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. 2014 Jun 16;9(6):e99887.
doi: 10.1371/journal.pone.0099887. eCollection 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

Rodrigo Maciel da Costa Godinho  1 , Flavio Lemos Matassoli  1 , Carolina Gonçalves de Oliveira Lucas  1 , Paula Ordonhez Rigato  2 , Jorge Luiz Santos Gonçalves  3 , Maria Notomi Sato  2 , Milton Maciel Jr  4 , Ligia Maria Torres Peçanha  3 , J Thomas August  5 , Ernesto Torres de Azevedo Marques Jr  6 , Luciana Barros de Arruda  1
Affiliations

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 et al. PLoS One. .

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
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 (gagN) 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 gagN or LAMP/gag. After 24 hours, the cells were pulsed with S35 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 β-actin CT number. Data is representative of three independent experiments. * p<0.05.
Figure 2
Figure 2. Schematic representation of the constructed plasmids containing different domains of LAMP-1 associated to p55gag.
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).
Figure 3
Figure 3. LAMP-mediated increased Gag expression is dependent 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 β-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.
Figure 4
Figure 4. Truncated LAMP/Gag chimeras poorly colocalize with endogenous LAMP.
Mouse DCEK cells were transfected with the indicated plasmids. After 48-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 representative of four independent experiments.
Figure 5
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 centrifugation. 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 LAMPlum/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 LAMPT2-lum/gag, LAMP/gag or LAMPlum/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.
Figure 6
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 µg/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-γ production was analyzed by ELISPOT assay. The bars indicate the average of SFC/106 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.
Figure 7
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-γ, or with PercP-anti-CD4, APC-anti-IL-2 and PE-anti-TNF-α, and analyzed by FACS. Dot blots indicate CD4 and IFN-γ staining (A) or TNF-α and IL-2 staining among CD4+ cells (B). C) Culture supernatants were collected and the amount of TNF-α was analyzed by ELISA. D–E) Cells were stained with PercP-anti-CD4, PE-anti-CCR7 and APC-anti- IFN-γ. CD4+ cells were gated and the percentage and/or mean fluorescence intensity (MFI) of CD4+CCR7+IFN-γ+ cells were analyzed by FACS. D) Histograms indicate CCR7 expression among CD4+ cells. pITR vectors are in black; gagN 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-γ expression among CD4+ cells. Numbers indicate the percentage of IFN-γ+ cells among CCR7hi and CCR7l°CD4+ gated cells. The data are representative of three independent experiments. * p<0,05.
Figure 8
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 gagN, as controls. Fifteen days after the second immunization, total splenocytes were cultured with CD8-restricted gag peptide and cytokine production was analyzed. A) TNF-α secretion was analyzed by ELISA. B) The cells were incubated with PercP-anti-CD8, PE-anti-CCR7 and FITC-anti-IFN-γ and the expression of CCR7 and IFN-γ among CD8+ cells were analyzed by FACS. The data are representative of two independent experiments. * p<0,05.
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References

    1. Borrow P, Lewicki H, Hahn BH, Shaw GM, Oldstone MB (1994) Virus-specific CD8+ cytotoxic T-lymphocyte activity associated with control of viremia in primary human immunodeficiency virus type 1 infection. J. Virol. 68 (9): , 6103–6110. - PMC - PubMed
    1. Ferre AL, Lemongello D, Hunt PW, Morri MM, Garcia JC, et al.. (2010) Immunodominant HIV-specific CD8+ T-cell responses are common to blood and gastrointestinal mucosa, and Gag-specific responses dominate in rectal mucosa of HIV controllers. J. Virol. 84 (19), : 10354–10365. - PMC - PubMed
    1. Owen RE, Heitman JW, Hirschkorn DF, Lanteri MC, Biswas HH, et al.. (2010) HIV+ elite controllers have low HIV-specific T-cell activation yet maintain strong, polyfunctional T-cell responses. AIDS. 24(8): ,1095–10105. - PMC - PubMed
    1. Koup RA, Safrit JT, Cao Y, Andrews CA, McLeod G, et al.. (1994) Temporal association of cellular immune responses with the initial control of viremia in primary human immunodeficiency virus type 1 syndrome. J. Virol. 68 (7): , 4650–4655. - PMC - PubMed
    1. Pontesilli O, Klein MR, Kerkhof-Garde SR, Pakker NG, de Wolf F, et al.. (1998) Longitudinal analysis of human immunodeficiency virus type 1-specific cytotoxic T lymphocyte responses: a predominant gag-specific response is associated with nonprogressive infection. J. Infect. Dis. 178(4): , 1008–1018. - PubMed

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