajara, Jalisco 44270, México
Full list of author information is available at the end of the article
Manuel-Cabrera et al. Virology Journal 2012, 9:75
http://www.virologyj.com/content/9/1/75
© 2012 Manuel-Cabrera et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.

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virus (TMV) [10,11], cowpea mosaic virus [12-15],
cucumber mosaic virus (CMV) [16], alfalfa mosaic virus
[17], potato virus × [18], and papaya mosaic virus
(PapMV) [19]. Until now, only one potyvirus, plum pox
virus, has been used as a platform for displaying foreign
amino-acid sequences on its surface [20,21].
One limitation of the translational fusion approach is
the size of the sequence that can be inserted without
compromising the capsid protein self-assembly, which is
fundamental to stimulating a good immune response.
Generally, this size cannot exceed 20 amino acids,
although larger sequences should be exposed [22]. One
alternative to translational fusions is coupling the viruses
to peptides or complete antigens through chemical cross-
linkers that bind specifically to groups present in the side
chains of some amino acids. With this strategy, several
plant viruses have been used for the surface display of
exogenous proteins. In the cowpea mosaic virus, an ico-
sahedral virus model that has been genetically modified
for accurate chemical conjugation, 100% occupancy of
CP monomers by complex molecules was demonstrated,
with the retention of the biological activity of the
attached proteins [23]. Another study has shown that
TMV is an effective vaccine carrier for stimulating pep-
tide-specific immunity to both single and multivalent
vaccines [24]. The presentation of whole protein on
TMV has also been demonstrated, expanding the utility
of TMV as a vaccine scaffold by the genetic manipulation
of both TMV and the presented antigen [25]. There is
apparently no limitation on the antigen size with this
approach and a variety of epitopes can be exposed on a
single viral particle. However, this assumption must be
evaluated for each specific case.
When we analyzed several reported CP sequences from
TEV, we realized that the TEV CP amino terminus is rich
in positively charged residues, predominantly lysines.
Lysine residues are often utilized for chemical coupling
via their epsilon amino groups. If these lysine residues
were exposed on the viral surface, they would be available
for chemical conjugation with a variety of antigens. In this
study, we demonstrated that TEV CP lysines exposed on
the particle surface can be used for antigen coupling
through chemical conjugation. We also evaluated the
immune response to the virus in a mouse model. Based on
these findings, we propose that TEV be evaluated as an
adjuvant for subunit vaccines.
Results and discussion
Why TEV is a good candidate vaccine adjuvant
We consider that TEV offers several advantages as a car-
rier for antigen presentation. There has been no report
that TEV or any other plant virus replicates effectively in
humans or animals. Moreover, yields of TEV from
infected tobacco plants are high and its purification is
relatively easy, providing enough material for large-scale
formulations. Most importantly, by substituting a few
amino acids in the CP and the helper-component protei-
nase, nonaphid-transmissible mutants can be generated
to prevent the virus spreading [26,27].
TEV CP in silico model
The TEV used in this study, designated TEV-NAY, was a
field isolate from Nayarit, Mexico. The TEV-NAY isolate
was propagated and purified as described in the Methods
section and its genetic material was isolated. Its CP cis-
tron was amplified, cloned, and sequenced. As shown in
Figure 1A, the TEV CP amino region, spanning the first
29 amino acids, contains approximately five lysines in
nine residues [4,5]. Lysine reacts well with N-hydroxy
succinimide (NHS) esters without the assistance of
neighboring amino acids [28], and NHS esters are used
extensively for chemical crosslinking.
From the various sequences of several Potyvirus CPs,
we generated ab initio models using the Rosetta software
(http://www.pyrosetta.org/) and selected the sequence
that most broadly represented the biochemical features
previously reported [29,30]. The best model was used as
the template to generate the homologous model (http://
salilab.org/modeller/) of the TEV-NAY CP sequence.
This model provides valuable structural information that
strongly suggests the availability of the epsilon amino
groups of surface-exposed lysine 12 (K12), K13, K14,
K17, and K20 for chemical conjugation to a variety of
antigens (Figure 1B).
TEV has amino groups exposed on its surface
Based on the observations described above, we indirectly
tested whether the virions from the TEV-NAY isolate car-
ried exposed amino groups on their surfaces. First, the
integrity of the pure TEV virions was confirmed by elec-
tron microscopy (data not shown). Second, the availability
of the amino groups on the TEV particles was evaluated
with Sulfo-NHS-SS-Biotin. This reagent is composed of
NHS, which covalently binds the amino groups exposed on
the surface of any protein, attached to biotin via a spacer
arm. The biotin can be removed from the reagent with a
reducing agent (Figure 2A). After TEV was incubated with
Sulfo-NHS-SS-Biotin, the sample was subjected to sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-
PAGE) under reducing or nonreducing conditions and
stained with Coomassie Blue or transferred to a membrane
for western blot analysis to verify biotin binding. Coomas-
sie Blue staining revealed a band slightly larger than the
band present in the untreated TEV sample (Figure 2B),
which we presumed corresponded to TEV CP, which has a
calculated mass of 32 kDa [31]. The observed increase in
mass suggests the coupling of TEV CP with the reagent,
because one molecule of Sulfo-NHS-SS-Biotin attached to
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the 32 kDa TEV CP represents only a 606.9 Da increase in
molecular weight. This increase results in a discernible
shift on gel electrophoresis, allowing us to assess the extent
of TEV CP conjugation. Because no unconjugated TEV
CP band was observed after the Sulfo-NHS-SS-Biotin
treatment, we assumed that 100% occupancy was achieved
after incubation with a 20-fold molar excess of Sulfo-
NHS-SS-Biotin for 1 h at room temperature. This con-
trasts with a study of a modified TMV [25], in which a
240-fold molar excess of NHS-PEO4-biotin was required
to obtain fully biotinylated TMV. To confirm that Sulfo-
NHS-SS-Biotin was indeed bound to the TEV after treat-
ment, a western blot analysis was performed using horse-
radish peroxidase (HRP)-conjugated streptavidin as the
conjugate (Figure 2C). Under nonreducing conditions, the
main band detected corresponded to TEV CP, but a
90 kDa band was also clearly observed, which was also
present under nonreducing conditions after Coomassie
Blue staining. This larger band might correspond to either
different oligomeric states of CP after SDS treatment or to
an association between the terminal CPs and either helper
component protein, viral genome-linked protein, or cyto-
plasmic inclusion protein, which are thought to be present
at one end of CP in a subpopulation of virions [32-34].
Under reducing conditions, the TEV CP signal was con-
siderably reduced because of the partial separation of bio-
tin from Sulfo-NHS-SS-Biotin. An interesting observation
was the presence of a second band smaller than the major
conjugation product after gel electrophoresis and western
blotting, which could indicate the coupling of more than
one biotin to a single TEV CP. We expected the TEV CP
signal to disappear completely under reducing conditions,
and this may have been achieved by incubating the sample
with reducing agent for a longer period of time or by
using dithiothreitol rather than 2-mercaptoethanol.
Another interesting observation was that there was no
20
TEV CP S G T V D A G A D A S K K K D H K D D K
↓
40
TEV CP V A E Q V S R D R D V N A G T S G T F S
60
TEV CP V P R I N A M A T K L Q Y P R M R K E V
A
B
Figure 1 Partial amino acid sequence and structural model of TEV capsid protein. A) Predicted sequence for the first 60 amino acids of
TEV-NAY capsid protein (CP). Solid arrow indicates the first 29 amino acids of CP, located on the virion’s surface. Solid boxes indicate lysines
present in the surface-exposed region of CP that are available for chemical conjugation. B) Predicted structure for TEV-NAY CP, with the solvent-
accessible N-terminal lysine-rich stretch. A closer view of localized lysines (K12, K13, K14, K17, and K20) is shown in the rectangle.
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signal for the 90 kDa form of CP under reducing condi-
tions. In summary, our results strongly suggest that TEV
particles are amenable to chemical coupling through their
surface-exposed amino groups.
TEV induces antibody production
We next immunized female BALB/c mice (Figure 3) to
evaluate the immune response induced by TEV in this
model. Mice were injected with 25 μg of TEV or viral dilu-
ent (20 mM Tris, pH 8.0) on days 1 and 14 and were bled
on days -7, 13, and 27. Their sera were analyzed for the
relative levels of anti-TEV antibodies. Three immunoglo-
bulin G (IgG) isotypes against TEV were measured: IgG1,
IgG2a, and IgG2b. The isotype with the highest titer was
IgG2a, followed by IgG1 and IgG2b (Figure 4). Our results
are similar to those obtained with the intraperitoneal
immunization of mice with PapMV, a potexvirus that, like
TEV, is a flexible rod, but it is 1.5 times wider and two
times shorter than TEV [35]. In another report, very simi-
lar results were observed when PapMV-virus-like particles
harboring a hepatitis C virus epitope were administered
subcutaneously, but the response evaluated in that study
was against the hepatitis C virus epitope [36]. Another
study reported that TMV, chemically decorated with
either green fluorescent protein (GFP) or the canine oral
papillomavirus L2 antigen, induced antigen-specific anti-
bodies in mice and guinea pigs immunized with low doses
of TMV complex without adjuvant [25]. Our results show
that TEV is a strongly immunogenic particle, and can
induce an antibody response in which the IgG2a subclass
is the major antibody product, a bias that can be indicative
of a stronger Th1 immune response.
TEV induces T-lymphocyte proliferation
To examine the capacity of TEV to induce a T-lymphocyte
response, immunized and control mice were killed 28 days
after their first immunization, and the mononuclear cells
were isolated from their spleens with a Ficoll-Paque den-
sity gradient. The isolated cells were then grown in med-
ium and either stimulated or not stimulated with TEV for
three days. After this incubation period, the cells were har-
vested and the CD3+/CD4+(T-helper lymphocyte) and
CD3+/CD8+(T-cytotoxic lymphocyte) subpopulations
were analyzed by flow cytometry. Only cell cultures from
MWM (kDa) 1 2 3 4
97
66
45
31
21.5
14.5
6.5
116
200
188
62
49
38
28
18
14
6
MWM (kDa) 1 2 3
A
C B
Figure 2 Availability of surface-exposed amino groups on TEV capsid protein for chemical conjugation to biotin-tagged reagent. A)
Chemical structure of biotin-tagged reagent, Sulfo-NHS-SS-Biotin: the NHS ester group reacts specifically with the amino groups of amino acids;
the spacer arm contains an S-S bond that can be cleaved by treatment with the reducing agent 2-mercaptoethanol; and biotin is attached on
the other side of the spacer arm. B) 12% SDS-PAGE of untreated virus under nonreducing and reducing conditions (lanes 1 and 2, respectively)
and biotinylated virus under nonreducing and reducing conditions (lanes 3 and 4, respectively) stained with Coomassie Blue. C) Western blot of
treated and untreated samples. Lane 1: untreated virus under reducing conditions; lanes 2 and 3, biotinylated virus under nonreducing and
reducing conditions, respectively. A streptavidin-horseradish peroxidase conjugate was used as the detection reagent. MWM, molecular weight
marker. Bold arrows indicate main biotinylation products.
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the immunized mice that were further stimulated in vitro
with TEV showed a significant increase in both subpopu-
lations (Figure 5). This indicates that a memory of TEV
was established in both T-helper and T-cytotoxic lympho-
cytes. A similar study hypothesized that CD4+T cells
improved the efficacy of TMV-antigen complex and thus
inhibited tumorigenesis [24]. It has also been observed
that the formation of a complex between TMV and a
cargo molecule is required for the robust induction of acti-
vated antigen-specific CD8+T cells [25].
TEV induces interferon g (IFNg) secretion in cultured
splenocytes
To determine whether TEV preferentially induces a Th1
or Th2 response, two cytokines, IFNg and interleukin 4
(IL4), were measured in the supernatants of cultured sple-
nocytes. IFNg and IL4 are mediators of the Th1 and Th2
responses, respectively. The results, shown in Figure 6,
indicate that TEV induces a bias towards the Th1
response, because TEV treatment caused IFNg secretion.
It is noteworthy that this response was observed only in
the splenocytes from TEV-vaccinated animals that were
further stimulated with TEV. No levels of IL4 were
detected after any treatment. A previous study of CMV,
an icosahedral virus, showed that CMV itself induced
IFNg secretion in peripheral blood mononuclear cells that
had not been primed against the virus, indicating that
CMV induces a dominant Th1 immune response [37].
Furthermore, a bivalent formulation of TMV conjugated
to toxin-derived peptides, T-helper epitopes, or peptides
for enhanced antigen uptake, significantly improved the
immune responses in mice, as measured by the levels of
IFNg-secreting cells, and their survival after lethal chal-
lenge with tumor cells without adjuvant [24]. The results
of the present study indicate that TEV, which induces the
secretion of a molecule that mediates the Th1 response,
could be used as a vaccine adjuvant when a Th1 response
is fundamental to the generation of protective immunity.
Conclusions
The following conclusions can be drawn from the results
presented here. First, the amino groups of CP lysines are
exposed on the surfaces of infectious TEV particles, as
revealed by in silico modeling and biotinylation experi-
ments. This is a remarkable property that may be unique
to TEV. Such amino groups are probably available for
chemical coupling to antigens of choice. Therefore, no
modification of the native TEV CP is necessary because
Bleeding 1
(Day -7)
First
Immunization
IP via
(Day 0)
TEV 25μg
Boosting
IP via
(Day 14)
TEV 25 μg
Bleeding 2
(Day 13)
Bleeding 3
(Day 27)
Sacrifice and
splenectomy
(Day 28)
Figure 3 Immunization and serum collection schedule. To evaluate the TEV-associated immune response in a mouse model, two groups of
five female Balb/c mice were tested, designated C and TEV. Pure TEV (25 μg) or an equal volume of 20 mM Tris (pH 8.0) were administered to
the TEV or C group, respectively. The mice were immunized intraperitoneally (IP) on day 0 and boosted on day 14. During the experiment, the
mice were bled three times, on days -7 (B1), 13 (B2), and 27 (B3). The animals were killed at the end of the experiment on day 28, before
splenectomy.
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the amino-acid side chains commonly targeted for conju-
gation are naturally present in TEV field isolates.
Whether these groups can be coupled to large antigens
will primarily depend on the spacer arm length present
in the crosslinker utilized for this purpose and the mass
and tertiary structure of the antigen, and is yet to be
determined. We expect no change in the virus structure
after biotin coupling, although we did not explicitly
assess this in this study. In previous work with a modified
TMV, the complete biotinylation of the CP did not alter
the size distribution of the virus. Nonetheless, when bio-
tinylated TMV was loaded with green fluorescent pro-
tein-streptavidin at 26% capacity, a notable reduction in
rod length was documented, leading to the conclusion
that the load capacity of the virus depends on the size of
the antigen [25]. Second, TEV induces T-cell and anti-
body responses when administered alone to mice. Third,
TEV induces IFNg secretion, which is a mediator of the
Th1 response. Therefore, TEV could be a useful adjuvant
against some intracellular pathogens where this type of
response is critical for adequate protection. Further
experiments are required to determine whether TEV can
induce type-I interferon production in immune cells
through the interaction of its single-stranded RNA with
Toll-like receptor 7 [38]. In summary, we propose a thor-
ough evaluation of TEV as a vaccine adjuvant, to increase
the density of the potentially displayed antigens.
Methods
Virus preparation
The virus used in this study was a field isolate from
Nayarit, Mexico, designated TEV-NAY, and was propa-
gated in about 20 Burely B49 Nicotiana tabacum plants.
When the symptoms of infection were fully expressed
0.000
0.200
0.400
0.600
0.800
1.000
1.200
B1 B2 B3
O. D. at 370 nm
IgG1
C TEV
*
*
A
0.000
0.200
0.400
0.600
0.800
1.000
1.200
1.400
1.600
B1 B2 B3
O. D. at 370 nm
IgG2a
C TEV
*
*
B
0.000
0.100
0.200
0.300
0.400
0.500
0.600
0.700
B1 B2 B3
O. D. at 370 nm
IgG2b
C TEV
*
C
*
Figure 4 Analysis of the antibody responses with ELISA. Serum samples were collected from each group (C and TEV) at different times (see
the immunization and bleeding schedule in Figure 3), diluted 1:40 with PBS, and tested for anti-TEV IgG1 (A), IgG2a (B), and IgG2b (C) antibody
isotypes. Aliquots (5 μg per well) of TEV were fixed to the plate. Readings were performed with the TMB colorimetric substrate at 370 nm until
reaction saturation. The results are expressed as the means ± standard deviations (SD) of the corresponding measurements. Statistically
significant differences are indicated with an asterisk (*P < 0.05).
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systemically, 200 g of leaf tissue was ground in a blender
with two volumes of 20 M HEPES (pH 7.5), butanol (18%
final concentration), and sodium sulfite (0.1% final con-
centration) for 2 min. The glass-fiber-filtered extract was
centrifuged for 5 min at 1500 × g. The first precipitation
was performed with PEG 8000 (4% w/v), Triton X-100
(1% w/v), and NaCl (0.1 M final concentration), after stir-
ring for 1 h at 4°C and centrifugation at 3000 × g. Using a
glass homogenizer, the pellet was resuspended in 20 mM
HEPES (pH 7.5) in one quarter of the original volume.
The second precipitation was performed with 8% PEG
8000 and no Triton X-100. The pellet was resuspended in
3 mL of the same HEPES buffer and placed on 3.5 mL of
CsCl for a final centrifugation at 153,400 × g at 4°C for 10-
12 h. The viral band was dialyzed against 0.01 M HEPES
overnight at 4°C. A yield of up to 18 mg per 100 g of
infected tissue was usually obtained. Viral integrity and
purity were verified with electron microscopy.
Nucleic acid purification, amplification, and sequencing
Total RNA from TEV-infected tobacco plants was used
as the template to amplify the viral CP cistron, in an RT-
PCR reaction. The primers were designed according to
the available TEV sequences from NCBI GenBank, direc-
ted towards the two sets of five amino acids flanking the
viral CP cistron. The amplified product was cloned in the
pGEM-T Easy vector (Promega, USA) and sequenced
with an ABI Prism Sequencer (Applied Biosystems,
USA). The nucleotide sequence was compared with the
sequences available at NCBI to confirm its identity.
In silico modeling of TEV CP
Several potyviruses (papaya ringspot virus, sugarcane
mosaic virus, bean common mosaic virus, bean common
mosaic necrosis virus and TEV) were used to generate
50,000 models per sequence using the PyRosetta software,
with the 5 Å grouping restriction of the root mean square
deviation between the Cas, and selecting the model most
representative of the largest group. The lateral chains were
added, refined, and relaxed later on the model. Validation
and ranking with the ConSurf and ConQuass software,
respectively, were then performed. The final selection was
made with consideration of previously reported biochem-
ical data, the secondary structures predicted by PSIpred,
0.00
2.00
4.00
6.00
8.00
10.00
12.00
14.00
16.00
18.00
C- C+ TEV- TEV+
RELATIVE % OF DOUBLE LABELED CELLS
CD3+ CD4+
CD3+ CD4+
*
A
0.00
1.00
2.00
3.00
4.00
5.00
6.00
C- C+ TEV- TEV+
RELATIVE % OF DOUBLE LABELED CELLS
CD3+ CD8+
CD3+ CD8+
*
B
Figure 5 In vitro proliferation assay of T-cell subpopulations derived from spleen cells of TEV-vaccinated mice. Mononuclear cells were
isolated from the spleens of TEV-immunized (TEV) and nonimmunized (C) mice. To evaluate lymphocyte proliferation, the cultured cells of both
groups were either unstimulated (-) or stimulated with 5 μg of virus (+). The collected cells from each treatment were triple-stained with fluorescent
antibodies (PE-Cy5-labeled anti-CD3, FITC-labeled anti-CD4, and PE-labeled anti-CD8) and analyzed in a flow cytometer. The graphs show the relative
percentages of double-stained cells, CD3+/CD4+for helper T cells (A) and CD3+/CD8+for cytotoxic T cells (B). The results are expressed as the means ±
standard deviations (SD) of the corresponding measurements. Statistically significant differences are indicated with an asterisk (*P < 0.05).
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and the model ranking by Procheck. This final model was
used as the template to generate homologous models of
the TEV-NAY CP sequence using Modeller.
TEV surface amino group availability assay
Using a biotin-tagged reagent, Sulfo-NHS-SS-Biotin
(Thermo Scientific Pierce, USA), an experiment was per-
formed to determine the presence of amino groups
exposed on the surface of TEV particles, following the
manufacturer’s instructions. Briefly, TEV was dialyzed
overnight against 2,000 volumes of phosphate-buffered
saline (PBS; pH 7.2) at 4°C, using Slide-A-Lyzer Dialysis
Cassette Kit for 0.5-3.0 mL samples (Thermo Scientific
Pierce). The dialyzed TEV was then mixed with a 20-fold
molar excess of Sulfo-NHS-SS-Biotin prepared from a
fresh 10 mM Sulfo-NHS-SS-Biotin solution, and incu-
bated for 1 h at room temperature. The sample was then
immediately loaded onto a gel for SDS-PAGE under non-
reducing or reducing conditions (5% 2-mercaptoethanol)
and stained with Coomassie Blue. A portion of the sam-
ple was subjected to SDS-PAGE as described above, and
blotted onto Hybond ECL nitrocellulose membrane (GE
Healthcare, USA). The blot was incubated with HRP-
labeled streptavidin (KPL, USA) and then visualized with
HRP Color Development Reagent (BioRad, USA), as
recommended by the manufacturer.
Mouse immunization and bleeding
Ten four-week-old female BALB/c mice (Harlan, Mexico)
were randomly divided into two groups of five animals
each, and maintained throughout the experimentation
period in the Vaccine Evaluation Module-Animal Experi-
mentation Laboratory, Centro de Investigación y Asisten-
cia en Tecnología y Diseño del Estado de Jalisco. The
animals were cared for according to good laboratory prac-
tice guidelines. Before the experiments, the animals were
allowed to adapt to these conditions for one week.
After the adaptation period, the mice were bled from the
tail and individual preimmune sera were collected. One
week later, the mice were immunized intraperitoneally.
The immunization scheme is shown in Figure 3. On day 0,
the first immunization (priming) was performed; one
group, designated TEV, was inoculated with 25 μg of virus
solution, and the other group, used as the control and
designated C, was inoculated with the same volume of
buffer solution (20 mM Tris, pH 8.0). The mice were bled
a second time 13 days after priming. A second identical
immunization (booster) was performed on day 14. The
0
100
200
300
400
500
600
C- C+ TEV- TEV+
Concentration (pg/ml)
IFN-γ
A
0
100
200
300
400
500
C- C+ TEV- TEV+
Concentration (pg/ml)
IL-4
B


Figure 6 IFNg and IL4 levels in stimulated splenocytes from mice immunized with TEV. IFNg and IL4 levels in the supernatants of cultured
splenocytes from immunized or nonimmunized mice following restimulation with TEV or no treatment were measured by ELISA. The graphs
show the IFNg (A) and IL4 concentrations (B) in picograms per milliliter (pg/mL) for each treatment: nonimmunized/unstimulated (C-),
nonimmunized/stimulated (C+), immunized/unstimulated (TEV-), and immunized/stimulated (TEV+). The cytokine levels were calculated using
standard curves generated with known concentrations of recombinant proteins. The data are presented as the mean cytokine concentrations ±
SD of triplicate measurements. “Undetectable” (§) means below the lowest point on the standard curve.
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mice were bled a third time 27 days after priming. The
individual serum samples were stored at -70°C until
analysis.
Determination of antibody titers with enzyme-linked
immunosorbent assays (ELISAs) of sera
The sera obtained from the blood samples collected in the
immunization experiments were used to determine the
relative levels of mouse IgG1, IgG2a, and IgGb isotypes,
using a mouse monoclonal antibody isotyping kit (Sigma,
USA), following the manufacturer’s procedure for antigen-
mediated ELISA, with slight modifications. Briefly, 5 μg of
TEV diluted in coating buffer (100 mM carbonate buffer,
pH 9.6) was added to each well of a 96-well MaxiSorp
Immuno Plate (NUNC, USA) and incubated at 4°C over-
night. The plate was then blocked with 5% skim milk in
PBS-0.05% Tween 20 for 2 h at room temperature. Indivi-
dual serum samples were diluted 1:40 in PBS and loaded
into the wells in duplicate and the plate was incubated for
1 h at room temperature. Anti-mouse-isotype antibody
diluted 1:1000 in PBS was then added and the samples
incubated for 1 h. The plate was then incubated with
1:5000 HRP-conjugated anti-goat IgG antibody (Sigma,
USA). Finally, TMB liquid substrate (Sigma, USA) was
added for color development, which was monitored in an
xMark Microplate Absorbance Spectrophotometer (Bio-
Rad, USA) at 370 nm every 5 min until reaction saturation.
Isolation and purification of splenic lymphocytes
All the animals were killed on day 28, and their spleens
were collected under sterile conditions inside a laminar
flow hood, washed several times with PBS (pH 7.4), and
maintained in RPMI-1640 medium (Gibco, USA) supple-
mented with 10% fetal bovine serum (Gibco, USA), penicil-
lin-streptomycin-neomycin antibiotic mixture (Gibco,
USA), and GlutaMAX (Gibco, USA) until homogenization.
The spleens were homogenized by hand in a PYREX homo-
genizer and the cell suspensions were passed through a
40 μm cell strainer (BD Falcon, USA) to eliminate cellular
and tissue debris. The filtered homogenates from either the
TEV or C treatments were pooled and the suspensions
obtained were loaded onto a density gradient of 1.077 g/mL
Ficoll-Paque PLUS (GE Healthcare, USA) in a 1:1 ratio and
centrifuged at 2,200 × g for 25 min at room temperature.
The mononuclear-cell-enriched fraction was collected, and
washed once with RPMI-1640 medium. The cells were
counted in a Neubauer chamber with 0.4% Trypan Blue
Stain solution (Gibco, USA).
T-cell proliferation assay
Each pool of cells (TEV or C) was dispensed into six
wells of a flat-bottom 24-well cell culture dish (Costar,
USA) to a final concentration of 5 × 106cell/500 μL of
RPMI-1640 medium. Three wells were stimulated with
5 μg of TEV, and the other three wells were not stimu-
lated. The plate was incubated at 37°C for 72 h under 5%
CO2. After 24 h, 500 μL of fresh RPMI-1640 medium
was added to each well. After 48 h, 200 μL of the super-
natant was collected from each well for cytokine profiling
(below) and replaced with 200 μL of fresh medium. At
72 h, the cells were collected by centrifugation at 250 × g
for 5 min, washed once with PBS, and immediately
stained for flow cytometry.
To identify the T-cell subpopulations in the splenic
cell cultures, a set of three different antibodies was used:
phycoerythrin (PE)-Cy5-labeled anti-mouse CD3ε (Bio-
Legend, USA), fluorescein isothiocyanate (FITC)-labeled
anti-mouse CD4 (BioLegend, USA), and PE-labeled anti-
mouse CD8a (BioLegend, USA). Cultured splenic cell
replicates were pooled, divided into aliquots (106cells in
a volume of 100 μL) and triple-labeled following the
manufacturer’s instructions. The cells were then washed
once with PBS, fixed in an appropriate volume of 0.05%
paraformaldehyde in PBS, and stored at 4°C until analy-
sis in triplicate in a Beckman Coulter EPICS XL-MCL
Flow Cytometer. A control unstimulated sample from
nonvaccinated mice with no fluorescent marker or with
a single fluorescent marker was used to identify the cell
population or to adjust the color compensation settings
for the multicolor analysis, respectively. All data were
analyzed with the Beckman Coulter System II software.
Determination of IFNg and IL4 levels in the culture
supernatants
This assay was conducted to identify any Th1/Th2 bias in
response to TEV, based on the expression of the IFNg
and IL4 cytokines. For this purpose, Quantikine Mouse
IFN-g and Mouse IL-4 ELISA Kits (R&D Systems, USA)
were used to analyze the supernatants from the T-cell-
proliferation assay cultures, according to the instructions
of the manufacturer. The supernatants (200 μL) of the
cell cultures were collected after 48 h, centrifuged at
3,000 × g for 5 min, and stored at -70°C until analysis.
The cytokine levels were determined using standard
curves generated with known concentrations of the
recombinant proteins. The results are expressed in pico-
grams per milliliter (pg/mL).
Statistical analysis
Differences between groups were analyzed with Duncan’s
test. Probability values (P values) less than 0.05 were con-
sidered to be significant. Excel and StatGraphics were
used for the statistical analysis.
Acknowledgements
This work was funded by Consejo Nacional de Ciencia y Tecnología SEP-
CONACYT Project 36833. We are indebted to CONACYT for the scholarship
to CAMC.
Manuel-Cabrera et al. Virology Journal 2012, 9:75
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Author details
1Unidad de Biotecnología Médica y Farmacéutica, Centro de Investigación y
Asistencia en Tecnología y Diseño del Estado de Jalisco, Normalistas 800,
Colinas de la Normal, Guadalajara, Jalisco 44270, México.2División de
Inmunología, Centro de Investigación Biomédica de Occidente, Instituto
Mexicano del Seguro Social, Sierra Mojada 800, Independencia, Guadalajara,
Jalisco 44340, México.3Departamento de Ingeniería Genética, Centro de
Investigación y de Estudios Avanzados del Instituto Politécnico Nacional,
Unidad Irapuato, km 9.6 Libramiento Norte, Carretera Irapuato-León,
Irapuato, Guanajuato 36821, México.4Laboratorio Nacional de Genómica
para la Biodiversidad, Centro de Investigación y de Estudios Avanzados del
Instituto Politécnico Nacional, Unidad Irapuato, km 9.6 Libramiento Norte,
Carretera Irapuato-León, Irapuato, Guanajuato 36821, México.
Authors’ contributions
CAMC bled the mice, performed the conjugation experiment, antibody
titering, and cytokine profiling, and also helped to draft the manuscript.
AMA performed the mouse splenectomies and established and maintained
the spleen cell cultures. RHG immunized the mice. PCOL performed and
interpreted the flow cytometry. GCC and MCT established the in silico TEV
CP model. LSR propagated and purified the TEV and helped to draft the
manuscript. AGO conceived the study, participated in its design and
coordination, and drafted the manuscript. All the authors have read and
approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 23 October 2011 Accepted: 27 March 2012
Published: 27 March 2012
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doi:10.1186/1743-422X-9-75
Cite this article as: Manuel-Cabrera et al.: Immune response to a
potyvirus with exposed amino groups available for chemical
conjugation. Virology Journal 2012 9:75.
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