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
Masoud Parsania1, Zuhair Muhammad Hassan2,*, 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 82883565; Fax: +98 88006544; E-mail: email@example.com
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); DulbeccoÕs Minimal Eagle Medium, (DMEM); Fetal Calf Serum, (FCS); glycoprotein of B, (gB); glycoprotein of D, (gD); Herpes Simplex Virus type 1, (HSV-1)
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.
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 DulbeccoÕs 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 -70ûC.
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).
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 4ûC.
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 H2So4 and 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.
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
First injection of the materials to the mice in different groups
Second injection of the materials to the mice in different groups
Third injection of the materials to the mice in different groups
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 animalÕs 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 manufacturerÕs 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.
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.
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).
Figure 2. Lymphocyte proliferative responses after in vitro stimulation with the heat inactivated KOS strain of HSV-1. Two weeks after the third immunization, each group of mice (n=5) was sacrificed and the splenocytes were stimulated with three moi of the heat inactivated KOS strain of HSV-1. After 48 h of stimulation, MTT was added and the OD was determined after a further 4 h inoculation. The samples were assayed in triplicate.
♦ P< 0.0001, KOS immunized group showed highest lymphocyte proliferation.
♦♦ P< 0.05, pgB-Bax50 group showed significantly lower lymphocyte proliferation comparing to the other gB encoding plasmid immunized groups.
♦ P< 0.0001 KOS immunized group showed highest IFN-γ production (A).
♦♦ pgB-Bax50 was significantly lower than the pgB, pgB-Bax10 and pgB-bax25 groups (P < 0.05, P< 0.05, P = 0.003, respectively) (A).
♦, ♦♦ P< 0.0001 KOS and pgB-Bax25 groups induced significantly higher level of IL-4 production comparing to the pgB, pgB-Bax10 and pgB-bax50 groups (B).
D. Intraperitoneal acute HSV-1 challenge
The mice were challenged with 1×106 pfu of the wild-type strain HSV-1 intraperitoneally two weeks after the third immunization and the survival rate was recorded for 14 days. The survival rates are shown in Figure 4. The mice immunized with pgB-Bax25 and the mice immunized by live virus (strain KOS) showed a 100% survival rate, as compared with 80% survival rate in the group immunized with pgB and pgB-Bax10. All the mice in the negative control groups immunized with PBS, pcDNA3, Den (data not shown) and pbax died after the viral challenge. A significant decrease in the resistance to virus challenge was recorded in the pgB-Bax50 group compared with the pgB-Bax25 group (P = 0.025).
In the present study, we investigated the effect of apoptosis on the efficacy of a DNA vaccine against HSV-1. Our results showed that bax-encoding plasmid enhanced immune responses during the DNA vaccination when a suitable dose was used.
The apoptotic death of the DNA vaccine transfected host cells could be very beneficial when attempting to improve the efficiency of genetic immunization (Leitner and Restifo, 2003; Bergmann-Leitner and Leitner, 2004). Kinseye and colleagues have explored that co-injection of the plasmids encoding gp 120 of HIV and Bax elicited both humoral and cytotoxic immunity (Kinsey et al, 2004). In the present study, our finding showed that simple intradermal co-injection of the pro-apoptotic bax gene together with a plasmid encoding gB of HSV-1 increased the protective immune responses to the antigen. The obtained data indicated that serum antibody level and production of IL-4 from the splenocytes after antigenic stimulation in the pgB-bax25 group were significantly higher than in the pgB group.
Figure 4. Survival of the immunized mice after wild-type strain HSV-1 challenge. All the groups immunized with pgB (n=8) and the positive and negative control groups (n=5) were immunized as described in the Materials and Methods. Two weeks after the third immunization, the mice were challenged with 1×106 pfu of the wild-type strain HSV-1 by intraperitoneal route. The survival rate was monitored daily for 14 days after the challenge.
♦ P = 0.025, comparing the pgB-Bax25 group with the pgB-Bax50 group.
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 hostÕs 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.
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).
Ada G, Ramshaw I (2003) DNA vaccination. Expert Opin Emerg Drugs 8, 27-35.
Albert ML, Sauter B, Bhardwaj N (1998) Dendritic cells acquire antigen from apoptotic cells and induce class Ι-restricted CTLs. Nature 392, 86-9.
Balenga NA, Zahedifard F, Weiss R, Sarbolouki MN, Thalhamer J, Rafati S (2006) Protective efficiency of dendrosomes as novel nano-sized adjuvants for DNA vaccination against birch pollen allergy. J Biotechnol 124, 602-14.
Bamdad T, Roostaee MH, Sadeghizadeh M, Mahboudi F, Kazemnejad A, Soleimanjahi H (2005) Immunogenicity and protective effect of a DNA construct encoding certain neutralizing epitopes of herpes simplex virus type-1 glycoprotein B. Folia Biol (Praha) 51, 109-13.
Bergmann-Leitner ES, Leitner WW (2004) Danger, death and DNA vaccines. Microbes Infect 6, 319-27.
Bernstein DI, Stanberry LR (1999) Herpes simplex virus vaccines.Vaccine 17, 1681-9.
Bower JF, Sanders KL, Ross TM (2005) C3d enhances immune responses using low doses of DNA expressing the HIV-1 envelope from codon-optimized gene sequences. Curr HIV Res 3, 191-8.
Donnelly, Ulmer JB, Shiver JW, Liu MA (1997) DNA vaccines. Annu Rev Immunol 15, 617-48.
Flo J, Beatriz Perez A, Tisminetzky S, Baralle F (2000) Superiority of intramuscular route and full length glycoprotein D for DNA vaccination against herpes simplex 2. Enhancement of protection by the co-delivery of the GM-CSF gene. Vaccine 18, 3242-53.
Giese M (1998) DNA-antiviral vaccine: new development and approaches a review. Virus Genes 17, 211-232.
Kim JJ, Trivedi NN, Nottingham LK, Morrison L, Tsai A, Hu Y, Mahalingam S, Dang K, Ahn L, Doyle NK, Wilson DM, Chattergoon MA, Chalian AA, Boyer JD, Agadjanyan MG, Weiner DB (1998) Modulation of amplitude and direction of in vivo immune responses by co-administration of cytokine gene expression cassettes with DNA immunogens. Eur J Immunol 28, 1089-103.
Kinsey BM, Marcelli M, Song L, Bhogal BS, Ittmann M, Orson FM (2004) Enhancement of both cellular and humoral responses to genetic immunization by co-administration of an antigen-expressing plasmid and a plasmid encoding the pro-apoptotic protein Bax. J Gene Med 6, 445-54.
Koelle DM, Corey L (2003) Recent progress in herpes simplex virus immunobiology and vaccine research. J Clin Microbiol 16, 96-113.
Leitner WW, Restifo NP (2003) DNA vaccines and apoptosis: to kill or not to kill? J Clin Invest 112, 22-4.
Lemieux P (2002) Technological advances to increase immunogenicity of DNA vaccines. Expert Rev Vaccines 1, 85-93.
Li X, Marani M, Mannucci R, Kinsey B, Andriani F, Nicoletti I, Denner L Marcelli M (2001) Overexpression of BCL-X(L) underlies the molecular basis for resistance to staurosporine-induced apoptosis in PC-3 cells. Cancer Res 61, 1699-706.
Li X, Marani M, Yu J, Nan B, Roth JA, Kagawa S, Fang B,Denner L, Marcelli M (2001) Adenovirus-mediated Bax overexpression for the induction of therapeutic apoptosis in prostate cancer. Cancer Res 61, 186-91.
Nass PH, Elkins KL, Weir JP (2001) Protective immunity against herpes simplex virus generated by DNA vaccination compared to natural infection. Vaccine 19, 1538-46.
Nimal S, McCormick AL, Thomas MS, Heath AW (2005) An interferon gamma gp120 fusion delivered as a DNA vaccine induces enhanced priming. Vaccine 23, 3984-90.
Nimal S, Thomas MS, Heath AW (2007) Fusion of antigen to Fas-ligand in a DNA vaccine enhances immunogenicity. Vaccine 25, 2306-2315.
Osorio Y, Ghiasi H (2003) Comparison of adjuvant efficacy of herpes simplex virus type 1 recombinant viruses expressing TH1 and TH2 cytokine genes. J Virol 77, 5774-83.
Pachl C, Burke RL, Stuve LL, Sanchez-Pescador L, Van Nest G, Masiarz F, Dina D (1987) Expression of cell-associated and secreted forms of herpes simplex virus type 1 glycoprotein gB in mammalian cells. J Virol 61, 315-25.
Robinson HL, Pertmer TM (2000) DNA vaccines for viral infections: basic studies and applications. Adv Virus Res 55, 1-74.
Rover P, Vallinoto C, Bondanza A, Corsti MC, Rescigno M, Ricciardi-Castagnioli P, Rugarli C, Manfredi AA (1998) Bystander apoptosis triggers dendritic cell maturation and antigen-presenting function. J Immunol 161, 4467-71.
Sarbolouki MN, Sadeghizadeh M, Yaghoobi MM, Karami A, Lohrasbi T (2000) Dendrosomes: A novel family of vehicles for transfection and therapy. J Chem Tech Biotech 75, 919-922.
Sasaki S, Amara RR, Oran AE, Smith JM, Robinson HL (2001) Apoptosis-mediated enhancement of DNA-raised immune responses by mutant caspases. Nat Biotechnol 19, 543-7.
Sasaki S, Amara RR, Yeow WS, Pitha PM, Robinson HL (2002) Regulation of DNA-raised immune responses by cotransfected interferon regulatory factors. J Virol 76, 6652-9.
Sin JI, Kim JJ, Boyer JD, Ciccarelli RB, Higgins TJ, Weiner DB (1999) In vivo modulation of vaccine-induced immune responses toward a Th1 phenotype increases potency and vaccine effectiveness in a herpes simplex virus type 2 mouse model. J Virol 73, 501-9.
Soleimanjahi H, Roostaee MH, Rasaee MJ, Mahboodi F, Bamdad T (2003) Immunogenecity and efficacy of baculovirus-derived glycoprotein D of herpes simples virus type-1 in mice. Arch Razi Ins 55, 19-28.
Stanberry LR, Spruance SL, Cunningham AL, Bernstein DI, Mindel A, Sacks S, Tyring S, Aoki FY, Slaoui M, Denis M, Vandepapeliere P, Dubin G, GlaxoSmithKline (2002) Herpes Vaccine Efficacy Study Group. Glycoprotein-D-adjuvant vaccine to prevent genital herpes. N Engl J Med 347, 1652-61.
Ulmer JB, DeWitt CM, Chastain M, Freidman A, Donelly JJ, McClements WL, Caulfield MJ, Bohannon KE, Volkin DB, Evans RK (1999) Enhancement of DNA vaccine potency using conventional aluminum phosphate adjuvants. Vaccine 18, 18-28.
Wang S, Liu X, Fisher K, Smith JG, Chen F Tobery TW, Ulmer JB, Evans RK, Caulfield MJ (2000) Enhanced type І immune response to a hepatitis B DNA vaccine by formulation with calcium- or aluminum phosphate. Vaccine 18, 1227-35.
Xiao S, Chen H, Fang L, Liu C, Zhang H, Jiang Y, Hong W (2004) Comparison of immune responses and protective efficacy of suicidal DNA vaccine and conventional DNA vaccine encoding glycoprotein C of pseudorabies virus in mice. Vaccine 22, 345-51.
Zuhair Muhammad Hassan