Gene Ther Mol Biol Vol Vol 12, 7-14, 2008

Pro-apoptotic gene enhances the immunogenicity of glycoprotein B gene of herpes simplex virus-1

Research Article

Masoud Parsania¹, Zuhair Muhammad Hassan^2,*, Taravat Bamdad¹, Maryam Kheirandish³, Mohammad Hassan Pouriayevali¹, Rohollah Dorostkar Sari¹, Mohammad Nabi Sarbolouki⁴, Abbas Jamali¹, Mehdi Mahdavi²

¹Department of Virology, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran;

²Department of Immunology, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran;

³Research Center, Iranian Blood Transfusion Organization, Tehran, Iran;

⁴Institute of Biochemistry and Biophysics, Tehran University, Tehran, Iran

__________________________________________________________________________________

*Correspondence: Zuhair Muhammad Hassan, Department of Immunology, School of Medical Sciences, Tarbiat Modares University, P. O. Box: 14115-111, Tehran, I.R.Iran; Tel: +98[21] 82883565; Fax: +98[21] 88006544; E-mail: hasan_zm@modares.ac.ir

Key words: Apoptosis; Bax; DNA vaccine; HSV-1

Abbreviations: 3-(4,5-dimethylthiazol-2-yl)-2,5-dipheyltetrazolium bromide, (MTT); Antigen Presenting Cells, (APCs); Cytotoxic T Lymphocyte, (CTL); Dulbecco’s Minimal Eagle Medium, (DMEM); Fetal Calf Serum, (FCS); glycoprotein of B, (gB); glycoprotein of D, (gD); Herpes Simplex Virus type 1, (HSV-1)

Received: 24 December 2007; Revised: 15 April 2008

Accepted: 28 April 2008; electronically published: 6 May 2008

Summary

Increasing apoptosis in transfected host cells has caused a significant enhance in the immunogenicity of DNA vaccine. It has been known that pro-apoptotic protein Bax induces apoptosis and adjuvant effect of Bax is achieved when suitable dose of the Bax gene is used as a molecular adjuvant. We compared three doses of Bax encoding plasmid (pbax) including 10, 25 and 50 μg of plasmid DNA, intradermally co-injected with glycoprotein B (gB) of Herpes Simplex Virus (HSV)-1encoded plasmid (pgB) into the C57BL/6 mice. Then, the responses of the mice to viral challenge and serum antibody levels, as well as lymphoproliferative responses and cytokine production by splenocytes were studied. Our findings showed that the mice immunized with 25 μg pbax together with pgB had more efficient protection than the mice immunized with 10 and 50 μg of pbax together with pgB. Analysis of the cellular and humoral responses showed that the mice immunized with 25 μg pbax and pgB induced higher levels of antibody as well as stronger lymphocyte proliferative responses and higher levels of Interleukin-4 compared to those mice received 10 and 50 μg of pbax together with pgB. It is concluded that co-immunization with 25 μg of pbax and pgB increased the induced immune responses comparing to 10 and 50 μg of pbax and pgB.

I. Introduction

Gene vaccination or plasmid DNA immunization is a promising strategy for the development of new vaccine to elicit immune responses against an encoded antigen that leads to protective humoral or cell-mediated immune responses (Donnelly et al, 1997; Giese 1998; Lemieux et al, 2002).

There are many approaches being tried to enhance the immuogenicity of DNA vaccines. These include the use of conventional adjuvant (Ulmer et al, 1999; Wang et al, 2000), the optimization of antigen expression, the use of various cytokines or other immunologically active molecules which may be encoded within the same vector (Kim et al, 1998; Bower et al, 2005; Nimal et al, 2005) and also the combined use of DNA vaccine and live virus (Ada and Ramshaw, 2003).

Studies have linked the immunostimulatory properties of apoptotic cell death to enhanced antigen presentation and cytotoxic T Lymphocyte (CTL) responses (Albert et al, 1998; Rover et al, 1998). More recent studies have shown that apoptotic death can be triggered by a variety of mechanisms, accompanied by the production and release of various factors that help the immune system to make a decision about the handling of the dead cells. The apoptotic cells are recognized by professional Antigen Presenting Cells (APCs) through an assay of the molecules found on the surfaces of dying cells through the receptors such as CD36, CD14, CD91, or class A scavenger receptor. After ingestion and degradation of the antigen-loaded apoptotic cells, the APCs migrate to a lymphatic organ and present the antigen of interest to CD4+ and CD8+ T cells. Various reports have shown that the immunogenicity of antigenic material associated with dead or dying cells enhances DNA vaccine efficacy (Leitner and Restifo, 2003; Bergmann-Leitner and Leitner, 2004).

Herpes simplex virus type 1 (HSV-1) is common throughout the world. HSV-1 can cause a variety of clinical illnesses including oral-facial infections, cutaneous infections, neonatal herpes, herpes encephalitis and disseminated infections. Many HSV infections, however, are either asymptomatic or unrecognized. Despite efforts over many years to develop prophylactic protection against HSV infection, there is no effective vaccine available yet (Bernstein and Stanberry, 1999; Koelle and Corey, 2003). The HSV glycoproteins of B and D (gB and gD) are attractive choices for vaccination, because they are targets for both humoral and cell mediated immunities (Stanberry et al, 2002). With regard to HSV, several reports have demonstrated that immunization of animals with the expression of plasmids encoding HSV glycoproteins of B and D induces virus specific humoral and cellular responses and protects animals from experimental HSV challenge (Flo et al, 2000; Koelle and Corey, 2003).

The efficacy of dendrosome (novel dendritic spheroid nanoparticle gene porters) has been assessed in transferring cDNA into various cell lines and target cells in BALB/c mice. Previous studies have shown the efficacy of dendrosome (Den)123 nanoparticles in delivering the DNA vaccine (Sarbolouki et al, 2000; Balenga et al, 2006).

Bax gene is a pro-apoptotic member of bcl-2 family. Transfection of cells with a plasmid encoding Bax has been shown to cause the transfected cells to undergo programmed cell death over a period of days by triggering the mitochondrial pathway of apoptosis (Li et al, 2001; Kinsey et al, 2004). Increasing apoptosis in DNA vaccine transfected host cells causes to enhance the immunogenicity of the DNA-encoded antigen. An enhancement of the DNA vaccine-induced immune response is only achieved when careful tittering of the Bax-encoding plasmid dose is used as a molecular adjuvant (Bergmann-Leitner and Leitner, 2004).

The present study investigated the potentials of apoptosis induced by Bax gene when co-delivered with gB of HSV-1 encoding plasmid. Thus, we three doses of Bax-encoding plasmid including 10, 25 and 50 μg of plasmid DNA with Den123 were evaluated for their ability to enhance immune responses, when co-administrated with gB of HSV-1 encoding plasmid.

ІІ. Materials and Methods

A. Cell line and viruses

Vero (African green monkey kidney cells) cell line was used for propagation of the viruses. The cells were cultured in Dulbecco’s Minimal Eagle Medium (DMEM; Gibco, UK) supplemented with 10% fetal calf serum (FCS; Gibco, Belgium). Wild-type strain HSV-1 was isolated from a cold sore lesion of a patient. The virus was confirmed as HSV-1 by an HSV-1 specific monoclonal antibody (Soleimanjahi et al, 2003). The wild-type strain HSV-1 and its KOS strain were grown and tittered on the Vero cell line and stored at -70˚C.

B. Mice

Male inbred C57BL/6 mice (6 to 7 weeks old) were purchased from Pasteur Institute (Tehran, I.R.Iran). All animal procedures were performed according to the approved protocols and in accordance with the recommendations for the proper use of laboratory animals.

C. Plasmid DNA constructs

Plasmid DNA encoding HSV-1 gB was constructed by the insertion of the gB of HSV-1 into pcDNA3 under the control of CMV promoter as described previously (Bamdad et al, 2005). The plasmid containing Bax; pcDNA3-Bax was kindly provided by Wolfgang W. Leitner from the National Cancer Institute (National Institute of Health, Bethesda, Maryland, USA).

D. Dedrosome123

Den123 was synthesized under sterile conditions as previously mentioned (Sarbolouki et al, 2000). It was suspended at a concentration of 10 mg/ml in phosphate-buffered saline (PBS) and left at ambient temperature for 15 min. The suspension was filtered through 0.22 μm filters (Schleicher and Schuell) and stored at 4˚C.

E. Preparation of plasmid-Den123 and immunization

Fifty to 100 μg of the plasmids were mixed with proper amounts of Den123 just before injections so that a plasmid to Den123 ratio of 150:1 was obtained for all the groups receiving DNA. The mixture was left for 5 min at ambient temperature. The final volume for DNA vaccination was 100 μl in sterile PBS, which was then injected intradermally into four different sites (left upper, right upper, left lower and right lower back of the mice). Ten to 13 mice per group were immunized and challenged in each experiment and the mice were grouped according to Table 1.

F. Antibody assay

Blood samples were collected two weeks after the last immunization by tail bleeding. Enzyme-linked immunosorbent assay (ELISA) was performed as previously described (Pachl et al, 1987; Sin et al, 1999). Briefly, 96-well microtiter plates (Immunoplates Maxisorp, Nunc) were incubated with lectin-purified KOS strain HSV-1 glycoprotein as a coating antigen (Nass et al, 2001) for 48 h at 4°C and blocked with PBS containing 2% bovine serum albumin (Gibco) for 2 h at 20°C. Sera diluted 1:50 in blocking solution including 0.05% Tween 20. Each serum sample was determined by duplicate and represented as mean of them. To determine IgG antibodies was detected with a horseradish peroxidase-goat anti-mouse IgG conjugate (1:10000 dilution; Sigma) incubated for 1 h. After extensive washing, color was developed with ortho-phenylenediamine dihydrochloride (Sigma) for 30 min in the dark, the reaction terminated with 3N H₂So₄and absorbance measured at 490 nm. The antibody response of each mouse was measured individually and represented as optical density (OD) value for a given serum dilution.

Table 1. The information of the immunized groups and the time table of immunization, sampling and viral challenge.

Groups	pgB	pgB-Bax10	pgB-Bax25	pgB-Bax50	pbax	pcDNA3	Den	PBS	KOS
Injected materials	50 μg pcDNA3-gB plus Den123	50 μg pcDNA3-gB and10 μg pcDNA3-Bax plus Den123	50 μg pcDNA3-gB and 25 μg pcDNA3-Bax plus Den123	50 μg pcDNA3-gB and 50 μg pcDNA3-Bax plus Den123	50 μg pcDNA3-bax plus Den123.	50 μg pcDNA3 plus Den123	Den123 in 100 μl sterile PBS	100μl sterile PBS	1×10⁶ pfu of HSV-1 strain KOS
Day 0	First injection of the materials to the mice in different groups
Day 14	Second injection of the materials to the mice in different groups
Day 28	Third injection of the materials to the mice in different groups
Day 42	Blood collection, harvest of splenocytes from 5 mice and viral challenge with wild-type strain HSV-1 to the other mice
Up to day66	Monitoring of the survival rate daily for 14 days after the challenge

G. Lymphocyte proliferative response

Spleen was removed from the immunized mice and homogenized in PBS (pH 7.4). The erythrocytes cell suspension was lysed with 0.75% Tris-NH₄CL (pH 7.4). After being washed three times with PBS, the splenocytes were resuspended at 2 × 10⁶ cells/ml with the supplemented RPMI 1640 containing 10% FCS, 14 mM Hepes, 50 mM 2-mercaptoethanol, 100 μg/ml streptomycin and 100 IU/ml penicillin. The splenocytes were then plated in 96-well flat-bottom plates at 100 μl per well (2 ×10⁵ cells per well). Subsequently, 100 μl per well of the medium with or without three multiplicity of infection (moi) of the heat inactivated KOS strain of HSV-1 were added to the plates and mixed. Phytohemagglutinin-A (5 μg/ml; Sigma) was used as a positive control. Each animal’s splenocytes were plated in triplicate. The proliferative response was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-dipheyltetrazolium bromide (MTT) assay according to the method described by Xiao and colleagues (Xiao et al, 2004). Stimulation Index (SI) was calculated as:

SI= OD of the wells containing inactivated virus-stimulated cells/ OD of the wells containing only the cells with medium.

H. Cytokine assays

The splenocytes were prepared, cultured and stimulated as described earlier. After 48 h, the culture supernatents were harvested to test the presence of IFN-γ and IL-4. Assays for IFN-γ and IL-4 were performed using ELISA procedures according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA). Absorbance was measured at 450 nm and the results were expressed as pg/ml IFN-γ or IL-4 in the samples, based on the standard curve.

I. Viral challenge

Two weeks after the last immunization, the mice were challenged by intraperitoneal route with 1 × 10⁶ plaque-forming unit (pfu) of wild-type strain HSV-1 (which is the minimum dose that causes 100% mortality in the unvaccinated mice) and the survivals monitored for two weeks.

J. Statistical analysis

Proliferation assay and the production of cytokines were analyzed by one-way ANOVA followed by tukey's test. Kaplan-Meier analysis and the log rank test were used for the survival rate. Values of P < 0.05 were considered as significant.

ІІІ. Results

A. Antibody responses

Antibody production was measured two weeks after the last immunization. As shown in Figure 1, all groups of the mice, immunized with the construct containing gB gene (pgB, pgB-bax10, pgB-bax25 and pgB-bax50), induced significantly higher levels of IgG production comparing to the negative control groups (PBS, pcDNA3, pbax and Den). The KOS immunized group showed the highest IgG level (P < 0.001). Furthermore, the pgB-Bax25 group showed significantly higher IgG level compairing to the other gB encoding plasmid immunized groups (P < 0.001). In contrast, the pgBax-50 group showed significantly lower IgG level comparing to the other gB encoding plasmid immunized groups (P < 0.05).

B. Lymphocyte proliferation

The proliferation of lymphocytes was estimated in all the groups for evaluation of cell-mediated immunity. The lymphocyte proliferative responses were analyzed two weeks after the third immunization. The obtained results indicated a significant increase in the stimulation index in the mice immunized with the construct containing gB (pgB, pgB-bax10, pgB-bax25, pgB-Bax50) comparing to the negative control groups i.e. pcDNA3, pbax and Den (Figure 2).

Although proliferative response in the pgB-bax25 group was enhanced comparing to the pgB group, but the difference was not significant. In contrast, proliferative response in the pgB-bax50 group was significantly lower than in the pgB, pgB-Bax10 and pgB-bax25 groups (P< 0.05). The highest stimulation index was observed in the splenocytes of the KOS immunized mice (P< 0.0001).

C. Cytokine assays

The shifting of immune response in all the groups was evaluated by measuring IFN-γ and IL-4 levels as indicators of Th1 or Th2 cell responses, respectively.

Cytokine assays were analyzed two weeks after the third immunization followed by in vitro restimulation of the splenocytes from the immunized mice. Production of IFN-γ and IL-4 in the supernatants of the inactivated virus-stimulated splenocytes from the immunized mice was assessed. As shown in Figure 3A, all groups of the mice immunized with the construct containing gB gene (pgB, pgB-Bax10, pgB-bax25 and pgB-bax50) induced significantly higher levels of IFN-γ production comparing to the negative control groups (PBS, pcDNA3, pbax and Den). The amount of IFN-γ in pgBbax50 group was significantly lower than in the pgB, pgB-Bax10 and pgB-bax25 groups (P < 0.05, P< 0.05 and P = 0.003, respectively). There was no significant difference in IFN-γ level between the pgB pgB-Bax10 and pgB-bax25 groups. The highest level of the IFN-γ production was observed in the KOS group (P< 0.0001). As shown in Figure 3B, significantly higher IL-4 levels were found in the supernatants of cultured splenocytes from the KOS and pgB-Bax25 groups comparing to the mice in the other groups (P < 0.0001). There was no significant difference in IL-4 level between the pgB, pgB-Bax10 and pgB-bax50 groups. The obtained results also indicated that the mice in the pgB-bax25 group induced enhanced Th2- type immune response.

class=Section5>

Figure 1. Antibody production measured two weeks after the last immunization by Enzyme-linked immunosorbent assay as described in the Materials and Methods. The means (the mean of five animals) of serum IgG antibody level were measured using the collected serum samples.

^♦ The KOS immunized group showed the highest IgG level (P < 0.001).

^♦♦The pgB-Bax25 group showed significantly higher IgG level comparing to the other gB encoding plasmid immunized groups (P < 0.001).

^♦♦♦ The pgB-Bax50 showed significantly lower IgG level comparing to the other gB encoding plasmid immunized groups (P < 0.05).

Surprisingly, Osorio and Ghiasi have shown that IL-4 has an important role in the enhancement of protective immunity against HSV-1 due to the raise in virus clearance (Osorio and Ghiasi, 2003). Also, in our experiment, enhancement of serum antibody level and IL-4 production in the pgB-bax25 group increased the resistance to virus challenge comparing to the pgB group. Our findings confirmed the shifting of immune response to Th2, supporting the previous data of Nimal and colleagues (Nimal et al, 2007).

Despite that there are much evidence demonstrating a significant correlation between apoptosis and increased immunogenicity in DNA vaccine (Sasaki et al, 2001; Nimal et al, 2007), some reports indicated that adjuvant activity of apoptosis must be restricted, because antigen expression must precede cell death, thereby, allowing the accumulation of antigenic material (Sasaki et al, 2001; Bergmann-Leitner and Leitner, 2004). Thus, an enhancement of DNA vaccine-induced immune response is only achieved when carefully titered dose of the bax-encoding plasmid is used as a molecular adjuvant (Bergmann-Leitner and Leitner, 2004). In this study, we compared three doses of bax-encoding plasmid including 10, 25 and 50 μg of plasmid DNA when co-administrated with 50 μg of gB encoding plasmid for the induction of protective immune responses. Our results showed a significant reduce in the cell mediated immunity as well as in the protection to virus challenge in the pgB-bax10 and pgB-bax50 groups comparing to the pgB-bax25 group. Based on the evidence cited above, in the case of pgB-bax50 group probably rapid apoptosis occurred in the transfected cells with 50 μg bax encoding plasmid, and interfered with antigen expression by co-administration with gB encoding plasmid, thus reducing the immune response against this antigen. Also it seems that in the pgB-bax10 group mild apoptosis occurred in the transfected cells with 10 μg bax encoding plasmid, thus adjuvant activity of apoptosis was insufficient. In pgB-bax10 group, immune responses such as serum antibody level, lymphocyte proliferative response and IFN-γ and IL-4 production slightly increased comparing to the pgB group, but the differences were not significant. However, it is suggested when 25 μg of bax-encoding plasmid was used, the expression of antigen occurred before the generation of apoptotic bodies and caused a great enhancement in the immunogenicity of DNA vaccine. In their recent report, Sasaki and colleagues showed that antigen-laden apoptotic bodies created by the vectors co-expressing influenza virus hemagglutinin and nucleoprotein genes as well as mutant caspase genes, markedly increased immune responses (Sasaki et al, 2001). They also reported that the adjuvant activity was restricted partially to the inactivated caspases that allowed immunogen expression before the generation of apoptotic bodies. Further, they demonstrated that immunomodulatory effect can be achieved depending on the dose, the kinetics of apoptosis induced and the ratio of the antigen plasmid to apoptosis plasmid (Sasaki et al, 2002). We and others have found that when bax encoding plasmid co-administrated with DNA vaccine by simple co-injection, the dose of the apoptosis gene encoding plasmid must be optimized (Kinsey et al, 2004).

One of the concerns about the safety of DNA vaccine has been insertional mutagenesis (Robinson and Pertmer, 2000; Sasaki et al, 2001; Li et al, 2001). Expression of the bax gene necessarily leads to self-limiting, because most of the transfected cells die within a few days (Li et al, 2001; Xiao et al, 2004). Thus, triggering of apoptosis in the cells transfected with a DNA plasmid eliminates a lingering safety concern of many critics of DNA vaccines, the unlikely but theoretically possible integration of the DNA into the host’s genome, resulting in tumor genesis.

In conclusion the results of our study showed that co-immunization with 25 μg of bax-encoding plasmid and gB-encoding plasmid increased the induced immune responses comparing to 10 and 50 μg of bax-encoding plasmid and gB-encoding. Finally we recommend to evaluate the degree of apoptosis in the transfected cells, with the aim of confirming and determining the exact effects of apoptosis on enhancing the efficacy of DNA vaccine.

Acknowledgements

We would like to thank Dr Wolfgang W. Leitner (National Cancer Institute, National Institute of Health, USA) for his kind gift of the bax cDNA construct and a critical review of the article. This work has been supported financially by Tarbiat Modares University (Tehran, Iran).

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Zuhair Muhammad Hassan