Induction of HIV-specific memory T-cell responses by topical DermaVir vaccine

Vaccine 25 (2007) 3070–3074

Induction of HIV-specific memory T-cell responses by topical DermaVir vaccine Sandra A. Calarota a , David B. Weiner b , Franco Lori a,∗ , Julianna Lisziewicz c a

Research Institute for Genetic and Human Therapy (RIGHT), 27100 Pavia, Italy & Bethesda, MD 20814, USA b Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA c Genetic Immunity, H-1027 Budapest, Hungary & Washington, DC 20007, USA Available online 22 January 2007

Abstract In vivo antigen expression by plasmid DNA could provide a potent and cost-effective vaccine platform if its immunogenicity were improved to induce antigen-specific memory T-cell responses. To study these immune responses, we compared naked DNA vaccine with topical DermaVir formulated with the same HIV-1 (Gag) DNA in the mouse model. Topical DermaVir induced HIV-specific effector memory CD8+ T-cell responses, which were 2.4-fold higher than those elicited by intramuscular injection of naked DNA. DermaVir, but not naked DNA vaccination, induced robust HIV-specific central memory T-cells responses, which are likely to be more efficient in mediating protective immunity. DermaVir formulation combined with topical administration provides an improved immunogenicity of antigen-expressing DNA vaccines. © 2007 Elsevier Ltd. All rights reserved. Keywords: Topical HIV-1 DermaVir; Dendritic cells; Memory T-cell responses

1. Introduction DNA vaccination offers a potent and cost-effective vaccine platform technology with the unique advantage of allowing repeated administrations since immunity is solely elicited to the encoded antigen(s). Although it has been demonstrated that naked DNA vaccines can safely elicit antigen-specific immune responses, it is evident that their potency should be improved to induce antigen-specific memory T-cell responses. To achieve such responses, DermaVir, a topical DNA-based vaccine, has been developed. The plasmid DNA expressing the gene(s) of interest is formulated with a chemical polymer, polyethylenimine-mannose (PEIm) and glucose to build a nanoparticle [1]. This mannosylated nanoparticle effectively delivered HIV-1 genes-expressing plasmid DNA to cultured dendritic cells (DC) and induced HIV-specific ∗ Corresponding author at: Research Institute for Genetic and Human Therapy (RIGHT), 4400 East-West Hwy, Suite 1126, Bethesda, MD 20814, USA. Tel.: +1 202 338 9580. E-mail address: [email protected] (F. Lori).

0264-410X/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2007.01.024

cellular immunity in vitro and ex vivo in macaques [2]. Since ex vivo DC vaccination was not feasible to be administered in large number of individuals, we have developed an in vivo DC vaccination technology with the DermaVir nanoparticles. This in vivo DC vaccination is taking advantage from high abundance of DC precursors, Langerhans cells (LC), in the epidermis and their role in natural antigen entry through the skin. Following skin injury induced by exfoliation, epidermal LC pick up DermaVir nanoparticles and the subsequent DNA gene expression triggers antigen production and maturation of LC to DC. Vaccine-containing DC migrate to the draining lymph nodes and present the DNA-derived antigens to na¨ıve T-cells [1]. Topical DermaVir immunization with simian–human immunodeficiency virus (SHIV) genes-encoding plasmid, induced both CD8+ and CD4+ SIVspecific T-cell responses in na¨ıve rhesus macaques [3], and enhanced SIV-specific T-cell responses and controlled viral rebound during treatment interruptions in chronically SIVinfected rhesus macaques [4]. Here we compared the induction of HIV-specific memory T-cell responses by DermaVir and DNA vaccines using a

S.A. Calarota et al. / Vaccine 25 (2007) 3070–3074

DNA expressing HIV-1 Gag in mice. We show that central memory T-cell responses detected in DermaVir-immunized mice was significantly higher compared to mice immunized with naked DNA given by intramuscular (i.m.) injection.

2. Materials and methods 2.1. Plasmid DNA and immunizations Female 6–8 week-old Balb/C mice (The Jackson Laboratory, Bar Harbor, ME) were immunized with an HIV-1 Gag-encoding plasmid DNA (pGag) [5] or control vector (pVax). For topical immunization, DermaVir was formulated with 25 ␮g DNA/200 ␮l with PEIm and dextrose and applied as previously described [3]. For naked DNA i.m. injection, mice received 25 ␮g DNA/100 ␮l formulated in 0.15 M citrate buffer and 0.25% bupivicaine (Sigma–Aldrich, St. Louis, MO). 2.2. Study design Mice (four per group) were immunized at weeks 0 and 2, with pGag or control pVax, either as naked DNA i.m. or formulated with DermaVir given topically, alone or in a DNA prime followed by an intraperitoneal (i.p.) boost with 2.2 × 106 PFU of an HIV-1 Gag-expressing vaccinia vector [6] (VacV-Gag; provided by Dr. S. Isaacs, University of Pennsylvania, PA) at week 6. 2.3. Splenocytes and peripheral cells isolation Spleens were removed and a single-cell suspension was prepared for each group of mice. The pooled cells were incubated ACK lysing buffer (Biosource, Rockville, MD) to lyse red blood cells, washed and resuspended in culture medium (RPMI 1640 medium containing 2 mM l-glutamine and supplemented with 10% FBS, 100 IU/ml penicillin, 100 ␮g/ml streptomycin). Retro-orbital bleeding was performed at several time points and a single-cell suspension was prepared for each group of mice. The pooled blood was incubated with ACK lysing buffer, washed and resuspended in culture medium. 2.4. Standard IFN-γ ELISPOT assay MultiScreen-IP 96-well plates (Millipore, Bedford, MA) were coated with anti-mouse IFN-␥ mAb (Mabtech, Cincinnati, OH) overnight at 4 ◦ C. Plates were washed and blocked for 2 h at room temperature with culture medium. Cells (2 × 105 per well) were added in triplicate (splenocytes) or duplicate wells (peripheral cells) and stimulated with HIV-1 Gag peptide pools (10 ␮g/ml). Peptides (123, 15mer overlapping by 11 amino acids, divided into four pools with 30–31 peptides each) corresponding to the complete HIV-1 Consensus Subtype B Gag sequence were obtained

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from the NIH AIDS Research and Reference Reagent Program. ConA (5 ␮g/ml, Sigma–Aldrich) and culture medium were used as positive and negative control, respectively. After an incubation of 24 h at 37 ◦ C, plates were incubated overnight at 4 ◦ C with biotinylated detection mAb (Mabtech). Streptavidin–alkaline phosphatase (Mabtech) was added for 1 h at room temperature. Spots were visualized with BCIP/NBT (Sigma) substrate and counted using an automated ELISPOT reader system (CTL analyzers, Cleveland, OH). Assays were also performed after depletion of CD8+ Tcells from splenocytes by using CD8a (Ly-2) MicroBeads (Miltenyi Biotec, Auburn, CA) following manufacturer’s instructions, using autoMACS (Miltenyi Biotec). The mean number of spots from triplicate or duplicate wells was adjusted to 1 × 106 cells. 2.5. Cultured IFN-γ ELISPOT assay Splenocytes (1 × 106 per ml) were plated in each of four wells in a 48-well plate, one HIV-1 Gag peptide pool was added per well, and cultured at 37 ◦ C for 6 days. Cells from each well were then washed twice, counted and suspended at 1 × 106 per ml. Cells (100 ␮l/well) were tested in the same way as the standard IFN-␥ ELISPOT assay in response to the corresponding HIV-1 Gag peptide pool or control medium only.

3. Results and discussion We first compared the immunogenicity of topical DermaVir with naked DNA i.m. immunization. Immunizations were performed at weeks 0 and 2 with pGag (25 ␮g DNA/dose) either formulated as DermaVir nanoparticle given topically, DermaVir(pGag) or as naked DNA given by i.m. injection, DNA(pGag); a group of mice remained na¨ıve (Fig. 1A). A pool of splenocytes from each experimental group was assayed for antigen-specific effector memory T-cell responses by standard IFN-␥ ELISPOT in response to four HIV-1 Gag peptide pools at week 3. No IFN-␥ spots were detected in na¨ıve mice, while mice immunized with DermaVir(pGag) mounted a 2.4-fold higher IFN-␥ response than mice immunized with DNA(pGag) (Fig. 1B, left panel). Depletion of CD8+ T-cells resulted in loss of IFN-␥ production, indicating that mostly CD8+ T-cells mediated the IFN-␥ response (Fig. 1B, right panel). The immunogenicity of an initial DNA priming substantially affects the magnitude of the immune response following a recombinant viral boost. The prime is intended to initiate antigen-specific memory T-cells and the boost to expand the memory response. To examine memory T-cell responses, we compared topical DermaVir and naked DNA i.m. during a prime phase followed by a VacV-Gag boost. Mice were primed at weeks 0 and 2 and boosted i.p. with a VacV-Gag at week 6 (Fig. 2A). A pool of peripheral cells from each experimental group was evaluated for IFN-␥ production in

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Fig. 1. Antigen-specific T-cell responses induced by topical DermaVir or naked DNA immunization. (A) Balb/C mice (four per group) were na¨ıve or primed with pGag naked DNA given i.m., DNA(pGag), or pGag DermaVir given topically, DermaVir(pGag), at weeks 0 and 2 (25 ␮g DNA/immunization). (B) Antigen-specific effector memory T-cell response in total (left panel) and CD8+ T-cell depleted (right panel) splenocytes was evaluated 1 week after the second immunization by standard IFN-␥ ELISPOT assay in response to four HIV-1 Gag peptide pools. Net number of spots was determined by subtracting the number of spots formed in control medium wells.

response to the four HIV-1 Gag peptide pools by standard ELISPOT at weeks 3, 6 (before boost) and 7 (1 week after the boost). Although after priming the antigen-specific IFN␥ response detected in the blood was of low magnitude, mice receiving DermaVir still showed a higher response than those primed with naked DNA (Fig. 2B and C). Vaccinia boost increased the IFN-␥ response in mice primed with DermaVir or naked DNA; the number of IFN-␥ spots detected in peripheral blood at week 7 was quite similar (Fig. 2D). However, antigen-specific IFN-␥ response was detected in control mice primed with control DNA(pVax) after VacV-Gag boost (Fig. 2D). At week 8, splenocytes from all experimental groups were analyzed to detect long-lasting immunity. VacV-Gag boost enhanced antigen-specific effector memory T-cell responses, as assayed by standard IFN-␥ ELISPOT, in all groups (Fig. 3A, left panel); CD8+ T-cells mostly mediated the responses (Fig. 3A, right panel). Cultured IFN-␥ ELISPOT assays were also carried out at week 8 (Fig. 3B). Priming with DermaVir(pGag) resulted in a 5.1-fold increase of memory T-cell responses compared to control DermaVir(pVax). In contrast, a 1.3-fold increase in memory T-cell responses was observed after DNA(pGag) priming as compared with DNA(pVax). After background responses from each control pVax group were subtracted from the corresponding

Fig. 2. Antigen-specific effector memory T-cell responses in peripheral cells induced by topical DermaVir prime or naked DNA prime/VacV boost regimen. (A) Balb/C mice (four per group) were primed with control pVax or pGag, formulated with DermaVir given topically (left panel) or as naked DNA given i.m (right panel) at weeks 0 and 2 (25 ␮g DNA/immunization) and boosted i.p. with 2.2 × 106 PFU VacV-Gag at week 6. IFN-␥ production in response to four Gag peptide pools was evaluated by standard ELISPOT at weeks (B) 3, (C) 6 (before boost), and (D) 7 (after boost) in peripheral cells obtained by retro-orbital bleeding. Net number of spots was determined by subtracting the number of spots formed in control medium wells.

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Fig. 3. Antigen-specific memory T-cell responses in splenocytes induced by topical DermaVir prime or naked DNA prime/VacV boost regimen. Balb/C mice (four per group) were primed with control pVax or pGag, formulated with DermaVir given topically or as naked DNA given i.m. at weeks 0 and 2 (25 ␮g DNA/immunization) and boosted i.p. with 2.2 × 106 PFU VacV-Gag at week 6. (A) Antigen-specific effector memory T-cell responses evaluated at week 8 by standard IFN-␥ ELISPOT in response to four Gag peptide pools in total (left panel) and CD8+ T-cell depleted splenocytes (right panel). (B) Antigen-specific central memory T-cell responses evaluated at week 8 by cultured IFN-␥ ELISPOT in response to four Gag peptide pools. Asterisk indicates that Gag-specific memory response (calculated by subtracting the background responses detected in each control group from the corresponding experimental group) is significantly different (p = 0.01) between DermaVir(pGag)-primed and DNA(pGag)-primed mice.

experimental group, Gag-specific memory T-cell response detected in topically DermaVir-immunized mice was significantly higher compared to mice immunized with naked DNA i.m. (p = 0.01; Mann–Whitney U-test). Our data demonstrate that both topical DermaVir and naked DNA i.m. immunizations induce HIV-specific effector memory T-cell responses but DermaVir elicits significantly more central memory Tcells than naked DNA. These results explain the very effective DermaVir priming for a recombinant vaccinia boost. The differences between DermaVir and DNA immunizations might be related with the cellular uptake of plasmid DNA and its expression. Following the administration of naked DNA by i.m. injection, the expression of the DNA-

encoded antigen occurs primarily in transfected myocytes, or by direct transfection of small numbers of DC present at the site of inoculation or by cross-presentation of extracellular antigen [7]. In contrast, DermaVir targets specifically LC that are distributed in a dense network throughout the epidermis (500–1000 cells/mm2 ), that authentically process the antigen and migrate rapidly to the draining lymph nodes; thus, the expression of the DNA-encoded antigen occurs in the DC of the draining lymph nodes [1]. Here, we analyzed HIV-specific memory T-cell responses by cultured IFN-␥ ELISPOT. This assay was originally developed to identify protective T-cell responses against malaria infection [8,9]. Specifically, these studies have shown that the

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cultured IFN-␥ ELISPOT, but not the standard assay, identifies long-lasting protective anti-malarial T-cell responses. In the HIV-1 field, recent studies indicate that antigen-specific central memory T-cells induced by a vaccine regimen are essential for a better outcome and survival after pathogenic challenge in a macaque model [10,11], consistent with the concept that effective and long-term antigen-specific immune responses should rely on cells that can rapidly expand after antigen challenge, that is central memory Tcells. Here, we show a substantial improvement in DNA vaccination demonstrating the generation of antigen-specific central memory T-cells by DermaVir. These cells are likely to be more efficient in mediating protective immunity, the goal of prophylactic vaccination, and in suppressing HIV-1 for therapeutic immunization.

[3]

[4]

[5]

[6]

[7]

[8]

Acknowledgement [9]

This study was supported by NIH grant R01 AI05698201 to F. Lori.

References [1] Lori F, Trocio J, Bakare N, Kelly LM, Lisziewicz J. DermaVir: a novel HIV immunization technology. Vaccine 2005;23:2030–4. [2] Lisziewicz J, Gabrilovich DI, Varga G, Xu J, Greenberg PD, Arya SK, et al. Induction of potent human immunodeficiency virus type 1-specific

[10]

[11]

T-cell-restricted immunity by genetically modified dendritic cells. J Virol 2001;75:7621–8. Lisziewicz J, Trocio J, Whitman L, Varga G, Xu J, Bakare N, et al. DermaVir: a novel topical vaccine for HIV/AIDS. J Invest Dermatol 2005;124:160–9. Lisziewicz J, Trocio J, Xu J, Whitman L, Ryder A, Bakare N, et al. Control of viral rebound through therapeutic immunization with DermaVir. AIDS 2005;19:35–43. Kim JJ, Nottingham LK, Sin JI, Morrison L, Oh J, Dang K, et al. CD8 positive T cells influence antigen-specific immune responses through the expression of chemokines. J Clin Invest 1998;102:1112–24. Kwak H, Mustafa W, Speirs K, Abdool AJ, Paterson Y, Isaacs SN. Improved protection conferred by vaccination with a recombinant vaccinia virus that incorporates a foreign antigen into the extracellular enveloped virion. Virology 2004;322:337–48. Giri M, Ugen KE, Weiner DB. DNA vaccines against human immunodeficiency virus type 1 in the past decade. Clin Microbiol Rev 2004;17:370–89. Reece WH, Pinder M, Gothard PK, Milligan P, Bojang K, Doherty T, et al. A CD4+ T-cell immune response to a conserved epitope in the circumsporozoite protein correlated with protection from natural Plasmodium falciparum infection and disease. Nat Med 2004;10:406–10. Keating SM, Bejon P, Berthoud T, Vuola JM, Todryk S, Webster DP, et al. Durable human memory T cells quantifiable by cultured enzymelinked immunospot assays are induced by heterologous prime boost immunization and correlate with protection against malaria. J Immunol 2005;175:5675–80. Letvin NL, Mascola JR, Sun Y, Gorgone DA, Buzby AP, Xu L, et al. Preserved CD4+ central memory T cells and survival in vaccinated SIV-challenged monkeys. Science 2006;312:1530–3. Mattapallil JJ, Douek DC, Buckler-White A, Montefiori D, Letvin NL, Nabel GJ, et al. Vaccination preserves CD4 memory T cells during acute simian immunodeficiency virus challenge. J Exp Med 2006;203:1533–41.