Efficiency of a myogenic DNA vaccine is strictly dependent upon cellular localization of HIV-1 Pr55gag

Vaccine 20 (2002) 1980–1984

Note

Efficiency of a myogenic DNA vaccine is strictly dependent upon cellular localization of HIV-1 Pr55gag Alexandra Bojak, Jens Wild, Hans Wolf, Ralf Wagner∗ Institute of Medical Microbiology, University of Regensburg, Franz-Josef-Strauß Allee 11, 93053 Regensburg, Germany Received 7 June 2001; accepted 28 November 2001

Abstract Most studies on DNA-based immunization have used viral promoters to drive antigen expression. Recently, the use of tissue-specific DNA vaccines has been favored regarding safety issues. In this study, we determined the impact of antigen localization and tissue-specific expression on the induction of humoral as well as cellular immune responses in a BALB/c mouse model. Thereby, we show that using the muscle-specific muscle creatine kinase (MCK) promoter/enhancer the efficiency of immune stimulation is strictly dependent on the ability of HIV-1 Pr55gag to be released from cells. By contrast, localization of Pr55gag and derivatives thereof plays only a minor role when antigen is constitutively expressed using the ubiquitous viral CMV promoter. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: DNA vaccine; Tissue-specific; HIV-1

1. Introduction Increasing evidence from studies on long-term nonprogressing human immunodeficiency virus type-1 (HIV-1) infected individuals revealed that besides neutralizing antibodies cytotoxic T lymphocytes (CTL) may play an important role in controlling HIV-1 infection [1,2]. The development of an efficient HIV-1 vaccine may therefore depend on the induction of effective CTL and/or T-helper responses against conserved viral proteins, such as the HIV-1 group-specific antigen Pr55gag (Gag). In contrast to several traditional approaches such as protein vaccination, intramuscular administration of a DNA vaccine represents a simple and effective technique of inducing both humoral and cellular immune responses (for review, see [3]). We recently reported the construction of a Rev-independent, codon optimized synthetic gag gene allowing high level gene expression in various cell lines in vitro. Furthermore, we demonstrated that intramuscularly delivery of naked DNA into mice induces strong humoral as well as cellular immune responses [4,5]. Utilization of synthetic genes instead of viral wild-type sequences provides a major improvement regarding current ∗ Corresponding author. Tel.: +49-941-944-6452; fax: +49-941-944-6402. E-mail address: [email protected] (R. Wagner).

safety requirements to common DNA candidate vaccines. In addition, a controlled and tissue restricted antigen expression may be considered to further improve the safety of DNA immunization. Therefore, we investigated the use of the muscle creatine kinase (MCK) promoter, which has been demonstrated to be restricted to differentiated, multinucleated myofibers [6], instead of the commonly used viral, constitutive CMV promoter to drive antigen expression. In this study, we evaluated the immunogenicity of various myogenic DNA vaccines encoding for modified Gag antigens, which either are (i) membrane-attached or (ii) cytoplasmatic or (iii) could be released from cells as virus-like particles (VLPs). 2. Materials and methods 2.1. DNA plasmids The Gag expression vector pc-syngag has been previously described [4]. The synthetic gag coding region (syngag), the corresponding myristoylation defective mutant gene (Gly2 -Ala2 ) and the synthetic p24CA coding region (133–363 aa) was cloned into pcDNA3.1 backbone containing either the CMV or the MCK promoter/enhancer element to yield the expression vectors illustrated in Fig. 1. Expression and release of virus-like particles (VLPs) was

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Fig. 1. Schematic representation of Gag expression plasmids. Open boxes represent various HIV-1 gag codon optimized synthetic gene sequences: full length syngag, mutated myristylation defective syngagMyr- and p24 capsid protein. CMV, cytomegalievirus immediate early promoter/enhancer; MCK, minimal 1350 bp muscle creatine kinase promoter; represents the mutated codon (Gly2 →Ala2 ).

analyzed by Western blot analysis of transiently transfected human lung carcinoma cell line H1299 and C2C12 murine myoblasts (data not shown). 2.2. Plasmid immunization protocols Female BALB/c mice (Charles River, Sulzfeld, Germany) were i.m. immunized three times at 3 weeks intervals with 100 ␮g endotoxin-free plasmid DNA each. Blood samples were obtained by tail bleeding at indicated time points. For assaying cellular parameters, spleens from immunized mice were removed and splenic mononuclear cells were isolated as described in detail elsewhere [5]. 2.3. Evaluation of Gag-specific immune responses Total anti-Gag antibodies as well as antibody isotypes IgG1 and IgG2a were quantified by an end-point dilution ELISA assay on samples from individual animals as previously described [5]. The cytokine profile upon in vitro re-stimulation of splenocytes (2 ×106 cells/ml) with Pr55gag VLPs [7], recombinant p24CA , or MHC-class I restricted p24 peptides, A9Ip24 (197–205 aa) and E19Fp24 (291–300 aa) measured by ELISA was carried out following the manufacturers instructions (Becton Dickonson). For measurement of Gag-specific CTL activity (A9Ip24 ) a 4 h 51 Cr release assay was performed as described in detail elsewhere [5]. 3. Results 3.1. Evaluation of humoral immune responses following immunization of mice Sera collected from individual mice between single DNA inoculations were examined for the development of HIV-1 Gag-specific antibodies (total IgG) by ELISA (Fig. 2).

Mice immunized with CMV promoter driven expression constructs show nearly identical Gag-specific antibody titers independent from the nature and localization of the expressed antigen (Fig. 2a). In comparison, humoral responses induced by injection of myogenic DNA vaccines were in general reduced (Fig. 2b). In detail, mice vaccinated with pMCK-syngag and pMCK-syngagMyr- developed comparable levels of Gag-specific antibody titers, whereas immunization with pMCK-synp24CA induced only weak responses. These data suggests, that using the tissue-specific MCK promoter to drive antigen expression, the nature of the antigen may be critical for the stimulation of an antigen specific humoral immune response. 3.2. T-cell responses following DNA immunization of mice The polarisation of T-cell responses was further determined by quantifying distinct antibody isotypes as a surrogate marker. Independently of promoter elements and expressed antigen DNA immunization resulted in a mixed Th1/Th2 type response since both IgG2a (Th1-type) and IgG1 (Th2-type) isotypes were found to predominate in individual mice (data not shown). Furthermore, the cytokine pattern found in the supernatants of splenocytes after in vitro re-stimulation with recombinant p24 antigens was clearly that of a type-1 cytokine response, characterized by elevated levels of IFN-␥ and IL-2. Cytokines specific for Th2-biased immune responses were only detectable at low levels in mice immunized with CMV driven expression vectors. Although the levels of IFN-␥ released from specifically re-simulated splenocytes of mice vaccinated with CMV-based vectors clearly exceeded those found in mice immunized with myogenic DNA vaccines, highest cytokine responses were observed when the non modified, full length and budding-competent Gag protein (encoded by pCMV-syngag) was expressed, respectively.

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Fig. 2. Kinetics of Gag-specific antibody induction following i.m. immunization of DNA plasmids. As indicated by arrows, BALB/c mice were immunized at 3 weeks interval with 100 ␮g of various Gag-specific DNA vaccines containing either the CMV (a) or the muscle-specific MCK (b) promoter enhancer. Sera from individual mice were assayed for total anti Gag antibodies (IgG) by an end-point ELISA. The reported titers correspond to the reciprocal of the highest serum dilution that gave a three times higher o.d. value than the corresponding dilution of a non-immune serum.

In addition, the vaccine construct encoding the p24 capsid moiety of p24 (pCMVsynp24CA ) turned out to be the less efficient approach to induce IFN-␥, irrespective of the type of promoter that was used. 3.3. Analysis of Gag-specific cytotoxic immune responses in vivo Highest Gag-specific CD8+ lytic activity was observed when the cytoplasmatic viral 24 kDa capsid protein is expressed form a CMV promoter driven DNA vaccine

construct. Reproducibly reduced CTL responses were found when the wt- or the myristylation deficient Pr55gag constructs were expressed from the pcDNA vector (pCMVsyngag, pCMV-syngagMyr-). However, the capacity of the wt-Gag protein to assemble at the inner leaflet of the cytoplasmic membrane and to be released from cells as virus-like particles by budding did not enhance the CTL responses, when a CMV promoter was utilized to express the indicated antigens (Fig. 3a). Conversely, in mice vaccinated with myogenic DNA plasmids, a significant CTL response was only induced utilizing the pMCK-syngag construct encoding for

Fig. 3. Cytotoxic T-cell responses following i.m. immunization of various DNA plasmids. BALB/c mice were immunized at 3 weeks interval with 100 ␮g of plasmids containing either the CMV (a) or the muscle-specific MCK (b) promoter/enhancer and expressing various Gag-specific antigens. One week after, the final immunization splenocytes from the mice in each group were harvested and pooled, and the CTL responses specific to HIV-1 p24 peptide (aa AMQMLKETI) were measured following antigen re-stimulation in vitro.

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Fig. 4. IFN-␥ release of in vitro re-stimulated splenocytes. BALB/c mice were immunized at 3 weeks interval with 100 ␮g of plasmids. One week after, the final boost immunization splenocytes were isolated, splenocytes were prepared and 2 × 105 cells were in vitro re-stimulated with 10 ␮M of MHC-class I restricted p24 peptide A9Ip24 and E10Fp24 for 36 h. Cytokine levels were measured from supernatants using a commercial ELISA.

the budding competent wild type Pr55gag protein (Fig. 3b). A moderate CTL response was also detectable in mice received the myogenic pMCK-syngagMyr- construct. The ranking regarding the capacity of the various constructs to induce a CTL response was further confirmed by the quantification of IFN-␥ release from splenocytes re-stimulated with MHC-class I restricted p24 peptides A9I and E10F (Fig. 4).

4. Discussion DNA immunization provides a simple and efficient method of inducing humoral as well as cellular immune responses. Nevertheless, the use of tissues-specific and controlled expression of foreign antigens would be highly appreciated from a safety point of view. Recently, we and others have shown that intramuscular administration of DNA plasmids, which utilize muscle-specific promoters to regulate gene expression is capable of eliciting an efficient immune response in the BALB/c mouse model [5,8,9]. Although the determined immune responses were usually weaker than those observed using the commonly used viral CMV promoter, the development of a tissue-specific DNA vaccines is—from a safety point of view—one of the major goals in current DNA vaccine design. In order to enhance the efficiency of myogenic DNA vaccines, we analyzed to what extent an altered cellular localization of a favorable antigen such as the HIV-1 Gag

protein is capable to modulate the immune response in vivo. Accordingly, this study aims towards determining and comparing the immunogenicity of three different HIV-1 Gag derivatives. Second, the influence of an ubiquitous (CMV IE) and a tissue-specific (MCK) promoter on the responses induced by the various antigens were determined. Herein, we demonstrated that the efficiency of immune induction utilizing myogenic DNA vaccines is strictly dependent on the nature of a given antigen. Due to the fact that the MCK promoter driven expression is exclusively restricted to differentiated muscle cells, the most potent immune induction was observed when antigen could be released from cells by budding—a necessary prerequisite to allow the uptake of the Gag antigen by professional antigen presenting cells (APC). These observations suggest that cross-priming effects, which mainly occurs when APC process secreted proteins from somatic cells may play a major role in immune induction by myogenic DNA vaccines [10–12]. In contrast, the use of the viral CMV promoter confers strong and promiscuous gene expression. Therefore, antigen expression following intramuscular immunization of DNA vaccines could occur both in muscle as well as other cells, including APC. The combination of cross-priming effects and direct transfecting of APCs may explain the observed high immunogenicity of CMV constructs. Interestingly, the expression of the p24 protein resulted in the most efficient CTL response suggesting that the cytoplasmatic protein p24 is more accessible to intracellular antigen processing and

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MHC-class I presentation than the Gag precursor protein itself. In conclusion, the use of myogenic DNA vaccines supporting tissue-specific and controlled antigen expression require optimization of the selected antigens to allow secretion or release from muscle cells and consecutive cross-priming events via APC.

Acknowledgements We thank Hanns Lochmüller and Nancy Larochelle (Genzentrum LMU, München) for providing parental MCK plasmids and muscle C2C12 cells. Purified recombinant HIV-1 p24 was obtained through the AIDS Research Reagent Program, Division of AIDS, NIAID and NIH. This work was supported by grant 01KI97 63/8 (BMBF) and the EU fifth-frame program EuroVac project 2.4 to R.W. References [1] Harrer W, Harrer T, Buchbinder S, Mann DL, Feinberg M, Yilma T, et al. HIV-1-specific cytotoxic T-lymphocyte response in healthy, long-term non-progressing seropositive persons. AIDS Res Hum Retrovir 1994;10(Suppl 2):77–8. [2] Wagner R, Leschonsky B, Harrer E, Paulus C, Weber C, Walker BD, et al. Molecular and functional analysis of conserved CTL epitope in HIV-1 p24 recognized from a long-term non-progressor: constrains on immune escape associated with targeting a sequence essential for viral replication. J Immunol 1999;162:3727–34.

[3] Donnelly JJ, Ulmer JB, Shiver JW, Liu MA. DNA vaccines. Annu Rev Immunol 2001;75(22):10991–11001. [4] Graf M, Bojak A, Deml L, Bieler K, Wolf H, Wagner R, et al. Concerted action of multiple cis acting sequences is required for Rev dependence of late human immunodeficiency virus type-1 gene expression. J Virol 2000;74(22):10822–6. [5] Deml L, Bojak A, Steck S, Graf M, Wild J, Schirmbeck R, Wolf H, Wagner R. Multiple effects of codon optimization on the expression and immunogenicity of DNA candidate vaccines encoding the human immunodeficiency virus type-1 Gag protein. J Virol, 2001, submitted for publication. [6] Johnson JE, Gartside CL, Jaynes JB, Hauschka SD. Expression of transfected mouse muscle-creatine kinase gene is induced upon growth factor deprivation of myogenic but not of non-myogenic cells. Dev Biol 1989;134(1):258–62. [7] Wagner R, Deml L, Schirmbeck R, Niedrig M, Reimann J, Wolf H, et al. Construction, expression and immunogenicity of chimeric HIV-1 virus-like particles. Virology 1996;220(1):128–40. [8] Gebhard JR, Zhu J, Coa X, Minnick J, Araneo BA. DNA immunization utilizing a herpes simplex virus type-2 myogenic DNA vaccine protects mice from mortality and prevents genital herpes. Vaccine 2000;18(17):1837–46. [9] Loirat D, Li Z, Mancini M, Tiollais P, Paulin D, Michel ML, et al. Muscle-specific expression of hepatitis B surface antigen: no effect on DNA-raised immune response. Virology 1999;260:74–83. [10] Fu TM, Ulmer JB, Caulfield MJ, Deck RR, Friedman A, Wang S, et al. Priming of cytotoxic T-lymphocytes by DNA vaccines: requirements for professional antigen presenting cells and evidence for antigen transfer from myocytes. Mol Med 1997;3(6):362–71. [11] Corr M, Lee DJ, Carson DA, Tighe H. Gene vaccination with naked plasmid DNA: mechanism of CTL priming. J Exp Med 1996;184(4):1555–60. [12] Corr M, von Damm A, Lee DJ, Tighe H. In vivo priming by DNA injection occurs predominantly by antigen transfer. J Immunol 1999;163(9):4721–7.