Intratumoral spread and increased efficacy of a p53-VP22 fusion protein expressed by a recombinant adenovirus

In vitro experiments have demonstrated intercellular trafficking of the VP22 tegument protein of herpes simplex virus type 1 from infected cells to neighboring cells, which internalize VP22 and transport it to the nucleus. VP22 also can mediate intercellular transport of fusion proteins, providing a strategy for increasing the distribution of therapeutic proteins in gene therapy. Intercellular trafficking of the p53 tumor suppressor protein was demonstrated in vitro using a plasmid expressing full-length p53 fused in-frame to full-length VP22. The p53-VP22 chimeric protein induced apoptosis both in transfected tumor cells and in neighboring cells, resulting in a widespread cytotoxic effect. To evaluate the anti-tumor activity of p53-VP22 in vivo, we constructed recombinant adenoviruses expressing either wild-type p53 (FTCB) or a p53-VP22 fusion protein (FVCB) and compared their effects in p53-resistant tumor cells. In vitro, treatment of tumor cells with FVCB resulted in enhanced p53-specific apoptosis compared to treatment with equivalent doses of FTCB. However, in normal cells there was no difference in the dose-related cytotoxicity of FVCB compared to that of FTCB. In vivo, treatment of established tumors with FVCB was more effective than equivalent doses of FTCB. The dose-response curve to FVCB was flatter than that to FTCB; maximal antitumor responses could be achieved using FVCB at doses 1 log lower than those obtained with FTCB. Increased antitumor efficacy was correlated with increased distribution of p53 protein in FVCB-treated tumors. This study is the first demonstration that VP22 can enhance the in vivo distribution of therapeutic proteins and improve efficacy in gene therapy.     

VP22 does not significantly enhance enzyme prodrug cancer gene therapy as a part of a VP22-HSVTk-GFP triple fusion construct

BACKGROUND: VP22 is a herpes simplex virus type 1 (HSV-1) tegument protein that has been suggested to spread from cell to cell, alone or as a part of fusion proteins. Creating controversy, some reports indicate that VP22 cannot facilitate significant intercellular spreading. To study the capacity of VP22 to cause spreading and enhance thymidine kinase/ganciclovir cancer gene therapy, we constructed a novel triple fusion protein containing VP22, HSV thymidine kinase and green fluorescent protein (VP22-Tk-GFP). This fusion protein has three functional domains in the same polypeptide, thus making it possible to reliably compare the causality between transduction rate and cell killing efficiency in vitro and in vivo. METHODS: VP22-Tk-GFP was cloned into lenti- and adenoviral vectors and used for expression studies, analyses for VP22-mediated protein spreading, and to study the effect of VP22 to thymidine kinase/ganciclovir-mediated cytotoxicity. The function of VP22-Tk-GFP was also investigated in vivo. RESULTS: The triple fusion protein was expressed correctly in vitro, but intercellular trafficking was not observed in any of the studied cell lines. However, under certain conditions, VP22-Tk-GFP sensitized cells more efficiently to ganciclovir than Tk-GFP. In vivo there was a trend for increased inhibition of tumor growth with VP22-Tk-GFP when ganciclovir was present, but the difference with Tk-GFP was not statistically significant. CONCLUSIONS: Based on our results, VP22 fusion proteins do not seem to traffic intercellularly at detectable levels in most tumor cell types. Even though VP22 enhanced cytotoxicity in one cell line in vitro, the effect in vivo was modest. Therefore, our results do not support the utility of VP22 as an enhancer of enzyme prodrug cancer gene therapy.     

Evaluation of VP22 spread in tissue culture

We compare methods of detection of intercellular transport of the herpes simplex virus protein VP22 and of a green fluorescent protein (GFP)-VP22 fusion protein. Spread of both proteins was observed by immunofluorescence (IF) using organic fixatives. Spread of both proteins was also detected by IF after paraformaldehyde (PFA) fixation and detergent permeabilization, albeit at reduced levels. However, while spread of GFP-VP22 was observed by examining intrinsic GFP fluorescence after methanol fixation, little spread was observed after PFA fixation, suggesting that the levels of the fusion protein in recipient cells were below the detection limits of intrinsic-fluorescence or that PFA fixation quenches the fluorescence of GFP-VP22. We further considered whether elution of VP22 from methanol-fixed cells and postfixation binding to surrounding cells contributed to the increased detection of spread observed after methanol fixation. The results show that while this could occur, it appeared to be a minor effect not accounting for the observed VP22 cell-to-cell spread in culture.     

Evaluation of the VP22 protein for enhancement of a DNA vaccine against anthrax

BACKGROUND: Previously, antigens expressed from DNA vaccines have been fused to the VP22 protein from Herpes Simplex Virus type I in order to improve efficacy. However, the immune enhancing mechanism of VP22 is poorly understood and initial suggestions that VP22 can mediate intercellular spread have been questioned. Despite this, fusion of VP22 to antigens expressed from DNA vaccines has improved immune responses, particularly to non-secreted antigens. METHODS: In this study, we fused the gene for the VP22 protein to the gene for Protective Antigen (PA) from Bacillus anthracis, the causative agent of anthrax. Protective immunity against infection with B. anthracis is almost entirely based on a response to PA and we have generated two constructs, where VP22 is fused to either the N- or the C-terminus of the 63 kDa protease-cleaved fragment of PA (PA63). RESULTS: Following gene gun immunisation of A/J mice with these constructs, we observed no improvement in the anti-PA antibody response generated. Following an intraperitoneal challenge with 70 50% lethal doses of B. anthracis strain STI spores, no difference in protection was evident in groups immunised with the DNA vaccine expressing PA63 and the DNA vaccines expressing fusion proteins of PA63 with VP22. CONCLUSION: VP22 fusion does not improve the protection of A/J mice against live spore challenge following immunisation of DNA vaccines expressing PA63.     

VP22-mediated intercellular transport correlates with enhanced biological activity of MybEngrailed but not (HSV-I) thymidine kinase fusion proteins in primary vascular cells following non-viral transfection

BACKGROUND: The intercellular transport properties of the herpes simplex virus (HSV) protein VP22 have been harnessed to enhance the effectiveness of viral gene transfer. We investigated the intercellular transport and biological effects of VP22 fused with the dominant negative c-Myb chimera, MybEngrailed (MybEn) and HSV-I thymidine kinase (TK), in primary vascular smooth muscle cells (VSMC) following non-viral transfection. MATERIALS AND METHODS: Porcine VSMC transfected with plasmids encoding MybEn, TK and their respective N- and C-terminal VP22 fusion proteins were assayed for the extent and distribution of transgene expression (by immunohistochemistry), culture growth and apoptosis. RESULTS: The N-terminal MybEn fusion with VP22 (MybEnVP22) and both TK fusions, but not VP22MybEn, exhibited intercellular spread from primary transfected to up to 200 surrounding cells. pMybEnVP22-transfected cultures exhibited growth inhibition and apoptosis rates that were 10.6 +/- 3.6 and 3.2 +/- 1.0 fold higher than in pMybEn-transfected cultures; pVP22MybEn-transfected cultures showed no difference in these parameters. pTK-transfected cultures underwent 60-70% cell death in the presence of ganciclovir despite <2% primary transfection, which was not increased in cultures transfected with plasmids encoding VP22-TK fusions. CONCLUSIONS: The close correlation between immunocytochemical and biological assays suggests that intercellular transport is crucial to the enhanced biological activity of the MybEnVP22 fusion. The "intrinsic" bystander activity of TK was 4-fold greater than was "engineered" by VP22 fusion, probably reflecting the abundance of gap junctions between VSMC. VP22 fusion may enhance the efficiency of non-viral gene delivery when combined with the appropriate therapeutic transgene, target tissue and transfection method.     

MDV-1 VP22 conjugated VP2 enhancing immune response against infectious bursal disease virus by DNA vaccination in mice

VP22 of Marek’s disease virus serotype 1 (MDV-1) could function in protein transduction. In this study, an infectious bursal disease virus VP2 gene was fused to the carboxyl termini of VP22. It showed that the fusion protein did not spread into the bystander cells from the cells transfected with pVP22-VP2, as the VP22 alone could. The VP22 proteins were found to be translocated into all the nuclei in the neighboring COS-1 cells, as analyzed by a fluorescence assay. Although mice were immunized with the recombinant DNAs mixed with polyethylenimine (PEI) at a dose of 1:2, it failed to enhance the antibody response against IBDV VP2, as measured by the indirect ELISA assay, yet the cell mediated immune response was significantly increased. The ratio of CD8 /CD4 T cells was significantly increased in the immunized group with the fusion genes, compared with the group immunized with VP2 (P<0.05). Our results demonstrated that VP22 indeed enhances the cell-mediated response in the fused VP2 in a mice model system, possibly due to the fact that the IBDV VP2 could be carried into the surrounding cells at a limited level under pressure from MDV VP22.     

Enhancement of sindbis virus self-replicating RNA vaccine potency by linkage of herpes simplex virus type 1 VP22 protein to antigen

Recently, self-replicating and self-limiting RNA vaccines (RNA replicons) have emerged as an important form of nucleic acid vaccines. Self-replicating RNA eventually causes lysis of transfected cells and does not raise the concern associated with naked DNA vaccines of integration into the host genome. This is particularly important for development of vaccines targeting proteins that are potentially oncogenic. However, the potency of RNA replicons is significantly limited by their lack of intrinsic ability to spread in vivo. The herpes simplex virus type 1 protein VP22 has demonstrated the remarkable property of intercellular transport and provides the opportunity to enhance RNA replicon vaccine potency. We therefore created a novel fusion of VP22 with a model tumor antigen, human papillomavirus type 16 E7, in a Sindbis virus RNA replicon vector. The linkage of VP22 with E7 resulted in a significant enhancement of E7-specific CD8+ T-cell activities in vaccinated mice and converted a less effective RNA replicon vaccine into one with significant potency against E7-expressing tumors. These results indicate that fusion of VP22 to an antigen gene may greatly enhance the potency of RNA replicon vaccines.     

Glioma targeting peptide in combination with the P53 C terminus inhibits glioma cell proliferation in vitro

Glioma is a prevalent malignant primary brain tumor in adults, the treatment for which remains a challenge due to its high infiltration and recurrence. Hence, treatments that lead to the suppression of glioma cell migration and invasion may be used in addition to surgery to increase the therapeutic outcome. In this study, we aimed to construct a multifunctional protein that would exert an effect on glioma cell proliferation and migration. The protein is named GL1-P53C-11R and it consists of the glioma-targeting peptide GL1 (G), the P53 C terminus (Pc) and the cell-penetrating peptide arginine (R). GL1-P53C-R was expressed with the fusion protein ZZ and immunofluorescence analysis showed effective delivery of the fused ZZ-GL1-P53C-R protein represented as ZZ-GPcR. The ZZ-GPcR exhibited an inhibitory effect on the proliferation, migration and invasion of U87ΔEGFR cells. Western blotting results indicated that it caused significant changes in the expression levels of cell cycle and apoptotic proteins. Flow cytometric analysis showed increase apoptosis. Our findings suggest that the P53C in the fusion protein ZZ-GPcR can enter into glioma cells to exert its inhibitory effect.     

人细小病毒B19 VP1u多克隆抗体的制备及其保守区外N端氨基酸对sPLA2活性影响的初步研究

【目的】制备人细小病毒B19-VP1u的多克隆抗体,探究VP1u多克隆抗体及其保守区外N端氨基酸对病毒磷脂酶A2活性的影响。【方法】首先通过分子克隆方法构建相应原核表达载体;利用原核表达系统纯化含MBP标签的VP1u全长及N端系列截短突变融合蛋白;接着免疫新西兰大白兔制备全长VP1u蛋白的多克隆抗体;最后利用磷脂酶A2活性检测试剂盒检测了纯化蛋白的磷脂酶A2活性。【结果】Western blot及免疫荧光实验证实制备的多克隆抗体具有较高的特异性;磷脂酶A2活性检测发现全长VP1u-MBP融合蛋白具有一定的活性,该活性可以被VP1u的抗体抑制;N端保守区外截短系列蛋白的酶活检测发现,N端截掉12个氨基酸时酶活降低53%,截掉67个氨基酸时酶活性几乎完全丧失。【结论】首次发现VP1u保守区外N端氨基酸,尤其是第12个氨基酸前的区域以及第22-67个氨基酸之间的区域,对sPLA2活性的保持具有重要意义,推测该区域可能对维持正常的蛋白构象起重要的作用;而其特异性多克隆抗体的制备也为进一步研究B19病毒VP1u在病毒复制周期的作用奠定基础。     

伪狂犬病毒VP22蛋白C-端系列缺失突变体的构建及亚细胞定位分析

以绿荧光蛋白(GFP)为标记,构建了一系列伪狂犬病毒VP22蛋白的C-端缺失突变体与GFP融合表达的真核表达质粒,脂质体介导转染Hela细胞,通过荧光显微镜观察分析各个缺失突变体的亚细胞定位,发现伪狂犬病毒VP22蛋白与核定位有关的结构域在第60个到第90个氨基酸残基之间,第111个到第159个氨基酸残基有可能与形成细胞核内的颗粒有关,与微管蛋白结合有关的结构域可能在第187到第241个氨基酸残基之间。上述研究结果为进一步深入研究伪狂犬病毒VP22蛋白的结构与功能奠定了基础。     

The intranuclear location of simian virus 40 polypeptides VP2 and VP3 depends on a specific amino acid sequence.

A cDNA fragment coding for poliovirus capsid polypeptide VP1 was inserted into a simian virus 40 (SV40) genome in the place of the SV40 VP1 gene and fused in phase to the 3' end of the VP2-VP3 genes. Simian cells were infected with the resulting hybrid virus in the presence of an early SV40 mutant used as a helper. Indirect immunofluorescence analysis of the infected cells using anti-poliovirus VP1 immune serum revealed that the SV40/poliovirus fusion protein was located inside the cell nucleus. Deletions of various lengths were generated in the SV40 VP2-VP3 portion of the hybrid gene using BAL31 nuclease. The resulting virus genomes expressed spliced fusion proteins whose intracellular location was either intranuclear or intracytoplasmic, depending on the presence or absence of VP2 amino acid residues 317 to 323 (Pro-Asn-Lys-Lys-Lys-Arg-Lys). This was confirmed by site-directed mutagenesis of the Lys residue at position 320. Modification of Lys-320 into either Thr or Asn abolished the nuclear accumulation of the fusion protein. It is concluded that at least part of the sequence of VP2 amino acids 317 to 323 allows VP2 and VP3 to remain stably located inside the cell nucleus. The proteins are most probably transported from the cell cytoplasm to the cell nucleus by interaction, with VP1 acting as a carrier.     

Bovine herpesvirus tegument protein VP22 enhances thymidine kinase/ganciclovir suicide gene therapy for neuroblastomas compared to herpes simplex virus VP22

Herpesvirus tegument protein VP22 can enhance the effect of therapeutic proteins in gene therapy, such as thymidine kinase (tk) and p53; however, the mechanism is unclear or controversial. In this study, mammalian expression vectors carrying bovine herpesvirus 1 (BHV-1) VP22 (BVP22) or herpes simplex virus type 1 (HSV-1) VP22 (HVP22) and equine herpesvirus type 4 (EHV-4) tk (Etk) were constructed in order to evaluate and compare the therapeutic potentials of BVP22 and HVP22 to enhance Etk/ganciclovir (Etk/GCV) suicide gene therapy for neuroblastomas by GCV cytotoxicity assays and noninvasive bioluminescent imaging in vitro and in vivo. BVP22 enhanced Etk/GCV cytotoxicity compared to that with HVP22 both in vitro and in vivo. However, assays utilizing a mixture of parental and stably transfected cells indicated that the enhancement was detected only in transfected cells. Thus, the therapeutic potential of BVP22 and HVP22 in Etk/GCV suicide gene therapy in this tumor system is not due to VP22 delivery of Etk into surrounding cells but rather is likely due to an enhanced intracellular effect.     

Leishmania major: Protective capacity of DNA vaccine using amastin fused to HSV-1 VP22 and EGFP in BALB/c mice model

An intercellular spreading strategy using herpes simplex virus type 1 (HSV-1) VP22 protein is employed to enhance DNA vaccine potency of Leishmania major amastin antigen in BALB/c mice model. We evaluated the immunogenicity and protective efficacy of plasmid DNA vaccines encoding amastin-enhanced green fluorescent protein (EGFP) and VP22-amastin-EGFP. Optimal cell-mediated immune responses were observed in BALB/c mice immunized with VP22-amastin-EGFP as assessed by cytokine gene expression analysis using real time RT-PCR. Vaccination with the VP22-amastin-EGFP fusion construct elicited significantly higher IFN-gamma response upon antigen stimulation of splenocytes from immunized mice compared to amastin as a sole antigen. Mice immunized by VP22-amastin-EGFP showed partial protection following infectious challenge with L. major, as measured by parasite load in spleens. These results suggest that the development of DNA vaccines encoding VP22 fused to a target Leishmania antigen would be a promising strategy to improve immunogenicity and DNA vaccine potency.     

Ability of the Tat basic domain and VP22 to mediate cell binding, but not membrane translocation of the diphtheria toxin A-fragment

A number of proteins are able to enter cells from the extracellular environment, including protein toxins, growth factors, viral proteins, homeoproteins, and others. Many such translocating proteins, or parts of them, appear to be able to carry with them cargo into the cell, and a basic sequence from the HIV-1 Tat protein has been reported to provide intracellular delivery of several fused proteins. For evaluating the efficiency of translocation to the cytosol, this TAT-peptide was fused to the diphtheria toxin A-fragment (dtA), an extremely potent inhibitor of protein synthesis which normally is delivered efficiently to the cytosol by the toxin B-fragment.The fusion of the TAT-peptide to dtA converted the protein to a heparin-binding protein that bound avidly to the cell surface. However, no cytotoxicity of this protein was detected, indicating that the TAT-peptide is unable to efficiently deliver enzymatically active dtA to the cytosol. Interestingly, the fused TAT-peptide potentiated the binding and cytotoxic effect of the corresponding holotoxin. We made a fusion protein between VP22, another membrane-permeant protein, and dtA, and also in this case we detected association with cells in the absence of a cytotoxic effect. The data indicate that transport of dtA into the cell by the TAT-peptide and VP22 is inefficient.     

The amino-terminal half of rotavirus SA114fM VP4 protein contains a hemagglutination domain and primes for neutralizing antibodies to the virus.

We have previously reported the synthesis in Escherichia coli of polypeptide MS2-VP8', which contains the amino-terminal half of the SA114fM VP4 protein fused to MS2 bacteriophage polymerase sequences (C. F. Arias, M. Lizano, and S. López, J. Gen. Virol. 68:633-642, 1987). In this work we have synthesized the carboxy-terminal half of the VP4 protein also fused to the MS2 polymerase. This protein, designated MS2-VP5', was recognized by sera to the complete virion and was able to induce antibodies to the virus when administered to mice; however, these antibodies had no neutralizing activity. The two chimeric polypeptides were tested for their ability to agglutinate erythrocytes and to prime the immune system of mice. Bacterial lysates enriched for the MS2-VP8' hybrid polypeptide, but not those enriched for the MS2-VP5' protein or those containing proteins from the host E. coli strain, had hemagglutinating activity. This hemagglutination was inhibited by sera to SA114fM rotavirus. In addition, a single dose of the MS2-VP8' polypeptide was able to prime the immune system of mice for an augmented neutralizing antibody response when the animals were subsequently immunized with purified SA114fM virus.     

Improving vaccine potency through intercellular spreading and enhanced MHC class I presentation of antigen

The potency of naked DNA vaccines is limited by their inability to amplify and spread in vivo. VP22, a HSV-1 protein, has demonstrated the remarkable property of intercellular transport and may thus provide a unique approach for enhancing vaccine potency. Therefore, we created a novel fusion of VP22 with a model Ag, human papillomavirus type 16 E7, in a DNA vaccine that generated enhanced spreading and MHC class I presentation of AG: These properties led to a dramatic increase in the number of E7-specific CD8(+) T cell precursors in vaccinated mice (around 50-fold) and converted a less effective DNA vaccine into one with significant potency against E7-expressing tumors. In comparison, nonspreading VP22(1-267) mutants failed to enhance vaccine potency. Our data indicated that the potency of DNA vaccines may be dramatically improved through intercellular spreading and enhanced MHC class I presentation of Ag.     

Yeast expression of the VP8* fragment of the rotavirus spike protein and its use as immunogen in mice

The VP8* fragment from the rotavirus spike protein was expressed as a fusion protein with two different cell wall proteins of Saccharomyces cerevisiae, Icwp (Ssr1p) and Pir4, to achieve cell wall targeting or secretion to the growth medium of the fusion proteins. Two different host strains were used for the expression of the fusion proteins, a standard S. cerevisiae strain and a mnn9 glycosylation deficient strain, the later to reduce hyper-glycosylation. The Icwp-VP8* fusion could only be detected in the growth medium, indicating that the presence of the VP8* moiety interferes with the anchorage of Icwp to the cell wall. In the case of the Pir4-VP8* fusion proteins, we achieved cell wall targeting or secretion depending on how the gene fusion had been performed. In all cases, the fusion proteins expressed in the mnn9 strain showed a reduced level of glycosylation. Mice were inoculated intraperitoneally either with Pir4-VP8* or Icwp-VP8* fusion proteins purified from the growth medium of mnn9 strains expressing them or with whole cells of an mnn9 strain expressing a Pir4-VP8 fusion protein on its cell walls. Hundred percent of mice inoculated with the Pir4-VP8* fusion protein and 25% of those inoculated with the Icwp-VP8* fusion protein showed high titers of anti-VP8* antibodies. No specific immune response was detected in those mice inoculated with whole cells. Finally, susceptibility to rotavirus infection of the offspring of immunized dams was determined and protection was found in a percentage of approximately 60% with respect to the control group.     

PTX1(ERGIC2)-VP22 fusion protein upregulates interferon-beta in prostate cancer cell line PC-3

PTX1 is a gene identified by subtractive hybridization on the basis that it is expressed in normal prostate and not in prostate carcinoma. It is unrelated to the pituitary homeobox protein (Ptx1 or Pitx1), which regulates pituitary hormone gene expression, and its function is currently unknown. Recently, it was found to be a homolog of the yeast Erv41p, an endoplasmic reticulum (ER) resident protein involved in protein trafficking between ER and Golgi, and was renamed as ERGIC2. Ectopic expression of a partial sequence of PTX1 (Met84 - Leu225) as a VP22-fusion protein in prostate cancer cell line, PC-3, induced cellular senescence. Gene expression microarray analyses showed that interferon-beta (IFN-beta) and a number of IFN-inducible genes, among other genes, were upregulated by the PTX1-VP22 fusion protein. Upregulation of IFN-beta was confirmed by RTPCR and promoter-reporter assay. However, the upregulation of IFN-beta by the PTX1-VP22 fusion protein was not due to nuclear translocation of the PTX1 luminal domain.