Identification of new HIV-1 Gag-specific cytotoxic T lymphocyte responses in BALB/c mice
© Cellini et al; licensee BioMed Central Ltd. 2008
Received: 27 May 2008
Accepted: 14 July 2008
Published: 14 July 2008
As HIV-specific cytotoxic T cells play a key role during acute and chronic HIV-1 infection in humans, the ability of potential anti-HIV vaccines to elicit strong, broad T cell responses is likely to be crucial. The HIV-1 Gag antigen is widely considered a relevant antigen for the development of an anti-HIV vaccine since it is one of the most conserved viral proteins and is also known to induce T cell responses. In the majority of studies reporting Gag-specific cellular immune responses induced by Gag-based vaccines, only a small number of Gag T cell epitopes were tested in preclinical mouse models, thus giving an incomplete picture of the numerous possible cellular immune responses against this antigen. As is, this partial knowledge of epitope-specific T cell responses directed to Gag will unavoidably result in a limited preclinical evaluation of Gag-based vaccines.
In this study we identified new Gag CD8+ T cell epitopes in BALB/c mice vaccinated with the HIV-1 Gag antigen alone or in combination with the HIV-1 Tat protein, which was recently shown to broaden T cell responses directed to Gag. Specifically, we found that CTL responses to Gag may be directed to nine different CTL epitopes, and four of these were mapped as minimal CTL epitopes.
These newly identified CTL epitopes should be considered in the preclinical evaluation of T cell responses induced by Gag-based vaccines in mice.
Cellular immune responses are a critical part of the host defence against viruses, with cytotoxic T lymphocytes (CTLs) playing a key role in recognizing and eliminating infected cells. CTLs identify their targets as 8–10 amino acid long peptides which are derived from the intracellular degradation of viral antigens and presented in association with major histocompatibility complex class I (MHC-I) molecules at the surface of infected cells [1–3].
Several studies have indicated that HIV-specific T cell responses play a key role in limiting the progression of acute and chronic infection in humans [4, 5], and that long-term non-progressors have consistently higher levels of HIV-specific T cell responses than progressors . Thus, the ability of potential vaccines for HIV to elicit strong, broad T cell responses is likely to be a determining factor in their success.
We have recently reported that vaccines based on a combination of the HIV-1 Tat protein with heterologous antigens induce broader T cell responses against the co-administered antigen, thereby indicating Tat as a useful tool in the development of novel vaccination strategies against AIDS [7–9].
As the HIV-1 Gag antigen is one of the most conserved viral proteins, and is known to induce T cell responses, both in animal models and in humans, it is widely considered a relevant antigen for the development of an anti-HIV vaccine. Indeed, previous studies have shown that Gag-specific T cell responses contribute to clear primary viremia and control later viral replication, thereby slowing progression of the disease [4, 10–12].
Small animal models, in particular mice, represent a useful tool for studying the dynamics of immune responses induced after vaccination, although, the evaluation of cellular responses induced by vaccination is usually restricted to immunodominant T cell epitopes, which represent, only a minor part of the overall cellular immune response. In order to expand our limited knowledge of epitope-specific T cell responses directed to a given antigen, the aim of this study was to identify the repertoire of CD8+ T cell epitopes of the HIV-1 Gag antigen in BALB/c mice vaccinated with the HIV-1 Gag protein.
Results and Discussion
In vivomodulation of epitope-specific T cell responses against the HIV-1 Gag antigen by the HIV-1 Tat protein
We recently demonstrated in BALB/c mice vaccinated with the HIV-1 Gag protein  that Gag-specific T cell responses are directed to 7 different peptides (peptides: 42, 49, 50, 53, 65, 75 and 76). Only two (49 and 50) of these peptides were already known to contain the major Kd-restricted CTL epitope (AMQ, aa 197–205), while peptides 65 (aa 257–271) and 75 (aa 297–311) were known to contain T cell epitopes which had not been fully characterized [13, 14].
Identification of new CD8+ T cell responses against the HIV-1 Gag antigen
Gag peptides and predicted CTL epitopes
In this study we have defined new CD8-mediated Gag-specific T cell responses in BALB/c mice vaccinated with the Gag protein alone or with the combination Tat/Gag, which had previously been shown to be very efficient at inducing Th1 responses directed to Gag . We demonstrated that 9 out of 11 peptides stimulating IFN-γ T cell responses were recognized by CD8+ T cells. In addition, CD8+ T cells efficiently lysed peptide-pulsed target cells. Four peptides were also characterized as minimal CTL epitopes.
Induction of HIV-1-specific CTL responses has become a critical component in the design of recombinant subunit vaccines aimed at controlling HIV infection, and these CD8-restricted epitopes, which, with the exception of the AMQ peptide, were all identified here for the first time, should be exploited in preclinical models for a complete evaluation of cellular responses induced by Gag-based vaccines.
The biologically active HIV-1 Tat protein from the human T lymphotropic virus type IIIB isolate (BH10 clone) was expressed in E. Coli and purified by heparin-affinity chromatography and HPLC as previously described . The Tat protein was stored in lyophilized form at -80°C, reconstituted in degassed buffer before use, and handled as described to prevent oxidation and loss of biological activity . Different lots of Tat were used with reproducible results, and in all cases endotoxin concentration was undetectable (detection threshold: 0.05 EU/μg). HIV-1 GagSF2 protein was obtained from the NIH AIDS Reagent Program.
Peptides were synthesized by the solid phase method and purified by HPLC to >98% purity, as previously described . Structural verification was performed by elemental and amino acid analysis and mass spectrometry. Gag peptides, 15 amino acid long and overlapping by 10 to 11 amino acids, spanning the entire Gag (HIV-1 consensus subtype B Gag complete set # 8117) sequence, were provided by the NIH AIDS Reagent Program. Peptides were dissolved in DMSO at 10-3 M, kept at -20°C, and diluted in PBS before use.
Immunization of mice
BALB/c mice (H-2d) (Harlan, Udine, Italy) were immunized subcutaneously at a single site in the back with 5 μg of HIV-1 Gag protein alone or in combination with 5 μg of native monomeric biologically active Tat protein in Freund's adjuvant . Each group was composed of 5 animals. Immunogens were given subcutaneously in 100 μl injections, on days 1, 14 and 28, and mice were sacrificed 10 days after the final boost (day 38). During the course of the experiments, animals were checked twice a week at the site of injection, as well as for their general conditions (such as liveliness, food intake, vitality, weight, motility, sheen of fur). No signs of local or systemic adverse reactions were observed at any time in mice receiving the immunogens, as compared to control or untreated mice. Animal use was carried out according to European and institutional guidelines.
Splenocytes were purified from spleens squeezed onto filters (Becton Dickinson). Spleens of each experimental group were pooled. Following red blood cell lysis, cells were resuspended in RPMI 1640 supplemented with 10% FBS (Gibco) and immediately used for the analysis of antigen-specific cellular immune responses by cytotoxicity or Elispot assays . When indicated, splenocytes were also stimulated in vitro with 1 μg/ml of peptide and tested in cytotoxic assays after 5 days of culture. Purified CD8+ T cells were obtained using the BD™ IMag Mouse Lymphocyte Enrichment Set-DM filters (Becton Dickinson), according to the manufacturer's instructions. Purified cells were stained with FITC-conjugated anti-mouse CD8 mAb (Becton Dickinson). Flow cytometry analysis was performed with a FACScan (Becton Dickinson), and CD8-positive cells were > 90% in all cases.
P815 target cells were labeled with Na251CrO4 for 90 min at 37°C. Cytotoxicity tests were routinely run in triplicate at different effector:target ratios . Percent specific lysis was calculated as 100× (cpm sample - cpm medium)/(cpm Triton X-100 - cpm medium). Spontaneous release was always less than 20%.
Elispot (IFN-γ) was carried out using commercially available kits provided by Becton Dickinson, according to the manufacturer's instructions. Briefly, 96-well nitrocellulose plates were coated with 5 μg/ml of anti-IFN-γ overnight at 4°C . The following day, the plates were washed 4 times with PBS and blocked with RPMI 1640 supplemented with 10% FBS for 2 hours at 37°C. Splenocytes (2.5 × 105/200 μl) were added to the wells (duplicate wells) and incubated with peptides (10-6 M) for 16 hours at 37°C. Controls were represented by cells incubated with Concanavaline A (Sigma; 5 μg/ml) (positive control) or with medium alone (negative control). The spots were read using an Elispot reader (Aelvis, Germany). Responses at least 3 times higher than the mean number of spots in the control wells and ≥ 50 spots/well/106 cells were defined as positive. Results are expressed as net number of spot forming units (SFU)/106 cells: [mean number SFU of peptide treated wells minus mean number SFU of the negative control].
The authors wish to thank the NIH AIDS Repository Reagents and References Program for reagents, and Anna Forster for editorial assistance. This work was supported by grants from the Istituto Superiore di Sanità [National AIDS Project and the Italian Concerted Action on HIV-AIDS Vaccine Development (ICAV)], the 6th European Programme [AIDS Vaccine Integrated Project (AVIP)], and the Ministero dell'Istruzione, dell'Università e della Ricerca Scientifica (MIUR).
- Zinkernagel RM, Doherty PC: Immunological surveillance against altered self components by sensitised T lymphocytes in lymphocytic choriomenengitis. Nature (London). 1974, 251: 547-548. 10.1038/251547a0.View ArticleGoogle Scholar
- Townsend ARM, Rothbard J, Gotch FM, Bahadur G, Wraith D, McMichael AJ: The epitopes of influenza nucleoprotein recognized by cytotoxic T lymphocytes can be defined with short synthetic peptides. Cell. 1986, 44: 959-968. 10.1016/0092-8674(86)90019-X.View ArticlePubMedGoogle Scholar
- Falk K, Rötzschke O, Stevanovic S, Jung G, Rammensee HG: Allele-specific motifs revealed by sequencing of self-peptides eluted from MHC molecules. Nature (London). 1991, 351: 290-296. 10.1038/351290a0.View ArticleGoogle Scholar
- Ogg GS, Jin X, Bonhoeffer S, Dunbar PR, Nowak MA, Monard S, Segal JP, Cao Y, Rowland-Jones SL, Cerundolo V, Hurley A, Markowitz M, Ho DD, Nixon DF, McMichael AJ: Quantitation of HIV-1-specific cytotoxic T lymphocytes and plasma load of viral RNA. Science. 1998, 279: 2103-2106. 10.1126/science.279.5359.2103.View ArticlePubMedGoogle Scholar
- Rosemberg ES, Billingsley JM, Caliendo AM, Boswell SL, Sax PE, Kalams SA, Walker BD: Vigorous HIV-1-specific CD4+ T cell responses associated with control of viremia. Science. 1997, 278: 1447-1450. 10.1126/science.278.5342.1447.View ArticleGoogle Scholar
- Deeks SG, Walker BD: Human immunodeficiency virus controllers: Mechanisms of durable virus control in the absence of antiretroviral therapy. Immunity. 2007, 27: 406-416. 10.1016/j.immuni.2007.08.010.View ArticlePubMedGoogle Scholar
- Ensoli B, Cafaro A, Caputo A, Fiorelli V, Ensoli F, Gavioli R, Ferrantelli F, Cara A, Titti F, Magnani M: Vaccines based on the native HIV Tat protein and on the combination of Tat and the structural HIV protein variant DeltaV2 Env. Microbes Infect. 2005, 7: 1392-1399. 10.1016/j.micinf.2005.07.016.View ArticlePubMedGoogle Scholar
- Caputo A, Gavioli R, Ensoli B: Recent advances in the development of HIV-1 Tat-based vaccines. Curr HIV Res. 2004, 2: 5038-5042. 10.2174/1570162043350986.View ArticleGoogle Scholar
- Gavioli R, Cellini S, Castaldello A, Voltan R, Gallerani E, Gagliardoni F, Fortini C, Cofano EB, Triulzi C, Cafaro A, Srivastava I, Barnett S, Caputo A, Ensoli B: The Tat protein broadens T cell responses directed to the HIV-1 antigens Gag and Env: Implications for the design of new vaccination strategies against AIDS. Vaccine. 2008, 30: 727-737. 10.1016/j.vaccine.2007.11.040.View ArticleGoogle Scholar
- Geldmacher C, Currier JR, Herrmann E, Haule A, Kuta E, McCutchan F, Njovu L, Geis S, Hoffmann O, Maboko L, Williamson C, Birx D, Meyerhans A, Cox J, Hoelscher M: CD8 T-cell recognition of multiple epitopes within specific Gag regions is associated with maintenance of a low steady-state viremia in human immunodeficiency virus type 1-seropositive patients. J Virol. 2007, 81: 2440-2448. 10.1128/JVI.01847-06.PubMed CentralView ArticlePubMedGoogle Scholar
- Sacha JB, Chung C, Rakasz EG, Spencer SP, Jonas AK, Bean AT, Lee W, Burwitz BJ, Stephany JJ, Loffredo JT, Allison DB, Adnan S, Hoji A, Wilson NA, Friedrich TC, Lifson JD, Yang OO, Watkins DI: Gag-specific CD8+ T lymphocytes recognize infected cells before AIDS-virus integration and viral protein expression. J Immunol. 2007, 178: 2746-2754.PubMed CentralView ArticlePubMedGoogle Scholar
- Almeida JR, Price DA, Papagno L, Arkoub ZA, Sauce D, Bornstein E, Asher TE, Samri A, Schnuriger A, Theodorou I, Costagliola D, Rouzioux C, Agut H, Marcelin AG, Douek D, Autran B, Appay V: Superior control of HIV-1 replication by CD8+ T cells is reflected by their avidity, polyfunctionality, and clonal turnover. J Exp Med. 2007, 204: 2473-2485. 10.1084/jem.20070784.PubMed CentralView ArticlePubMedGoogle Scholar
- Mata M, Travers PJ, Liu Q, Frankel FR, Paterson Y: The MHC class I restricted immune response to HIV-gag in BALB/c mice selects a single epitope that does not have a predictable MHC-binding motif and binds to Kd through interactions between a glutamine at P3 pocket D. J Immunol. 1998, 161: 2985-2993.PubMedGoogle Scholar
- Mata M, Paterson Y: Th1 T cell responses to HIV-1 Gag protein delivered bya Listeria monocytogenes vaccine are similar to those induced by endogenouslisterial antigens. J Immunol. 1999, 163: 1449-1456.PubMedGoogle Scholar
- Rammensee HG, Friede T, Stevanovic S: MHC ligands and peptide motifs: first listing. Immunogenetics. 1995, 41: 178-228. 10.1007/BF00172063.View ArticlePubMedGoogle Scholar
- Fanales-Belasio E, Moretti S, Nappi F, Barillari G, Micheletti F, Cafaro A, Ensoli B: Native HIV-1 Tat protein is selectively taken up by monocyte-derived dendritic cells and induces their maturation, Th-1 cytokine production and antigen presenting function. J Immunol. 2002, 168: 197-206.View ArticlePubMedGoogle Scholar
- Ensoli B, Buonaguro L, Barillari G, Fiorelli V, Gendelman R, Morgan RA, Wingfield P, Gallo RC: Release, uptake, and effect of extracellular human immunodeficiency virus type 1 Tat protein on cell growth and viral transactivation. J Virol. 1993, 67: 277-287.PubMed CentralPubMedGoogle Scholar
- Micheletti F, Canella A, Vertuani S, Marastoni M, Tosi L, Volinia S, Traniello S, Gavioli R: Supra-agonist peptides enhance the reactivation of memory cytotoxic T lymphocyte responses. J Immunol. 2000, 165: 4264-4271.View ArticlePubMedGoogle Scholar
This article is published under license to 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.