gene therapy.doc.2.doc

I. Introduction

Since the mid-1980’s, when HIV was first isolated from patients, a variety of vaccine constructs and vaccination strategies have been explored to combat this virus, although none has yet been demonstrated to be effective in preventing in vivo HIV infection.

Effective vaccines must stimulate the correct balance between cellular (Th1-driven) and humoral (Th2-driven) immune responses according to the particular infectious agent; strategies aimed toward triggering a both humoral and cellular responses are thus receiving considerable attention. Although the HIV-1 envelope (env) glycoprotein contains epitopes that activate T cell memory, and against which neutralizing antibodies as well as anti-HIV-1 cytotoxic T cells (CTL) are directed, vaccination with recombinant env glycoprotein induces only modest anti-HIV-1 CTL activity.

DNA immunization represents a novel approach to vaccine development and immunotherapy aimed toward immune response control. DNA vaccination induces antigen-specific cellular and humoral immune responses through the delivery of non-replicating transcription units, which drive the synthesis of specific foreign proteins within the inoculated host. Inoculation with plasmid DNA has been shown to be protective in several viral disease models (Tighe et al. 1998). DNA vaccines are simple to manufacture, provide prolonged antigen expression, and allow manipulation of protein antigenicity at the cDNA level with no need for protein production and purification. Unlike conventional protein vaccines, plasmid DNA vaccination leads to antigen processing and presentation by MHC class I and class II molecules and thus more closely resembles a viral infection. DNA vectors are particularly effective in inducing a strong MHC class I-restricted CTL response. Virus-specific CTL have been induced by gene vaccination with plasmid DNA encoding influenza (Raz et al., 1994) and HIV-1 viral proteins (Fuller et al., 1994); high CTL activity levels have been detected more than one year after intradermal gene vaccination with plasmid DNA encoding influenza virus nucleoptrotein (Raz et al., 1994). Intradermal gene vaccination in mice induces antigen-specific Th1 cells secreting high IFN-g levels, and stimulates IgG2a isotype antibody production (Raz et al., 1996; Sato et al., 1996). The safety, feasibility and immunogenicity of DNA vaccines are currently under evaluation in clinical trials. Immunization with DNA coding for HIV-1 gp160 elicits neutralizing antibodies and CTL responses in mice and in primates (Wang et al., 1995), and HIV-1-infected chimpanzees immunized with DNA expressing env substantially decreased the HIV-1 viral load (Ugen et al., 1997).

In general, the antibody response induced by gene vaccination is lower than that induced by protein vaccination, possibly due to the minute amount of gene product produced in vivo. The overall potency of naked DNA vaccines is less than that of recombinant vaccines; since DNA does not undergo replicative amplification as occurs with live recombinant viral and bacterial vaccines, the amount of antigen ultimately presented to the immune system may be limited. Although DNA vaccination appears to be a promising strategy, it may nonetheless require the use of immunomodulators, adjuvants such as cytokines, or specific immunization regimes to enhance immune responses.

Several groups have recently shown enhancement of the response triggered by DNA vaccination by coinjection of cytokine-encoding plasmids (Xiang & Ertl, 1995). Codelivery of vectors encoding a cytokine such as GM-CSF can augment antigen-specific responses following either intramuscular or epidermal plasmid DNA delivery (Xiang & Ertl, 1995). We have recently demonstrated that a recombinant vaccinia virus (VV) expressing a chimeric GM-CSF/gp120 fusion protein induces a significant increase in cellular immune responses against the HIV-1 env antigen (Rodriguez et al., 1998). It has also been shown that GM-CSF, given at priming in the form of a DNA/GM-CSF chimeric vaccine, increases the magnitude of the anamnestic response, irrespective of the antigen form used in the subsequent booster immunization (Gerloni et al., 1998). GM-CSF enhances viability and function of dendritic cells (DC) (Heufler et al., 1998; Witmer et al., 1987), activates cells of the dendritic lineage in vitro (Inaba et al., 1992), and potentiates antigen presentation in vivo (Disis et al., 1996). GM-CSF is neither a Th1 nor a Th2 cytokine and can exert its adjuvant effect without skewing the Th1/Th2 balance.

We previously described a vaccination protocol based on priming with influenza virus expressing a CD8⁺ T cell epitope of circumsporozoite (CS) protein from Plasmodium yoelii, followed by boosting with a VV recombinant expressing the entire CS protein which induces high protection levels against malaria in the murine model system (Li et al., 1993; Rodrigues et al., 1994). Other recent experiments in mice have shown that priming with DNA and boosting with recombinant vaccinia virus (VV) expressing a circumsporozoite protein is associated with high immunogenicity and protective efficacy against P. berghei (Sedegah et al., 1998). Delivering recombinant VV in the boost rather than in the priming dose may elude development of host immunity to the viral portion of the immunogen, which would reduce the response to the booster. The critical event in priming may depend on the processing properties of the immunogen expressed (influenza or DNA better than VV), whereas the secondary response is dependent on the quantity of immunogen expressed (VV better than influenza or DNA).

To improve the efficiency of a HIV-1 vaccine, we have implemented a vaccination regime consisting of priming with DNA vectors expressing gp120 and GM-CSF, followed by boosting with a recombinant VV expressing env. We also present compelling data that support the enhancement of both humoral as well as a cytotoxic T cell response by priming with DNA plasmids containing the appropriate antigen plus GM-CSF and boosting with a recombinant vaccinia virus containing the same antigen as that used for the initial immunization.

II. Results

A. Enhanced humoral immune response to HIV-1 gp120 by the priming/booster approach: vector DNA/VV recombinant

We have attempted to develop new strategies for triggering efficient immune responses by integrating current procedures with new techniques, using combinations of plasmids and live viruses. More specifically, we focused on the immune response to the HIV-1 env protein, using a DNA plasmid containing the env gp120 gene (pCdm7) to immunize mice intradermically; we then analyzed the antibody response to HIV-1 gp120. In a parallel set of experiments, we compared this response to that of mice injected intraperitoneally with a recombinant vaccinia virus encoding HIV-1 gp120. Finally, given the increase in immunogenicity of GM-CSF-linked antigens, we coimmunized mice with a plasmid DNA vector encoding murine GM-CSF.

Balb/c mice were primed and boosted with combinations of DNA vectors expressing HIV-1 gp120 or

Here we have analyzed the effect of different immunization protocols on triggering T cell activation. Two weeks after the last injection, splenocytes from immunized mice were tested for T cell proliferation. Antigen-specific T cell proliferation was determined after incubation of the splenocyte cultures with purified gp120. Cell proliferation was measured by addition of [³H]-thymidine to cultures and determination of the [³H]-thymidine incorporation ratio in gp120-stimulated cultures vs. unstimulated cultures, to derive the stimulation index (SI). The highest SI was obtained when the GM-CSF-DNA vector was coinjected with the gp120 DNA vector in the priming; this enhancement was observed regardless of whether the booster was given with vector DNA or VV-env (Fig. 3).

To determine the type of immune response, we analyzed the pattern of cytokine responsiveness. IL-4 and IFN-g levels were measured in in vitro gp120-stimulated splenocytes from immunized animals (Fig. 4). IFN-g levels in DNA/VV-immunized mice were significantly higher than in DNA/DNA-immunized mice. There was a significant increase in IFN-g production when GM-CSF-DNA was incorporated in the priming of mice immunized with DNA/DNA, but not when the priming/booster regime consisted of vector DNA/VV-env. In addition, IL-4 was only detectable in splenocyte cultures from DNA/VV-env-immunized mice; coinjection of GM-CSF-DNA at priming induced increased production of this cytokine.

C. DNA vaccination activates efficient CD8⁺ T cells

The ELISPOT assay uses peptides of defined MHC class I-restricted CTL epitopes to quantitate epitope-specific IFN-g release by individual CD8⁺ T cells in unstimulated splenocyte cultures, correlating with levels of cytotoxic activity (Hanke et al., 1998, Rodrigues et al, 1994). As detection of IFN-g-producing cells is more sensitive and quantitative than a ⁵¹Cr-release cytotoxicity assay, the ELISPOT assay is a useful method for evaluating the success of vaccination. The priming/boosting vaccination regime, using the DNA/VV-env, induces a dramatic stimulation of IFN-g-producing CD8⁺ cells, as compared to vaccination with the DNA/DNA approach (Fig. 5). A single VV-env inoculation was even more effective in inducing an env-specific CD8⁺ T cell response than the DNA/DNA double immunization regime, as seen by the larger number of IFN-g secreting cells among splenocytes from mice primed with GM-CSF-DNA and boosted with recombinant VV-env. In accordance with the observed increase in IFN-g production, there was a 10-fold increase in the CD8⁺ T cells number in mice primed with GM-CSF-DNA and the gp120-expressing DNA vector, and boosted with gp120-DNA. This enhancing effect of GM-CSF was not, however, observed in animals treated with the DNA/VV-env approach.

III. Discussion

The ideal HIV-1 vaccine would probably elicit both humoral and cellular immune responses. Such a dual response would aid in clearing virus before persistent infection is established, as well as in eliminating infected cells by recognizing processed viral fragments associated with host-specific MHC class I antigens and presented on the infected cell surface. The induction of antibodies against the gp120 surface protein of HIV-1 would be important, as this protein is the principal viral determinant interacting with host receptors and the major antigenic determinant to which neutralizing antibodies are directed. Moreover, HIV-specific antibodies that mediate ADCC are found very early in acute infection and correlate well with a decline in plasma virus load (D´Souza et al., 1996). On the other hand, the dramatic decrease in HIV-1 viral load following the initial appearance of CTL after primary infection, and the temporal association between HIV-specific CTL activity and stable viral load or CD4⁺ cell counts during asymptomatic stages are the best indicators of CTL efficiency.

Immunization with DNA vaccines induces protective CTL responses in many experimental models, and can prevent HIV-1 infection or trigger a neutralizing HIV-1 response in certain non-human systems (Wang et al., 1995; Ugen et al., 1997); the safety, feasibility and immunogenicity of DNA vaccines are currently under evaluation in clinical trials (unpublished data).

Although DNA vaccines generally elicit strong cytotoxic responses, the antibody response generated by gene vaccination is lower than that induced by protein vaccination. An additional boost may be required for the generation of a protective humoral immune response, and combined prime-boost immunization approaches using different vector combinations are under evaluation. Priming with a DNA vector followed by a boost with an attenuated VV (MVA) recombinant has recently been shown to result in unexpectedly high levels of protection against P. berghei in mice (Schneider et al., 1998). The rationale for using two distinct vaccine vehicles for the same antigen in combined prime/boost regimes lies in the observation that sequential vaccination with the same vehicle complex decreases vaccine immunogenicity; for example, prior host exposure to VV has been observed to reduce the immunogenicity of VV-based vaccines. VV recombinants expressing HIV-1 envelope antigens were among the first prototype vaccine constructs generated against AIDS (Chakrabarty et al., 1986; Hu et al., 1986). In animal models, these constructs elicit both humoral and cellular immune responses, which are nonetheless unable to control viral infection after HIV challenge (Earl et al., 1989).

A DNA prime-MVA boost regime has also been evaluated as a candidate HIV vaccine, given its ability to potentiate the induction of specific CTL (Hank et al., 1998); this study focuses, however, on cellular and not on antibody responses. Here we report the effect of the combined vaccine on both humoral and cellular responses, and take advantage of the immunostimulant properties of GM-CSF in the priming step. We and others have demonstrated that this cytokine increases the potency of immunization against tumor cells and protein antigens (Tao & Levy, 1993; Disis et al., 1996). Using a VV expressing the GM-CSF/gp120 fusion protein, we observed significant enhancement of the cellular immune response against the env antigen (Rodriguez et al., 1998).

Here we show an increase in both humoral and cellular immune responses using the DNA-prime/VV-boost regime, as compared to vaccination with DNA alone. This enhancement is especially relevant in the induction of IFN-g-producing CD8⁺ cells, for which a 100-fold increase was observed. The antibody response was also significantly augmented both in the titer of specific antibodies and in the viral neutralizing activity of the serum. Whereas sera from mice immunized with the DNA/DNA regime contained only IgM antibodies, IgG1 was the predominant isotype in sera from DNA/VV-immunized animals, although IgM and IgG2a were also detectable. This response appears to be paradoxical, since the induction of high CTL levels corresponds to a typical Th1-type response, whereas IgG1 antibody production is characteristic of a Th2-type response. In fact, we observed cytokines representative of both types of response (IFN-g and IL-4) in culture supernatants of splenocytes from mice receiving the combined immunization, whereas IFN-g but not IL-4 was present in DNA-immunized mice. It has been claimed that the effect of DNA is dominant, as preimmunization with plasmid DNA followed by boosting with protein in alum prevents the induction of the IgE antibody response or the activation of Th2 cells that would be expected in an alum-based vaccine (Raz et al., 1996). Our results do not confirm this observation, since boosting with gp120-expressing vaccinia virus induces a combined immune response characterized by IL-4 and IgG1 production, as well as by a large number of IFN-g-producing CD8⁺ cells. The only reports of significant antigen-specific IL-4 and IgG1 production following DNA immunization are associated with the gene gun route, since direct intramuscular or intradermal inoculation of naked DNA results specifically in Th1 responses (Feltquate et al., 1997). Immunization with the chicken ovalbumin (OVA) gene induces primarily an IgG1 rather than an IgG2a response, although the OVA-specific T cell response includes IFN-g-secreting Th1 cells (Tighe et al., 1998). An unbiased increase in all immune responses was also observed by increasing the period between immunizations, with significant enhancement of IFN-g, IL-4, IgG1 and IgG2a production (Prayaga et al., 1997). Immune responses of this nature may be more desirable for protection against mucosally-transmitted viral diseases and certain inflammatory autoimmune disorders such as rheumatoid arthritis.

Finally, we have observed that coimmunization of gp120-DNA with GM-CSF-DNA results in the enhancement of antigen-specific T cell proliferation rates in both DNA/DNA- and DNA/VV-immunized mice. The GM-CSF stimulation effect on the number of IFN-g-producing CD8⁺ cells can clearly be seen in DNA/DNA-immunized mice, but not in DNA/VV-vaccinated mice, probably due to a saturation effect of the VV boost. A modest but significant increase was seen, however, in the humoral response when GM-CSF-DNA is included in the DNA/VV immunization protocol.

Given the acceptability and potential safety of both DNA and VV vaccine vehicles in humans, it is reasonable to believe than this priming/booster regime based on these vectors may be effective not only for HIV, but also for other infectious agents and some forms of cancer.

IV. Materials and methods

A. Plasmids and recombinant virus

Plasmid DNA expressing gp120 was kindly provided by Dr. Andreas Bültmann (Max von Pettenkofer-Institut, Genzentrum, Munich, Germany) (André et al., 1998). The plasmid expressing GM-CSF was previously described (Rodriguez et al., 1998). Plasmids for immunization were purified using Qiagen Maxiprep Columns (Hilden, Germany). The VV-env recombinant virus has been previously described (Rodriguez et al., 1989).

B. Immunization of mice

Six-week-old female BALB/c mice received 50 mg of DNA in PBS intradermically. Fifteen days later, they received an intraperitoneal injection of 5 x 10⁷ plaque-forming units (pfu) of purified VV env.

C. Anti-gp120 ELISA

Anti-gp120 levels in immunized mouse serum were quantitated by titering on ELISA plates (Maxi-sorb, Nunc) coated with gp120 (IIIB; Intracel) at 1 mg/ml in PBS (100 ml/well) overnight at 4ºC. Remaining protein binding sites were blocked with 0.5% BSA in PBS (200 ml/well, 60 min, 37ºC). After washing plates with distilled water, the diluted sera were added to the wells and incubated for 60 min at 37ºC, followed by a peroxidase (PO)-labeled goat anti-mouse immunoglobulin antibody (GAM-PO, Tago Inc., Burlingame, CA) and OPD (Sigma Chemical Co., St. Louis, MO). The reaction was terminated with 3N sulphuric acid and optical density determined at 492 nm. The titer is expressed as the highest serum dilution giving an absorbance value three times higher than that of the preimmune serum.

D. Viral neutralization assays

HIV-1 strain IIIB cell-free virus (NL4-3 strain; 2 ng p24/10⁶ cells) was incubated with serial dilutions of mouse antisera for 1 h at 37ºC. PHA-activated PBMC were incubated with the virus-antiserum mix for 2 h, then washed three times. Triplicate 0.5 ml cultures were tested for p24 production at 5, 7 and 10 days post-infection. p24 antigen levels were measured by ELISA (Coulter, Miami, FL).

E. T cell proliferation assay

Spleens were removed from infected mice, single-cell suspensions prepared in complete medium (RPMI-1640 with 10% FCS, 2 mM L-glutamine and 10 µM 2-mercaptoethanol), and splenocytes (10⁶/well) dispensed into 96-well microtiter plates. Culture triplicates were challenged with 2 µg/ml of gp120 or concanavalin A (Con A, Sigma) and incubated for three days at 37ºC in 5% CO2, after which 1 µCi of [³H]-thymidine (5 Ci/mmol; Amersham) was added to each well. Cells were harvested after 16 h and [³H]-thymidine incorporation into DNA measured by liquid scintillation counting. Stimulation index (SI) was determined as the ratio: experimental count (mean of [³H]-thymidine incorporation in triplicate wells incubated with antigen) divided by the spontaneous count (mean of [³H]-thymidine incorporation in triplicate wells incubated with medium alone).

E. Cytokine assays

In vitro gp120-specific IFN-g and IL-4 production was measured using gp120-stimulated splenocytes as previously described (Rodriguez et al., 1998). Conditioned medium containing the secreted cytokines were collected from all cultures after 48 h.

F. ELISPOT assay

The ELISPOT assay was used to detect epitope-specific IFN-g-secreting cells (Miyahira et al., 1995). Briefly, nitrocellulose-bottomed 96-well plates were coated with anti-mouse IFN-g mAb R4-6A2 (8 mg/ml, Pharmingen, San Diego, CA). After overnight incubation at room temperature, wells were washed threes times with RPMI 1640, then 100 ml of medium supplemented with 10% FCS were added to each well, and plates incubated at 37ºC for 1 h. Duplicate cultures were prepared with serial doubling dilutions of immunized splenocytes, beginning with 10⁶ cells/well. P815 cells (H-2^d), used as antigen-presenting cells (APC), were pulsed with 10^-6 M of the synthetic peptide GPGRAFVTI, corresponding to the V3 loop of gp120, and treated with mitomycin C (30 mg/ml, Sigma). After several washes with culture medium, 10⁵ P815 cells were added to each well. Control P815 cells were not pulsed with the peptide. Plates were incubated for 26-28 h at 37ºC, washed with PBS containing 0.05% Tween-20 (PBS-T) and incubated overnight at 4ºC with biotinylated anti-mouse IFN-g mAb XMG1.2 (2 mg/ml, Pharmingen) in PBS-T. Plates were washed with PBS-T and PO-labeled avidin (Sigma; 100 ml, 1/800 dilution in PBS-T) was added to each well. One hour later, wells were washed with PBS/T and PBS. Spots were developed by adding 50 mM Tris-HCl, pH 7.5 containing 1 mg/ml of 3,3'-diaminobenzidine tetrahydrochloride (Sigma) and 0.015% H₂O₂. When the plates were completely dry, the number of spots was determined with the aid of a stereomicroscope.

Acknowledgments

The authors are grateful to L. Gómez for help with animal procedures and to C. Mark for excellent editorial assistance. This work was supported in part by grants from the CICYT of Spain SAF95-0072 and the Comunidad de Madrid 08.6/0020/97 (to M.E.). D.R. and J.R.R. were recipients of contracts from the CSIC of Spain. The Department of Immunology and Oncology was founded and is supported by the Spanish Research Council (CSIC) and Pharmacia & Upjohn

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Research Article

Received: 15 October 1998; accepted: 25 October 1998