Regulation of HIV-Gag Expression and Targeting to the Endolysosomal/Secretory Pathway by the Luminal Domain of Lysosomal-Associated Membrane Protein (LAMP-1) Enhance Gag-Specific Immune Response

Abstract

We have previously demonstrated that a DNA vaccine encoding HIV-p55gag in association with the lysosomal associated membrane protein-1 (LAMP-1) elicited a greater Gag-specific immune response, in comparison to a DNA encoding the native gag. In vitro studies have also demonstrated that LAMP/Gag was highly expressed and was present in MHCII containing compartments in transfected cells. In this study, the mechanisms involved in these processes and the relative contributions of the increased expression and altered traffic for the enhanced immune response were addressed. Cells transfected with plasmid DNA constructs containing p55gag attached to truncated sequences of LAMP-1 showed that the increased expression of gag mRNA required p55gag in frame with at least 741 bp of the LAMP-1 luminal domain. LAMP luminal domain also showed to be essential for Gag traffic through lysosomes and, in this case, the whole sequence was required. Further analysis of the trafficking pathway of the intact LAMP/Gag chimera demonstrated that it was secreted, at least in part, associated with exosome-like vesicles. Immunization of mice with LAMP/gag chimeric plasmids demonstrated that high expression level alone can induce a substantial transient antibody response, but targeting of the antigen to the endolysosomal/secretory pathways was required for establishment of cellular and memory response. The intact LAMP/gag construct induced polyfunctional CD4+ T cell response, which presence at the time of immunization was required for CD8+ T cell priming. LAMP-mediated targeting to endolysosomal/secretory pathway is an important new mechanistic element in LAMP-mediated enhanced immunity with applications to the development of novel anti-HIV vaccines and to general vaccinology field.

Full text

Regulation of HIV-Gag Expression and Targeting to the
Endolysosomal/Secretory Pathway by the Luminal
Domain of Lysosomal-Associated Membrane Protein
(LAMP-1) Enhance Gag-Specific Immune Response
Rodrigo Maciel da Costa Godinho
1.
, Flavio Lemos Matassoli
1.
, Carolina Gonc¸alves de Oliveira Lucas
1
,
Paula Ordonhez Rigato , Jorge Luiz Santos Gonc
2
¸alves , Maria Notomi Sato , Milton Maciel Jr.
3 2 4,5
,
Ligia Maria Torres Pec¸anha , J. Thomas August , Ernesto Torres de Azevedo Marques Jr.
3 5 5,6,7
,
Luciana Barros de Arruda
1
*
1Departamento de Virologia, Instituto de Microbiologia Paulo de Go
´es, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil, 2Laboratorio de Dermatologia e
Imunodeficiencias, LIM-56, Departamento de Dermatologia, Faculdade de Medicina, Universidade de Sa
˜o Paulo, Sa
˜o Paulo, Brazil, 3Departamento de Imunologia,
Instituto de Microbiologia Paulo de Go
´es, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil, 4Enteric Diseases Department, Infectious Diseases Directorate,
Naval Medical Research Center, Silver Spring, Maryland, United States of America, 5Department of Pharmacology and Molecular Sciences, The Johns Hopkins School of
Medicine, Baltimore, Maryland, United States of America, 6Department of Infectious Diseases and Microbiology, Center for Vaccine Research, Pittsburgh, Pennsylvania,
United States of America, 7Departamento de Virologia, Fiocruz – Pernambuco, Recife, Brazil
Abstract
We have previously demonstrated that a DNA vaccine encoding HIV-p55gag in association with the lysosomal associated
membrane protein-1 (LAMP-1) elicited a greater Gag-specific immune response, in comparison to a DNA encoding the
native gag. In vitro studies have also demonstrated that LAMP/Gag was highly expressed and was present in MHCII
containing compartments in transfected cells. In this study, the mechanisms involved in these processes and the relative
contributions of the increased expression and altered traffic for the enhanced immune response were addressed. Cells
transfected with plasmid DNA constructs containing p55gag attached to truncated sequences of LAMP-1 showed that the
increased expression of gag mRNA required p55gag in frame with at least 741 bp of the LAMP-1 luminal domain. LAMP
luminal domain also showed to be essential for Gag traffic through lysosomes and, in this case, the whole sequence was
required. Further analysis of the trafficking pathway of the intact LAMP/Gag chimera demonstrated that it was secreted, at
least in part, associated with exosome-like vesicles. Immunization of mice with LAMP/gag chimeric plasmids demonstrated
that high expression level alone can induce a substantial transient antibody response, but targeting of the antigen to the
endolysosomal/secretory pathways was required for establishment of cellular and memory response. The intact LAMP/gag
construct induced polyfunctional CD4
+
T cell response, which presence at the time of immunization was required for CD8
+
T
cell priming. LAMP-mediated targeting to endolysosomal/secretory pathway is an important new mechanistic element in
LAMP-mediated enhanced immunity with applications to the development of novel anti-HIV vaccines and to general
vaccinology field.
Citation: Godinho RMdC, Matassoli FL, Lucas CGdO, Rigato PO, Gonc¸alves JLS, et al. (2014) Regulation of HIV-Gag Expression and Targeting to the
Endolysosomal/Secretory Pathway by the Luminal Domain of Lysosomal-Associated Membrane Protein (LAMP-1) Enhance Gag-Specific Immune Response. PLoS
ONE 9(6): e99887. doi:10.1371/journal.pone.0099887
Editor: Xiao-Fang Yu, Johns Hopkins School of Public Health, United States of America
Received February 5, 2013; Accepted May 19, 2014; Published June 16, 2014
Copyright: ß2014 Godinho et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by Grants R37-AI41908 and R21-AI44317 from the National Institute of Allergy and Infectious Diseases, National Institutes of
Health; and by Brazilian Ministry of Health, CAPES, CNPq, FAPERJ, and FINEP. R.M.C. Godinho and F.L. Matassoli were the recipients of a CNPq fellowship; C.G.O.
Lucas was the recipient of a FAPERJ fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* Email: arruda@micro.ufrj.br
.These authors contributed equally to this work.
Introduction
The magnitude and quality of the cellular and humoral
immunological responses are crucial attributes for the develop-
ment of an anti-HIV vaccine. Viral components can elicit a
substantial immune response, as observed in long-term nonpro-
gressors patients, and HIV-Gag structural protein seems to be
particularly important in this context [1–3]. The presence of
cellular immune responses directed towards this protein has been
associated to the control of HIV infection both in the acute and
asymptomatic stages and a strong anti-Gag CTL response is
inversely correlated with the viral load in HIV-infected patients
[3–6]. In addition, Gag is a well-conserved protein among
different virus strains and subtypes, indicating that this protein is
a target for the development of vaccines [7–9].
PLOS ONE | www.plosone.org 1 June 2014 | Volume 9 | Issue 6 | e99887

DNA plasmid based immunization has been shown to be a
promising strategy in inducing immune response in different
models [10–15]. The development of an anti-HIV DNA vaccine,
however, is hampered by the fact that the expression of some viral
proteins is dependent on viral regulatory elements. Specifically, the
expression of HIV-Gag is critically dependent on Rev and Rev
Responsive Elements (RREs) interactions for an efficient mRNA
stability and translocation to the cytoplasm. Consequently, Gag
protein expression is severely impaired in mammalian cells [16].
Several strategies have been used to overcome this Rev-
dependence, like codon optimization and mutation of inhibitory
sequences elements (INS) present along gag-pol sequence [17–19].
DNA immunization with these optimized sequences has been
shown to elicit antibody and cytotoxic responses [20–22].
Stimulation of CD4
+
helper T cells is essential for the induction
of sustained CTL and antibody responses [23]. In this regard, an
impaired ability to generate CD8
+
T cells has been noticed in
DNA vaccination systems, unless a CD4
+
T cell response is also
stimulated [24,25]. Since Gag-specific CD4
+
and CD8
+
T cells
proliferative responses are related to lower viral loads, an
enhanced CD4
+
T cell activation may be particularly critical for
an effective HIV vaccine and for maintaining functional CD8
+
T
cell during chronic viral infection [5,6,26,27].
The intracellular localization of an antigen can influence the
magnitude and quality of humoral immune response and can also
target the response to CD8
+
or CD4
+
T cells. In this regard,
antigen targeting to different cellular processing compartments
may improve its presentation by MHC I or MHC II molecules
and enhance specific immune response [28–32]. In addition, the
secretion of cellular proteins was reported to modulate the
immunological responses. For instance, it was observed that a
secreted form of HIV-Gag can induce a higher cellular response
after DNA immunization than plasmids encoding a cytoplasmic
form of this antigen [33]. Exosomes are endosome-derived vesicles
commonly exploited by several cell types to secrete proteins. These
vesicles are characterized by the presence of molecules related to
the lysosomes, like CD81 tetraspanin, CD63, LAMP-1 and
LAMP-2 [34–36]. Depending on the cell type, the exosomes
may be originated from the invagination of the MIIC compart-
ments and may also present proteins related to antigen presen-
tation, such as MHC II, and co-stimulatory molecules, like CD86,
what has been associated to increased vaccine efficiency [37,38].
Indeed, exosomes derived from antigen presenting cells, such as B
lymphocytes and dendritic cells are capable of antigen presenta-
tion and stimulation of T cells [39,40]. Also, exosome derived from
other cell types had been demonstrated to be directed to APCs,
mediating antigen cross priming [41,42].
We have previously shown that association of HIV-p55gag with
mouse lysosomal associated protein-1 (LAMP-1), in a form of
DNA vaccine chimera, promoted an enhanced Gag-specific
immune response, in comparison to native gag DNA [28,43,44].
In vitro studies had also demonstrated that LAMP/Gag chimera
was highly expressed and colocalized with MHCII in transfected
cell lines [43]. However, the mechanisms regulating protein
expression and intracellular targeting, as well as the relevance of
each phenomenon in the enhanced immune response were not
addressed yet. In the present study, we investigated the LAMP
sequences necessary to modulate protein expression and intracel-
lular targeting, and addressed which of these effects was associated
to the increased immune response induced by LAMP/gag DNA
vaccine. We observed that the association with LAMP-1 increases
chimeric gag mRNA levels. The increased Gag expression in the
LAMP/Gag context was dependent on LAMP-1 luminal domain
and a minimum of 247aa of this region was necessary to increase
antigen expression. The luminal domain also showed to be
essential to target Gag to lysosomes and to induce Gag secretion.
Increased expression was sufficient to induce a high acute antibody
response in immunized mice. However, the enhanced CD4
+
and
CD8
+
T cells activation, and prolonged antibody responses
seemed to depend on Gag targeting to the endolysosomal/
secretory pathway. We believe that the mechanistic study of the
immune response induced by LAMP/gag is an essential step for the
development of novel anti-HIV vaccines and may also contribute
to the development of other LAMP-based vaccines.
Materials and Methods
Plasmids
Eukaryotic expression plasmids were constructed using nucle-
otides 1–1503 of the HIV-1 HXB2 p55gag gene (GenBank
K03455) (HIV sequence Database, 1997, Los Alamos National
Laboratory Theoretical Biology and Biophysics, Los Alamos,
NM), inserted into pITR vector [45], which contains adeno-
associated virus inverted terminal repeats (AAV-ITR) flanking the
expression elements (CMV promoter and BGH polyadenylation
signal). The LAMP/gag construct was made by inserting the
p55gag (XhoI and EcoRI) sequence between the luminal domain
(lum, between NheI and XhoI) and the transmembrane domain
and cytoplasmic tail (TM-Cyt, between EcoRI and KpnI) of
mouse LAMP-1 (GenBank J03881), as described previously [43].
The LAMP
lum
/gag construct was made by the same strategy,
without the TM-Cyt insert.
The LAMP
REV-lum
/gag construct was made by replacing the
luminal domain of LAMP-1 by its oriented reverse sequence. The
LAMP-1 luminal domain in the reverse orientation was made by
PCR, using the sequence 59ccg.ctc.gag.atg.gcg.gcc.ccc.ggc.
gcc.cgg.c 39, (with the XhoI site) as the sense primer and the
sequence 59cta.gct.agc.cat.gtt.gtt.acc.atc.ctg.aac 39, (with the NheI
site) as the anti-sense primer. In this plasmid, a kozak sequence
was added in the 59end of p55gag sequence. The plasmids
containing the truncated LAMP-1 luminal domain were con-
structed by maintaining one third (372 bp; 124aa) or two thirds
(741 bp; 247aa) from the 59end of the luminal domain. Therefore,
the same sense primer used for the construction of LAMP/gag
plasmid was used to make the truncated ones. The anti-sense
primers used to make these plasmids were the following sequences:
LAMP
TM-Cyt
/gag (24aa of LAMP lum): 59ccg.ctc.gag.agc.t-
ga.ggc.gcc.atg.tgc 39; LAMP
T1-lum
/gag (124aa of LAMP lum):
59ccg.ctc.gag.att.ggg.aaa.atg.ttc.tgt.atc 39; LAMP
T2-lum
/gag (247aa
of LAMP lum): 59ccg.ctc.gag.gaa.cgc.tct.ggt.cac.cgt.ctt 39. All the
plasmids were produced by transforming DH5aE. coli (Invitro-
gen, Carlsbad, CA) and purified with endotoxin-free columns
(Qiagen Inc., Valencia, CA).
Pulse and chase and immunoprecipitation
Cells from 293 cell line (HEK293, ATCC, Rockville, MD) were
cultured in 6 well plates, at 5610
5
cells/well, in RPMI-1640
medium, containing 10% FCS, 2 mM L-glutamine and 100 U/ml
of penicillin/streptomycin (Invitrogen). The cellswere transfected
with pITR gag or pITR LAMP/gag (2 wells/plasmid), using
Lipofectamine-2000 transfection reagent (Invitrogen), according to
the manufacturers’ protocol. Following 24 hours of culture, the
medium was changed by a methionine-free RPMI medium
(Invitrogen) and the cells were starved for 45 min/37uC. Then,
25 mCi of S
35
labeled methionine (Amersham Pharmacia Biotech)
were added to each well and the plates were incubated for
45 min/37uC. The cells were washed, the medium changed by a
cold RPMI, and the cells and supernatants of these cultures were
LAMP-1 Luminal Domain Enhances Anti-Gag Immune Response
PLOS ONE | www.plosone.org 2 June 2014 | Volume 9 | Issue 6 | e99887

collected after 20 min, 1 hour and 6 hours. The cell samples were
lysed with lysis buffer and both the cell lysates and supernatants
were immunoprecipitated with anti-Gag antibody. Initially, the
samples were pre-cleared by incubation with fixed SaC (Calbio-
chem-Novabiochem Corporation, San Diego, CA) for 1 hour, on
ice, pelleted and incubated with normal goat serum (NGS) and
SaC for 1 hour more, on ice. The pellets were incubated with
mouse anti-Gag at 20 mg/ml in PBS, containing 1% BSA,
overnight at 4uC and, then, incubated with 10 mg of purified goat
anti-mouse IgG, on ice, for 1 h. The SaC was, then, added and the
samples incubated for 20 min/ice, after what, the samples were
washed twice with Pastan buffer (50 mM Tris, 5 mM EDTA,
100 mM NaCl, 2 M KCl, pH 7.5). The pellets were resuspended
in TEN buffer (100 mM Tris, 5 mM EDTA, 150 mM NaCl,
pH 8.0) with 1% NP-40, centrifuged and the obtained pellets were
resuspended in SDS-PAGE sample buffer. Each sample was
resolved in SDS-polyacrylamide gels and transferred to Immobi-
lon P membranes (Millipore, Bedford, MA). A molecular weight
marker was used as a standard (Amersham Pharmacia Biotech).
The amount of radioactivity in the bands corresponding to native
Gag or LAMP/Gag was measured in a phosphoimager.
Analysis of gag mRNA by quantitative real time RT-PCR
HEK293 cells were transfected with the indicated plasmids.
After 24 hours, mRNA was obtained either from total cell
preparation or from isolated nuclear and cytoplasmic fractions
using trizol reagent (Invitrogen), as indicated by the manufacturer.
To isolate nuclear and cytoplasmic fractions, the cells were
incubated for 10 minutes with lysis buffer (50 mM TrisHCl
pH 8.0, 100 mM NaCl, 5 mM MgCl
2
, 0.5% vol/vol Nonidet p40)
followed by centrifugation for 10 minutes at 10.000 RPM at 4uC.
The pelleted fraction corresponded to the nucleus and the
supernatant to the cytoplasm. The obtained mRNA was treated
with DNAfree kit (Applied Biosystems, Carlsbad, CA, USA) to
remove any plasmid contamination. The cDNA synthesis was
conducted using the AMV first strand cDNA synthesis kit
(Invitrogen), according to manufacturer’s protocol. The cDNA
from total cell lysate or nucleus and cytoplasm fractions were
quantified for HIV-gag presence using the SYBR green method
(Applied Biosystems), according to the manufacturer’s instructions
using the specific HIV-gag primers P24-7r (59CCC.TGA.-
CAT.GCT.GTC.ATC.A39) and P24inf (59GTC.CAA.AAG.C-
GA.ACC.CAG.ATT.GTA.A 39). For cycling and quantification a
StepOne equipment and software (Applied Biosystems) were used.
Analysis of protein expression by western blotting
HEK293 cells were transfected with the indicated plasmids, as
described above. After 48 h of culture, the supernatant and cells
were harvested, the cells were disrupted with lysis buffer (10 mM
Tris-HCl (pH 7.5) with 150 mM NaCl, 1% sodium deoxycholate,
0.1% SDS, 1% Triton X-100 and premixed protease inhibitors
(Complete, Roche Applied Science, Mannheim, Germany), for
15 min on ice, and cellular debris was removed by centrifugation.
The amount of Gag protein in the cell lysate and supernatant
fractions was analyzed by western blotting. Initially, the samples
were normalized according to the total protein concentration, as
determined by BCA (Pierce, Rockford, IL). They were then
resolved on 10% polyacrylamide gels, transferred to Immobilon
membranes (Millipore, Bedford, MA), and blocked with PBS
containing 5% nonfat dried milk. Molecular weight markers
(Amersham Pharmacia Biotech, Piscataway, NJ) were used as
standards. After washing with PBS-0.05% Tween 20 (PBS-T), the
blot was probed with mouse anti-Gag (kindly provided by Dr.
James K. Hildreth, The Johns Hopkins School of Medicine,
Baltimore, MD) at a 1:50 dilution for 2 h, washed three times and
then incubated with peroxidase-conjugated goat anti-mouse IgG
antibody (Jackson ImmunoResearch Laboratories Inc., West
Grove, PA) at a 1:10,000 dilution for 1 h. The membranes were
also probed with anti-b-actin antibody (Santa Cruz Biotechnology,
Dallas, TX), followed by anti-mouse IgG, as a loading control.
Super Signal West Pico Chemiluminescent Substrate (Thermo
Scientific) was used for protein detection according to the
manufacturer’s instructions. The ratio of interest protein/consti-
tutive protein was determined using ScionImage software.
Exosome isolation
HEK293 cells were transfected with the indicated plasmids, as
described above and, after 48 hours the culture medium was
changed by serum-free Hybridoma-SFM (Invitrogen), containing
2 mM L-glutamine and 100 U/ml of penicillin/streptomycin.
After 48 hours more, exosomes were isolated, as described
elsewhere [46,47]. Briefly, the cells were separated and the
supernatant centrifuged for 10 min at 200 g(pellet P1). The
supernatant was removed and centrifuged twice for 10 min at
500 g(the pellets were pooled and are referred to as P2).
Supernatants were sequentially centrifuged at 2,000 gtwice for
15 min (the pooled pellets are referred to as P3), once at 10,000 g
for 30 min (pellet P4) and once at 70,000 gfor 60 min (pellet P5),
being P5 enriched in exosomes. The amount of Gag protein in
P1–P5 samples was analyzed by western blot or p24-ELISA (see
below). Western blot analyses were performed as described
previously. The membranes were probed with anti-Gag, or anti-
CD81 (1:500; Santa Cruz Biotechnology), or anti-CD63 (1:1000;
Santa Cruz Biotechnology); followed by anti-mouse IgG (Jackson
ImmunoResearch Laboratories) at a 1:10,000 dilution for 1 h. For
further purification of exosomes, P5 was resuspended in 5 ml of
2.5 M sucrose, 20 mM HEPES/NaOH, pH 7.2. A linear sucrose
gradient (2.0–0.25 M sucrose, 20 mM HEPES/NaOH, pH 7.2)
was layered on the top of the exosome suspension in a SW32Ti
tube (Beckman Instruments, Inc.). Gradients were centrifuged for
15 h at 100,000 g, after which 2-ml fractions were collected from
the bottom of the tube. To collect membranes from these fractions,
they were diluted with 3 mL of PBS and centrifuged for 60 min at
200,000 g, using a Sw55Ti rotor (Beckman Instruments, Inc.). The
fractions were washed once more and each one was analyzed by
dot blot, since this technique is more sensitive than western blot
and residual sugar did not interfere in the detection (see below).
Detection of p24 by ELISA
HEK293 cells were transfected with the indicated plasmids for
48 h and the content of p24Gag in the cell lysates and
supernatants was analyzed by ELISA. ELISA plates were coated
overnight with anti-Gag M1 antibody (kindly given by Dr. James
K. Hildreth, JHU), diluted in 50 mM Tris, pH 9.5, at 10 mg/ml.
The plates were washed with PBS-T and blocked with PBS 3%
BSA for 2 h, at 37uC. The samples and HIV p24 standard were
diluted in RPMI, supplemented with 10% FCS and 1% Triton X-
100, and incubated in the ELISA plates overnight, at 4uC. After
several washes with PBS-T, the plates were incubated with
biotinylated anti-p24Gag (kindly given by Dr. James Hildreth,
JHU), diluted at 1:4,000 in PBS with 5% normal goat serum, 1%
BSA and 0.05% Tween 20, for 2 h at room temperature (RT).
The plates were then incubated with HRP-streptavidin, for
30 min, at RT, washed and developed with TMB (BD PharMin-
gen, San Diego, CA, EUA). The reaction was stopped with 1 M
H
2
SO
4
and read at 450 nm analyzed using an ELISA reader
(BioRad Laboratories Inc., Hercules, CA, EUA).
LAMP-1 Luminal Domain Enhances Anti-Gag Immune Response
PLOS ONE | www.plosone.org 3 June 2014 | Volume 9 | Issue 6 | e99887

Confocal microscopy
For the analysis of the intracellular trafficking of the constructed
chimeras, we used the MHCII-expressing DCEK.ICAM.Hi7
mouse cells [43,48] (kindly given by Dr. Susan Swain, The
Trudeau Institute, Saranac Lake, NY). These cells were
maintained in RPMI-1640 medium, and were selected every
other week by adding to the culture 6 mg/mL micophenolic acid;
250 mg/mL xantin; 15 mg/mL hipoxantin (Sigma, St. Louis, MO)
and 800 mg/mL geneticin (Invitrogen). For immunofluorescence
assay, the cells were plated in 6 well plates over poly-D-lysin pre-
treated coverslips and were maintained overnight in RPMI
medium, at 37uC/5% CO
2
. The cells were, then, transfected
with the indicated plasmids, and, after 48 hours, the coverslips
were transferred to a 24 well plate, washed with PBS, fixed with
2% paraformaldehyde and blocked with 4% normal goat serum
and 0.1% saponin. To analyze Gag localization, the cells were
stained with mouse anti-Gag antibody at 1:50 dilution, washed
with 0.1% saponin and stained with texas-red conjugated anti-
mouse-IgG at 1 mg/mL (Jackson ImmunoResearch Laboratories
Inc.). Endogenous LAMP molecules were detected by incubating
the coverslips for 1 hour with rat anti-mouse LAMP-2 (ABL-93)
supernatant medium, diluted 1:50, and endogenous Golgi
compartments were detected by staining with ABL-85 supernatant
medium, diluted 1:50. The coverslips were, then, incubated for
1 hour with FITC-conjugated anti-rat IgG at 1 mg/mL (Jackson
ImmunoResearch Laboratories Inc.). The coverslips were washed
with PBS and mounted onto glass slides, using ProLong antifade
reagent (Molecular Probes, Eugene, Oregon). Confocal microsco-
py was performed using the Wallac confocal Laser Scanning
Microscope and the images were captured individually and
digitally coloured by using Photoshop 5.0 (Adobe, San Jose).
Mice CD4
+
T cell depletion
Female BALB/c mice, 6–8weeks age, were obtained from the
mice facility of the Instituto de Microbiologia, Universidade
Federal do Rio de Janeiro (IMPPG, UFRJ), Brazil. The animals
were bred and housed according to institutional policies for animal
care and usage and the protocol was approved by The Ethics
Committee of Animal Care and Use (Comite de Etica no Uso de
Animais-CEUA) from Centro de Ciencias da Saude, UFRJ
(Permit Number: IMPPG 025). The mice (4 mice/group) were
treated with purified rat IgG against mice CD4 molecule obtained
from GK1.5 hybridoma (ATCC TIB-207; kindly provided by Dr.
Jose´ Mauro Peralta, Universidade Federal do Rio de Janeiro,
Brazil). Each animal received an intraperitoneal injection of
100 mg/100 mL/mouse for 3 days followed by a 4 days’ rest. The
injections continued along the whole experiment with an interval
of 4 days between treatments. The efficacy of this procedure was
evaluated by flow cytometry achieving 70–85% T CD4
+
depletion
(data not shown).
Mice immunization
Female BALB/c mice, 6–8 weeks old, were immunized twice,
i.d., with the indicated plasmids at 50 mg/50 mL/mouse, at a 3-4
weeks interval.
Antibody response
Mice sera were obtained from the tail vein before the first
immunization (pre-bleed) and at different time points after the
second immunization, and the individual serum IgG levels were
measured by ELISA. Briefly, ELISA plates were coated with
50 mL of HIV
IIIB
lysate at 5 mg/ml (ABI, Rockville, MD) and
incubated at 4uC, overnight. The plates were blocked with PBS
containing 10% FCS for 2 hours/37uC, washed with PBS-T and
the serum samples were added, in serial dilutions, and incubated at
4uC/overnight. The plates were, then, incubated with HRP-
conjugated anti-mouse IgG (1:5,000; Jackson ImmunoResearch
Laboratories Inc.) for 2 hours/37uC, washed and developed using
TMB substrate (Pharmingen, San Diego, CA). After 30 minutes,
the reaction was stopped with 1 M H
2
SO
4
and read at 450 nm
using an ELISA reader (BioRad Laboratories Inc.).
T lymphocyte activation
ELISPOT assay. The activation of CD4
+
and CD8
+
T
lymphocytes was analyzed by ELISPOT assays, using the IFN-c
ELISPOT set from BD-Biosciences Pharmingen (San Diego, CA),
according to manufacturer’s protocol. Initially, ELISPOT plates
were coated with anti-IFN-cantibody at 5 mg/mL and incubated
at 4uC/overnight. The plates were blocked with RPMI 1640,
containing 10% FCS, for 2 hours at RT and then, total
splenocytes (10
6
cells/well), obtained from each immunized
mouse, were cultured in the presence of culture medium (RPMI
1640 medium supplemented with 5% FCS, 100 units/mL
penicillin/streptomycin, 2 mM L-glutamine, 50 mM 2-mercapto-
ethanol and 1 M HEPES buffer) or recombinant baculovirus
HIV
SF2
p55 Gag (5 mg/mL; NIH AIDS Research and Reference
Reagent Program), to analyze the CD4
+
response; or with the
MHC I restricted Gag epitope AMQMLKETI
65-73
(10 mg/mL),
to analyze the CD8
+
response, as indicated in the results. After
24 hours of culture, the plates were washed and incubated with
biotinylated anti-IFN-cantibody for 2 hours at room temperature,
followed by incubation with HRP-conjugated avidin, for 1 hour/
RT. The reaction was developed with AEC substrate (Calbio-
chem-Novabiochem Corporation, San Diego, CA). Analysis of the
IFN-clevels was performed using the Immunospot Analyzer
software (BD Biosciences, San Diego, CA). The data indicate the
average number of spot forming cells (SFC) obtained from
individual mice.
Analysis of cytokine production by ELISA. Splenocytes
(10610
6
cells/mL) were cultured in triplicate in a 96-well plate in
the presence of recombinant baculovirus HIV
SF2
p55 Gag, or 15-
mers spanning the whole Gag protein, or T CD4
+
-restricted Gag
peptide pools, or T CD8
+
-restricted Gag peptide pools [28]. All
peptide pools were at the same concentration (5 mg/mL; NIH
AIDS Research and Reference Reagent Program). As negative
and positive controls, cells were incubated with culture medium
alone or concanavalin A (ConA) (BD Biosciences), respectively.
Culture supernatants were harvested after 72 h for the quantita-
tion of secreted TNF-aor IFN-cusing an OPTEIA ELISA kit (BD
Biosciences).
Analysis of CCR7 expression and intracellular cytokine
staining by flow cytometry. Splenocytes harvested from the
immunized mice were incubated with the described Gag peptides,
in the presence of brefeldin A (eBiosciences Inc, San Diego, CA)
for 10 h, at 37uC. After incubation, cells were washed twice with
FACS buffer (HBSS, supplemented 2% FCS, 1 mM Hepes, 0.1%
NaN
3
), and nonspecific binding was blocked by incubating cells
with anti-FccR antibody (BD Pharmingen) at 10 mg/mL for
15 min at 4uC. The splenocytes (1610
6
cells/well) were stained in
duplicate with PerCP-conjugated rat anti-mouse CD4
+
antibody,
and/or PE-conjugated anti CCR7 (BD Pharmingen) at a dilution
of 1:100 for 30 min at 4uC. The cells were washed twice with
FACS buffer and resuspended in 200 mL of Cytofix/Cytoperm
solution at 4uC for 20 min. Cells were then washed twice with
Perm/Wash solution and stained with APC-conjugated anti-IFN-
c, or APC-conjugated anti-IL-2 and PE-conjugated anti-TNF-a
antibodies (BD Biosciences) diluted 1:100. Events acquisition was
LAMP-1 Luminal Domain Enhances Anti-Gag Immune Response
PLOS ONE | www.plosone.org 4 June 2014 | Volume 9 | Issue 6 | e99887

performed with a FACScalibur flow cytometer instrument and
data was analyzed with CellQuest software (BD Biosciences). A
minimum of 50,000 events were analyzed.
Statistical analysis
Statistical analysis of the results was based on unpaired t-tests
and chi-squared independence tests. p values ,0.05 were
considered statistically significant.
Results
Association of HIV-Gag with LAMP-1 increases gag mRNA
transcription
We have previously demonstrated that a DNA plasmid
construct containing the sequence of HIV-p55gag inserted between
the luminal and the transmembrane and cytoplasmic tail of
LAMP-1 was highly expressed after transfection of different cell
lines [28,43]. In order to verify whether the increased Gag
expression was related to a higher stability of LAMP/Gag protein
chimera, in comparison to native Gag, we performed pulse and
chase experiments and determined the degradation rate of each
protein. HEK293 cells were transfected with either native gag (gag
N
)
or LAMP/gag DNA plasmids. After 24 hours, the cells were pulsed
with S
35
and, then, chased for 20 minutes, 1 hour or 6 hours in
cold medium, and the samples were immunoprecipitated with
anti-Gag antibody. We observed that, while the amount of protein
produced following transfection with the native gag DNA was
much lower than the one produced by LAMP/gag [28,43]
(Figure 1A), their degradation curves were very similar
(Figure 1B). The degradation rate of LAMP/Gag chimera was
slightly higher, but this could be associated to the lysosomal
targeting and secretion of this protein, since LAMP/Gag was also
observed in the supernatant of transfected cells, whereas there was
no appreciable amount of native Gag in the supernatant
(Figure 1B, insert).
We then investigated if the modulation of Gag expression by
LAMP would be correlated either to mRNA transcription or
translocation to the cytoplasm. HEK293 cells were transfected
with gag
N
or LAMP/gag plasmids and the mRNA was extracted
from the whole cell lysate, or from isolated nuclear or cytoplasmic
fractions. HIV-gag mRNA was then quantified. We observed a
significant difference in the total mRNA concentration between
gag and LAMP/gag (Figure 1C), but the distribution between the
nucleus and the cytoplasm was very similar for both chimeric
genes (Figure 1D), indicating that there was no difference in the
translocation rate between these mRNAs. These results suggest
that the presence of LAMP signals in the chimeric construct
increased mRNA transcription or stability, resulting in higher
steady state levels of LAMP/gag mRNA.
The increased LAMP/Gag expression is mediated by
LAMP-1 luminal domain
We analyzed the role of the different LAMP-1 domains in the
regulation of Gag expression. Several DNA plasmids were
constructed by deleting either the transmembrane-cytoplasmic
tail (TM-Cyt) (plasmid LAMP
lum
/gag), or the luminal domain of
LAMP in the LAMP/gag chimera (plasmid LAMP
TM-Cyt
/gag)
(Figure 2). In the latter construct (LAMP
TM-Cyt
/gag), we
maintained 24aa of the 59terminal of LAMP, correspondent to
ER signal sequence. HEK293 cells were transfected with these
plasmids and Gag expression was analyzed by western blot
(Figure 3). The result presented in figure 3A confirms our previous
observation of an increased LAMP/Gag expression in comparison
to native Gag, and demonstrated that the deletion of LAMP
cytoplasmic domain did not affect Gag expression. In contrast,
deletion of the luminal domain led to a decreased Gag expression
to the level obtained with native Gag. Additionally, cell
transfection with a chimeric plasmid containing the LAMP
luminal domain in a reverse orientation at the 59end of the start
site of the gag gene also showed a decreased Gag expression,
comparable to native Gag (Figure 3B).
To identify the minimum sequence of LAMP luminal domain
necessary to increase Gag expression, we generated two other
truncated LAMP/Gag chimeras, with deleted sequences from the
39end of the LAMP luminal domain. One of the constructs,
LAMP
T1-lum
/gag, contained one third (372 bp–124aa) of the
luminal domain; and the other, LAMP
T2-lum
/gag, contained two
thirds (741 bp–247aa) of the luminal domain (Figure 2). HEK293
cells were transfected with these chimeras and their expression in
the cell lysate or culture supernatant were compared with the ones
Figure 1. Association of HIV-1 gag with LAMP-1 does not
protect Gag protein from degradation, but increases Gag
mRNA production. A) HEK 293 cells were transfected with native gag
(gag
N
) or LAMP/gag. After 48 hours, the expression of Gag protein was
analyzed by western blotting, by staining with mouse anti-Gag
antibody, followed by HRP-conjugated anti-mouse IgG. B) HEK 293
cells were transfected with gag
N
or LAMP/gag. After 24 hours, the cells
were pulsed with S
35
and, then, chased for 20 min, 1 h and 6 h. The
cells and supernatants were collected and immunoprecipitated with
anti-Gag antibody. The protein content in each sample was analyzed in
a phosphoimager and the curves represent the proportion of protein
observed at each time point in relation to the maximum value (100%);
the insert indicates the values obtained in the supernatant (sup) of
LAMP/gag transfected cultures. C and D) HEK 293 cells were transfected
with the indicated plasmids and, after 48 h, total RNA was obtained
from either whole cells (C) or from isolated nuclear (nuc) and
cytoplasmic (cyt) fractions (D). After reverse transcription using oligodT
primers, the amount of gag cDNA was evaluated by qPCR. Gag
concentration were normalized using the b-actin CT number. Data is
representative of three independent experiments. * p,0.05.
doi:10.1371/journal.pone.0099887.g001
Figure 2. Schematic representation of the constructed plas-
mids containing different domains of LAMP-1 associated to
p55
gag
.p55gag (black rectangles) sequence was inserted between the
intact or truncated luminal domain of LAMP (gray rectangle) and the
transmembrane and cytoplasmic tail of LAMP (TM/Cyt; striped
rectangles).
doi:10.1371/journal.pone.0099887.g002
LAMP-1 Luminal Domain Enhances Anti-Gag Immune Response
PLOS ONE | www.plosone.org 5 June 2014 | Volume 9 | Issue 6 | e99887

observed with the complete LAMP/gag construct and with the
construct containing only the whole luminal domain (LAMP
lum
/
gag). The amount of protein detected was proportional to the
length of LAMP luminal domain and a minimum of 247aa of the
luminal sequence was necessary to achieve an optimal expression
level (Figure 3C). In addition, cell transfection with the intact
LAMP/gag or with LAMP
lum
/gag induced the secretion of high
levels of Gag protein and, similarly, a minimum of 247aa was
required to induce Gag secretion, although at a lower level
(Figure 3C).
An intact LAMP luminal domain is necessary to target
Gag to lysosomes and exosome secretory pathway
We analyzed how much of the LAMP luminal domain was
required to promote Gag-targeting to the lysosomal compart-
ments. Mouse DCEK cells were transfected with the truncated
LAMP/Gag chimeras and their localization in lysosomes or Golgi
complex were analyzed by confocal microscopy, using anti-
LAMP-2 and anti-gp125 (ABL85 hybridoma) antibodies, respec-
tively. We observed that the truncated LAMP/Gag chimeras were
scarcely present at lysosomes in transfected cells, but they all
showed strong colocalization with Golgi apparatus (Figure 4),
suggesting that without essential sequences present in luminal
domain, the chimeras are retained at these compartments and are
not able to traffic through lysosomal/secretory pathway.
Since previous reports demonstrated the presence of LAMP in
exosomes in some cell types [34,49], we investigated whether
LAMP/Gag was being secreted in these vesicles. The supernatant
of LAMP/Gag-transfected cells was submitted to serial centrifu-
gations up to 70,000 g (pellet 5-P5), where the exosomes are
usually enriched. Western blot analysis demonstrated that Gag was
present in all supernatant fractions, including P5, where the CD81
tetraspanin was also enriched, strongly suggesting that this is
actually related to exosomes (Figure 5A). Still, since Gag protein
can form aggregates, and these could be pelleted in the same
fraction as the exosomes, we further purified the P5 fraction in
sucrose gradients and observed the presence of Gag in the
fractions related to membrane conjugates, confirming that
LAMP/Gag is partially secreted in exosomes (Figure 5B). To
further confirm the importance of the LAMP-1 luminal domain in
promoting Gag traffic through lysosomes/exosomes vesicles, we
also investigated fractionated supernatants form HEK293 cells
transfected with LAMP
lum
/gag, which contain only the luminal
domain of LAMP. Similar to complete LAMP/gag, LAMP
lum
/
Gag chimera was strongly expressed in all supernatant fractions,
including P5 (Figure 5C). The membranes were also probed with
anti-CD63, another exosome marker, which was also enriched in
P5 fraction, corroborating the previous data. These data suggest
that the whole LAMP luminal is necessary and maybe sufficient to
target Gag to the exosome secretory pathway.
We could not detect the LAMP
T2-lum
/Gag chimera in the
fractionated supernatants by western blot (data not shown).
Therefore, we also analyzed the Gag protein amount in the
cellular lysate and in all supernatant fractions isolated from
LAMP
T2-lum
/gag, LAMP/gag or LAMP
lum
/gag transfected cells by
p24 ELISA. Initially, we determined the proportion of Gag in the
total supernatant in comparison with cell lysates. After cell
transfection with LAMP
T2-lum
/Gag, 15% of total Gag was present
in the supernatants and around 85% was present in the cell lysates.
In contrast, samples obtained from LAMP/gag and LAMP
lum
/gag-
transfected cells showed around 70 and 75% of Gag in the
supernatants, respectively (Figure 5D). After fractionating the
supernatants, we observed that LAMP
T2-lum
/gag were highly
enriched in the P4 fraction, differently from LAMP/gag or
LAMP
lum
/gag, that were also enriched in P5 (Figure 5E).
Figure 3. LAMP-mediated increased Gag expression is depen-
dent on LAMP luminal domain. A–B) HEK293 cells were transfected
with the plasmids represented in Figure 2. After 48 h, the amount of
Gag protein was analyzed by western blotting, by staining with mouse
anti-Gag antibody, followed by HRP-conjugated anti-mouse IgG. The
membranes were also probed with b-actin, as a loading control. Bars
indicate the ration between Gag expression and b-actin, as measured
with ImageJ software. C) HEK293 cells were transfected with the
indicated plasmids and, 48 h later, the amount of Gag protein in the cell
lysates (cell) and culture supernatants (sup) were analyzed as in (A).
Data is representative of four independent experiments.
doi:10.1371/journal.pone.0099887.g003
Figure 4. Truncated LAMP/
Gag
chimeras poorly colocalize with
endogenous LAMP. Mouse DCEK cells were transfected with the
indicated plasmids. After 48 h, intracellular localization of Gag was
analyzed by confocal microscopy after incubating the cells with mouse
anti-Gag antibody, followed by incubation with texas-red anti-mouse
IgG. Colocalization with Golgi compartments was analyzed by staining
with a rat anti-mouse golgi gp125, followed by incubation with a FITC-
conjugated goat-anti-rat IgG. Colocalization with endogenous LAMP
was analyzed by staining with a rat anti-mouse LAMP-2, followed by
incubation with a FITC-conjugated goat-anti-rat IgG. Data is represen-
tative of four independent experiments.
doi:10.1371/journal.pone.0099887.g004
LAMP-1 Luminal Domain Enhances Anti-Gag Immune Response
PLOS ONE | www.plosone.org 6 June 2014 | Volume 9 | Issue 6 | e99887

LAMP luminal domain-mediated high expression and
targeting to lysosomal/secretory pathway promote an
enhanced anti-Gag immune response
We verified here that 247aa of the luminal sequence was
necessary to promote high levels of Gag expression but only the
complete and intact luminal domain was able to induce Gag-
targeting to the endolysosomal/secretory pathway. In order to
determine the effects of protein expression and cellular traffic on
immune responses, mice were immunized with the native gag and
with the different LAMP/gag constructs, and both the antibody
and the cellular responses were analyzed (Figure 6). The amount
of anti-HIV IgG was measured ten days after two DNA
immunizations, and we observed that the construct containing
247aa of the luminal domain (LAMP
T2-lum
/gag) induced an
antibody response level similar to the intact LAMP/gag (Figure 6A).
In contrast, both the CD4 and, mostly, the CD8 T cell response,
were remarkably impaired in the mice immunized with any of the
truncated LAMP/gag constructs, in comparison to the DNA
encoding the intact LAMP/gag (Figures 6B and 6C). These data
suggest that high protein expression is sufficient to elicit a
significant antibody response, but not cellular activation, which
seems to depend on intracellular traffic. Interestingly, immuniza-
tion of with LAMP
lum
/gag also increased production of IFN-cby
CD4
+
and CD8
+
T cells, as measured by ELISA and flow
cytometry. The T cell response observed in LAMP
lum
/gag
immunized mice were similar than the one induced by complete
LAMP/gag plasmid, which was much higher than the response
induced by native gag immunization (Figure S1A and B). These
data indicate that, in the system presented here, association of Gag
antigen with LAMP-1 luminal domain was sufficient to elicit a
potent Gag-specific T and B cell-mediated immune response.
Since T cell, particularly CD4
+
T cell priming, is important to
induce and maintain memory response in general, we questioned
whether the increased antibody production induced by truncated
LAMP
T2-lum
/Gag construct would be sustained for longer periods
of time.
Thirty days after the second immunization, the titer of anti-HIV
serum IgG antibodies was similar between LAMP
T1-lum
/gag,
LAMP
T2-lum
/gag and native gag immunized mice, and none of
them were comparable to the ones induced by intact LAMP/gag
construct. Consistent with our previous data, antigen-specific IgG
antibody response induced by the LAMP/gag chimera was
sustained at titers greater than 1:3,000 for at least three months
Figure 5. LAMP luminal domain induces LAMP/
Gag
secretion
through exosomes. A) HEK293 cells were transfected with LAMP/gag
and, after 48 hours, the supernatants were harvested and subjected to
differential centrifugations as described in Material and Methods. Cell
lysates (cell), the intermediate pellets obtained after each centrifugation
step (P1–P4), and the exosome fraction (P5) were obtained and Gag
expression was analyzed by western blotting. The membranes were
also probed with anti-CD81, as an exosome marker. B) P5 fraction
obtained as in (A) was brought to 2.5 M sucrose, overlaid with a
continuous sucrose gradient and subjected to equilibrium centrifuga-
tion. Fifteen fractions were collected and subjected to dot blot analysis
using anti-Gag antibody. Densitometry analysis was performed using
phosphoimager software and the results indicate the percentage of
each dot intensity in relation to the sum of all dots. Membrane-
associated fractions are indicated with a line in the bottom. C) HEK293
cells were transfected with LAMP/gag or HEK293 cells were transfected
with LAMP
lum
/gag. After 48 hours, the supernatants were harvested
and subjected to differential centrifugations as described. P1–P5
fractions were analyzed by western blotting, using anti-Gag or anti-
CD63 antibodies D–E) HEK293 cells were transfected with LAMP
T2-lum
/
gag, LAMP/gag or LAMP
lum
/gag. After 48 h, cells and supernatants were
harvested, fractionated as in (A), and the concentration of Gag was
measured by p24-ELISA. The proportion of Gag protein in the cell
lysates and total supernatant fraction is demonstrated in (D); the
percentage of Gag in each supernatant fraction in relation to total
supernatant is demonstrated in (E). Data is representative of three
independent experiments.
doi:10.1371/journal.pone.0099887.g005
Figure 6. High protein expression and targeting to secretory
pathway induced by LAMP luminal domain potentiate the
immune response to HIV-Gag. Balb/c mice were immunized twice
with the indicated plasmids, i.d, at 50 mg/mouse. A) The serum of each
mouse was collected before the immunization (pre-bleed) and 10 days
after the second immunization and the amount of anti-HIV IgG was
measured by ELISA. The curves indicate the average O.D. levels
obtained at different serum dilutions. B–C) Total splenocytes obtained
from individual mice were cultured with p55Gag protein (B), or with the
MHC I restricted Gag epitope AMQMLKETI65-73 (C) and IFN-c
production was analyzed by ELISPOT assay. The bars indicate the
average of SFC/10
6
cells, subtracting the values obtained with medium
only. D) Mouse serum of individual mice was collected at the indicated
time points after immunization with the indicated plasmids. The
amount of anti-HIV IgG was measured by ELISA as in (A). The bars
indicate the dilution point relative to 50% of the maximum O.D. (IgG
titer). The data are representative of three independent experiments. *
p,0.05.
doi:10.1371/journal.pone.0099887.g006
LAMP-1 Luminal Domain Enhances Anti-Gag Immune Response
PLOS ONE | www.plosone.org 7 June 2014 | Volume 9 | Issue 6 | e99887

after the immunization (Figure 6D). Taken together, these results
indicate that a combination of high expression and targeting to
secretory cellular pathway promote more complete and long
lasting responses.
Immunization with LAMP/gag induces polyfunctional T
cell response
An efficient vaccine response against HIV infection requires the
activation of polyfunctional T cells and the development of central
memory T cells [50–54]. Therefore, to further investigate the
effect of LAMP/gag immunization on the T cell response, we
analyzed the phenotype and the expression of different cytokines
by Gag-specific T cells (Figure 7). We observed that T cells
obtained from mice immunized with LAMP/gag produced IFN-c,
IL-2 and TNF-a, as detected by intracellular cytokine staining
(Figure 7A, B). TNF-asecretion was also evaluated by ELISA after
T cell culture with either CD4- or CD8-restricted peptides and we
confirmed that immunization with LAMP/gag induced an
enhanced secretion of this cytokine by both T cell subpopulations,
in comparison to immunization with native gag (Figure 7C).
Central memory T cells are characterized by the expression of
chemokine receptors targeting to lymphoid organs. Therefore, we
analyzed the expression of CCR7 in the CD4
+
T cells obtained
from native gag and LAMP/gag immunized mice and observed
that LAMP/gag induced an increased expression of this receptor in
CD4
+
T cells (Figure 7D). Since naı
¨ve T cells also express this
receptor we investigated whether the CCR7
+
were primed cells by
measuring IFN-cproduction. Indeed, 5.15% of CD4
+
T cells
obtained from LAMP/gag immunized mice were CCR7
hi
IFN-c
+
,
in comparison to only 3.14% of the cells obtained from mice
immunized with native gag (Figure 7E).
CD8
+
T cell activation induced by LAMP/gag
immunization is dependent on CD4
+
T cells
In an effort to elucidate the importance of CD4
+
T cell
activation in our vaccination model we evaluated whether the
activation markers detected in LAMP/gag immunized mice would
also be elicited in the absence of CD4
+
T cells. Mice were depleted
of CD4
+
T cells using specific anti-CD4 antibody and, then,
immunized with LAMP/gag. After 2 DNA immunizations, total
splenocytes were cultured with CD8-restricted Gag peptides and
the secretion of TNF-awas evaluated. We observed that T cells
obtained from animals immunized with LAMP/gag in the absence
of CD4
+
cells produced much lower amounts of TNF-a,
comparable to the levels induced by immunization with native
gag or pITR vector (Figure 8A). In addition, we observed a
decrease in the expression of CCR7 in the CD4-depleted mice and
a lower expression of IFN-camong CCR7
hi
CD4
2
or
CCR7
l
uCD4
2
cells (Figure 8B). The data demonstrated that
CD4
+
T cells were essential for the enhanced T cell response
elicited by LAMP/gag.
Discussion
Here, we dissected the roles of protein expression and cellular
localization on the anti-HIV immune response. We have
previously demonstrated that immunization of mice with a
chimeric DNA plasmid containing HIV-1 p55gag inserted between
luminal and transmembrane/cytoplasmic tail domains of LAMP-1
(LAMP/gag) elicited a much greater and prolonged Gag-specific
immune response, when compared to immunization with native
Figure 7. LAMP/
gag
immunization induces polyfunctional and
memory CD4
+
T cells. Balb/c mice were immunized twice with the
indicated plasmids. Fifteen days after the second immunization, the
splenocytes were cultured with CD4- or CD8-restricted Gag peptides
and the phenotype and cytokine production were analyzed. A–B) The
cells were stained with PercP-anti-CD4 and APC-anti-IFN-c, or with
PercP-anti-CD4, APC-anti-IL-2 and PE-anti-TNF-a, and analyzed by FACS.
Dot blots indicate CD4 and IFN-cstaining (A) or TNF-aand IL-2 staining
among CD4
+
cells (B). C) Culture supernatants were collected and the
amount of TNF-awas analyzed by ELISA. D–E) Cells were stained with
PercP-anti-CD4, PE-anti-CCR7 and APC-anti- IFN-c. CD4
+
cells were
gated and the percentage and/or mean fluorescence intensity (MFI) of
CD4
+
CCR7
+
IFN-c
+
cells were analyzed by FACS. D) Histograms indicate
CCR7 expression among CD4
+
cells. pITR vectors are in black; gag
N
are
in dashed line and LAMP/gag are in line histogram. The numbers
indicated MFI values. E) Dot blots indicate the percentage of CCR7 and
IFN-cexpression among CD4
+
cells. Numbers indicate the percentage
of IFN-c
+
cells among CCR7
hi
and CCR7
l
uCD4
+
gated cells. The data are
representative of three independent experiments. * p,0,05.
doi:10.1371/journal.pone.0099887.g007
Figure 8. Enhanced immune response induced by LAMP/
gag
immunization is dependent on CD4
+
T cells. Balb/c mice were
treated or not with anti-CD4 antibody and immunized with LAMP/gag,
as described in Material and Methods. Mice were also immunized with
pITR vector or gag
N
, as controls. Fifteen days after the second
immunization, total splenocytes were cultured with CD8-restricted
gag peptide and cytokine production was analyzed. A) TNF-asecretion
was analyzed by ELISA. B) The cells were incubated with PercP-anti-
CD8, PE-anti-CCR7 and FITC-anti-IFN-cand the expression of CCR7 and
IFN-camong CD8
+
cells were analyzed by FACS. The data are
representative of two independent experiments. * p,0,05.
doi:10.1371/journal.pone.0099887.g008
LAMP-1 Luminal Domain Enhances Anti-Gag Immune Response
PLOS ONE | www.plosone.org 8 June 2014 | Volume 9 | Issue 6 | e99887

gag [28,43,44]. Given that increased protein expression/stability
and differential antigen targeting can both influence the elicited
immune response [21,33,55], we evaluated the mechanisms
involved in these processes. We made several plasmid DNA
constructs, in which the sequence of HIVp55gag was associated
with intact or truncated domains of LAMP-1. The expression level
and intracellular localization of the chimeric antigens were
investigated in transfected cells and constructs inducing different
patterns of expression and cellular targeting were inoculated in
mice for analysis of the immune response.
We observed that the luminal domain was essential to LAMP-
mediated increased expression of Gag, which was associated to an
increased mRNA level in comparison to native gag. HIV structural
proteins, including Gag, are poorly translocated to the cytoplasm
[56]. The expression of Gag is hampered by the presence of
inhibitory elements that are binding sites for cellular factors
associated to mRNA instability, nuclear retention, and inefficient
translation [57,58]. During HIV infection, Gag expression
requires the activity of the viral Rev protein, which is postulated
to counteract the action of these factors [59]. Different strategies
had been used to overcome gag poor translation in order to obtain
an efficient protein expression [20-22,60]. These strategies were
supposed to either enhance translational efficiency, or to alter
RNA export to the cytoplasm. The effect of LAMP on Gag
expression, on the other hand, did not seem to be associated to
protein stability or mRNA export to the cytoplasm. Our data
suggests that addition of LAMP luminal domain upstream gag
promoted enhanced increased levels of mRNA either by increased
transcription or mRNA stability. Since no modification of the Gag
inhibitory elements or codon usage was attempted, it is possible
that the synthesis of LAMP/Gag chimera was being regulated by
cellular mechanisms involved in the LAMP expression directed by
signals present in the luminal domain.
The LAMP luminal domain also showed to be essential to Gag
targeting to lysosomal compartments. Our previous studies
showed that association of gag
N
with the transmembrane/
cytoplasmic domain of LAMP was not sufficient to target the
antigen to the MHCII-containing cellular compartments [43].
Therefore, we investigated the intracellular localization of the
chimeras containing truncated sequences of the luminal domain.
We observed that, different from native Gag, the truncated
chimeras were present in cellular vesicles and were able to traffic
to Golgi compartments. However, in spite of the presence of the
LAMP targeting signal, they barely reached the lysosomal
compartments, in contrast to the construct containing the whole
luminal domain, which had been largely demonstrated to
colocalize with endogenous LAMP [28,43,61].
The targeting of Gag mediated by the intact LAMP luminal
domain also culminated in the secretion of the chimera partly
associated to exosome-like vesicles. Several studies have associated
antigen-containing exosomes to enhanced antigen presentation
and activation of T cells. In fact, exosome-based cell free vaccines
demonstrated to induce specific T cell responses in vivo [37].
Exosomes derived from antigen presenting cells (APC) contain
MHC II and co-stimulatory molecules, and can directly stimulate
CD4
+
T cells [39,40,49]. Even when secreted by other cell types,
the exosomes may transfer intracellular antigens directly to APCs
and promote antigen cross-presentation [42]. This is particularly
interesting in the context of a DNA vaccine. Although inoculation
of DNA plasmids can induce immune response after direct
transfection of APCs, most of the inoculated DNA is probably
captured by other cell types. In this case, antigens that remain cell-
associated may not be efficiently delivered to APCs and need to be
secreted or transferred to APC by cross-priming to induce immune
response [62,63]. Indeed, association of tumor-derived antigens,
HIV-gp120, and other antigens with signal delivering to exosomes
was shown to increase T and B cell responses [37,38,64,65].
We could not detect native Gag in the supernatant of
transfected cells, in spite of its well-known ability to generate
secreted virus-like particles (VLP). It is possible that the amount of
synthesized protein in its native form, in the absence of Rev, was
not sufficient to allow VLP generation and release. Secreted HIV
Gag VLPs usually bud from the plasma membrane and not from
vesicles originated from endolysosomes [17]. Accordingly,
LAMP
T2-lum
/Gag chimera, which showed to be strongly ex-
pressed, was secreted by the transfected cells, although not
significantly detected in the exosome fraction. These data suggest
that the targeting of Gag to endolysosomal vesicle by the whole
luminal domain of LAMP influenced its secretion pathway.
Given that we constructed plasmids that differ in their
expression level and cellular traffic, we were able to evaluate the
relative importance of these features for the Gag-specific immune
response elicited by LAMP/gag DNA vaccines. Mice were
immunized with (i) native gag, (ii) the truncated LAMP/gag
chimeras (LAMP
T1-lum
/gag or LAMP
T2-lum
/gag), and (iii) the intact
LAMP/gag, and the T and B cell responses were analyzed. It was
observed that the anti-HIV antibody response elicited by these
plasmids was proportional to the length of the luminal domain.
Immunization with the truncated LAMP
T2-lum
/gag plasmid, which
induced the same expression level as the intact LAMP/gag, elicited
a similar level of anti-HIV serum IgG antibodies, indicating that
protein expression level was proportional to the magnitude of
acute antibody response. Analysis of the induced T cell response,
however, demonstrated that, although the level of activation was
also proportional to Gag expression, all the truncated plasmids
elicited a significantly lower IFN-cproduction by CD4
+
or CD8
+
T cells than the plasmid containing the whole LAMP luminal
domain.
Gag targeting to the endolysosomal compartments may not only
facilitate its presentation by MHC II molecules and increase CD4
+
T cells activation, but also, the secretion of Gag may improve
cross-priming and directly activation CD8
+
T cells. It has been
described that dendritic cells pulsed with a particulate form of the
hepatitis B antigen processed the antigen in the endolysosomal
compartment and efficiently primed CTL, inducing a higher T
cell response in comparison to equimolar concentration of the
peptide in a non-particulate form [66]. Although there is no
experimental evidence demonstrating Gag secretion in vivo, the fact
that the truncated LAMP
T2-lum
/gag plasmid did not induce the
same level of T cell response, in spite of a high level of Gag
expression, indicates that Gag traffic may be the main event
regulating the potent T cell activation mediated by LAMP/gag.
Accordingly, previous studies demonstrated that DNA vaccines
that generate Gag secreted as VLP, or in a soluble form, induce
different levels of T and B cell activation, which were also different
from the response induced by cytoplasmic Gag [33].
Since T cell activation is essential to induce memory response,
we evaluated whether the level of antigen-specific IgG antibodies
induced by LAMP
T2-lum
/gag would be sustained in spite of the low
T cell activation induced. Analyses of the IgG antibody titer at
different time points after immunization demonstrated that the
anti-HIV IgG levels induced by LAMP
T2-lum
/gag rapidly de-
creased, in contrast to the levels induced by intact LAMP/gag,
which was maintained for at least three months after vaccination.
These data demonstrated that the T cell activation associated to
differential antigen traffic was essential to promote prolonged
antibody response after immunization.
LAMP-1 Luminal Domain Enhances Anti-Gag Immune Response
PLOS ONE | www.plosone.org 9 June 2014 | Volume 9 | Issue 6 | e99887

CD4
+
T cells have also been reported to be essential for the
activation of CD8
+
T cells. We demonstrated here that LAMP/gag
immunization induced polyfunctional CD4
+
T cells able to
produce IFN-c, TNF-aand IL-2. Furthermore, those cells
presented a phenotype of central memory T cells, expressing
CCR7 and IFN-c. The importance of CD4
+
T cells for the whole
T cell activation was clearly evidenced after immunization of mice
depleted of CD4
+
T cells, where we observed almost total
abrogation of the TNF-aand IFN-cproduction by CD8
+
T cells.
Finally, we demonstrated that the association of Gag with
LAMP luminal domain, in the absence of transmembrane/
cytoplasmic domain, was sufficient to modulate Gag traffic and
anti-Gag immune response. Therefore, the traffic signals present
in this region seemed to be the main event regulating the immune
response.
In summary, we described the mechanisms involved in the
immune response induced by LAMP/gag chimeric DNA and
demonstrated that the luminal domain of LAMP is a key element
in this construct, inducing higher Gag expression, and its traffic
through the endolysosomal and secretory pathways. Analysis of the
immune response elicited by chimeric DNA plasmids containing
truncated sequences of LAMP-1 suggested that increased protein
expression was sufficient to induce an enhanced, but transitory
antibody response; however, increased T cell response depended
on antigen targeting. These findings further enhance our
knowledge regarding LAMP-mediated enhanced immunity and
may contribute not only for the development of novel anti-HIV
vaccines, but also to general vaccinology field.
Supporting Information
Figure S1 Association of p55gag with LAMP luminal
domain is sufficient to induce a T cell immune response.
A-B) Balb/c mice were immunized twice with the indicated
plasmids, and fifteen days later, total splenocytes were cultured
with p55Gag protein and IFN-csecretion was analyzed by ELISA
(A); or the cells were cultured with MHC I restricted Gag epitope
AMQMLKETI65-73 and the expression of IFN-camong CD8
+
cells were evaluated by FACS (B). The data are representative of
three independent experiments. * p,0,05.
(TIF)
Acknowledgments
The authors wish to thank Dr. James Hildreth (The Johns Hopkins
University School of Medicine) for kindly providing the anti-Gag
antibodies; and Dr. Susan Swain (The Trudeau Institute, Saranac Lake,
NY) for the fibroblast cell line DCEK.ICAM.Hi7. We also thank Dr. Jose
Mauro Peralta, responsible for the laboratory of Hybridoma and Cell
Culture (Instituto de Microbiologia Paulo de Go´ es, UFRJ, RJ, Brazil) for
the production and purification of anti-CD4 antibody (GK1.5). We
acknowledge Betty Earls Hart, Joa˜ o Baltazar and Sidney Gomes da Costa
for technical support. Several reagents were obtained through the AIDS
Research Reagents Program, Division of AIDS, NIAID, National Institutes
of Health: purified p55Gag protein and peptides.
Author Contributions
Conceived and designed the experiments: MNS LMTP JTA ETAM LBA.
Performed the experiments: RMCG CGOL FLM POR LBA. Analyzed
the data: MM LMTP JTA ETAM LBA. Contributed reagents/materials/
analysis tools: JLG. Wrote the paper: RMCG LBA.
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