Antitumor effects of non-replicative herpes simplex vectors expressing antiangiogenic proteins and thymidine kinase on Lewis lung carcinoma establishment and growth.

There is growing evidence that combinations of antiangiogenic proteins with other antineoplastic treatments such as chemo- or radiotherapy and suicide genes-mediated tumor cytotoxicity lead to synergistic effects. In the present work, we tested the activity of two non-replicative herpes simplex virus (HSV)-1-based vectors, encoding human endostatin::angiostatin or endostatin::kringle5 fusion proteins in combination with HSV-1 thymidine kinase (TK) molecule, on endothelial cells (ECs) and Lewis lung carcinoma (LLC) cells. We observed a significant reduction of the in vitro growth, migration and tube formation by primary ECs upon direct infection with the two recombinant vectors or cultivation with conditioned media obtained from the vector-infected LLC cells. Moreover, direct cytotoxic effect of HSV-1 TK on both LLC and ECs was demonstrated. We then tested the vectors in vivo in two experimental settings, that is, LLC tumor growth or establishment, in C57BL/6 mice. The treatment of pre-established subcutaneous tumors with the recombinant vectors with ganciclovir (GCV) induced a significant reduction of tumor growth rate, while the in vitro infection of LLC cells with the antiangiogenic vectors before their implantation in mice flanks, either in presence or absence of GCV, completely abolished the tumor establishment.

Characterization of herpes simplex virus type 1 recombinants that express and incorporate high levels of HCV E2-gC chimeric proteins.

We report the construction of two HSV-1 recombinants encoding chimeric forms of the E2 glycoprotein of HCV-1a composed of the ectodomain of E2 (aa384-611 or 384-711) fused to different parts of the transmembrane and cytoplasmic domain of the HSV-1 gC glycoprotein (gC). The parental HSV-1, known as KgBpK(-)gC(-), is deleted for gC and the main heparan sulphate (HS) binding domain of gB, and it exhibits impaired binding (ca. 80%) to HS compared to the wild type virus KOS [Laquerre, S., Argnani, R., Anderson, D.B., Zucchini, S., Manservigi, R., Glorioso, J.C., 1998. Heparan sulphate proteoglycan binding by herpes simplex virus type 1 glycoproteins B and C, which differ in their contributions to virus attachment, penetration, and cell-to-cell spread. J. Virol. 72, 6119-6130]. We show that gC:E2 proteins are efficiently expressed and transported to the cell surface. We also demonstrate that HSV-1 can incorporate both gC:E2 chimeric proteins into particles and show that incorporation of both chimeric molecules in the viral envelope partially restored binding (ca. 20%) of the HSV-1 recombinants to heparan sulphate. Finally, we showed that the gC:E2ScaI chimeric glycoprotein was able to bind a recombinant form of hCD81 and virion-expressed gC:E2ScaI permitted the binding of the HSV-1 recombinant virus to the hCD81 molecule.

Use of herpes simplex virus type 1-based amplicon vector for delivery of small interfering RNA.

Silencing of gene expression by small interfering RNAs (siRNAs) is rapidly becoming a powerful tool for genetic analysis of mammalian cells. The use of DNA-based plasmid vectors to achieve transient and stable expression of siRNA has been developed to avoid the problems of double-stranded oligonucleotides transfection. These vectors direct the transcription of small hairpin RNAs (shRNAs) from a polymerase-III (H1 or U6)-RNA gene promoter. However, numerous disadvantages remain, including low transfection efficiency and difficulty in transfecting primary cells. To overcome some of these problems, the use of viral vectors for siRNA delivery has been described. Retroviral, adenoviral, adeno-associated and herpes viral shRNAs delivery systems have been successfully used to silence genes, in vitro and in vivo. The use of a herpes simplex virus type 1 (HSV-1)-based amplicon vector for siRNA delivery into mammalian cells, using human polyomavirus BK (BKV)-transformed cells as a model system is described. The results demonstrate the ability of amplicon vectors to inhibit the expression of BKV T-Ag and tumorigenicity of BKV-transformed cells. We show that the use of the amplicon vector is highly efficient for the delivery of siRNA molecules. The unique ability of these vectors to deliver multiple copies of siRNA may provide a useful tool in the development of novel anticancer therapy.

Cationic liposomes as potential carriers for ocular administration of peptides with anti-herpetic activity.

In the present study the preparation, characterization and activity of cationic liposomes containing the secretory form of herpes simplex virus type 1 (HSV-1) glycoprotein B (gB1s) or two related polylysine rich peptides, namely DTK1 and DTK2, were described. The immunotherapeutic potential of these HSV antigens containing liposomes was examined with a rabbit ocular model of HSV-1 infection. Our study indicates that the liposomes (i) are able to encapsulate quantitatively gB1s and around 30% the DTK peptides, (ii) are characterized by dimensions compatible with ocular applications and (iii) can release the peptide comparably to the free solution. In addition, neutralization studies demonstrated that an anti-DTK specific polyclonal antiserum can inhibit HSV-1 infection, indicating that such peptides could be a good immunogen/antigen in an anti-HSV vaccine formulation. Although the vaccination protocol did not induce protection against the eye disease, a significative protection against a lethal ocular challenge was detectable together with the absence of reactivation episodes from latency on the survived animals. In this respect, the use of cationic liposomes coupled to gB1s and DTK peptides, as a local ocular vaccine, could represent an interesting approach in order to obtain a possible efficacy in protecting animals against a subsequent HSV-1 ocular challenge.

Replication-competent herpes simplex vectors: design and applications.

Replication-competent vectors are derived from attenuated viruses whose genes, that are nonessential for replication in cultured cells in vitro, are either mutated or deleted. The removal of one or more nonessential genes may reduce pathogenicity without requiring a cell line to complement growth. Herpes simplex viruses (HSV) are potential vectors for several applications in human healthcare. These include delivery and expression of human genes to cells of the nervous systems, selective destruction of cancer cells, prophylaxis against infection with HSV or other infectious diseases, and targeted infection to specific tissues or organs. This review highlights the progress in creating attenuated genetically engineered HSV vectors.

A direct gene transfer strategy via brain internal capsule reverses the biochemical defect in Tay-Sachs disease.

Therapy for neurodegenerative lysosomal Tay-Sachs (TS) disease requires active hexosaminidase (Hex) A production in the central nervous system and an efficient therapeutic approach that can act faster than human disease progression. We combined the efficacy of a non-replicating Herpes simplex vector encoding for the Hex A alpha-subunit (HSV-T0alphaHex) and the anatomic structure of the brain internal capsule to distribute the missing enzyme optimally. With this gene transfer strategy, for the first time, we re-established the Hex A activity and totally removed the GM2 ganglioside storage in both injected and controlateral hemispheres, in the cerebellum and spinal cord of TS animal model in the span of one month's treatment. In our studies, no adverse effects were observed due to the viral vector, injection site or gene expression and on the basis of these results, we feel confident that the same approach could be applied to similar diseases involving an enzyme defect.

Effects of defective herpes simplex vectors expressing neurotrophic factors on the proliferation and differentiation of nervous cells in vivo.

Neurotrophic factors (NTFs) are known to govern the processes involved in central nervous system cell proliferation and differentiation. Thus, they represent very attractive candidates for use in the study and therapy of neurological disorders. We constructed recombinant herpesvirus-based-vectors capable of expressing fibroblast growth factor-2 (FGF-2) and ciliary neurotrophic factor (CNTF) alone or in combinations. In vitro, vectors expressing FGF-2 and CNTF together, but not those expressing either NTF alone, caused proliferation of O-2A progenitors. Furthermore, based on double-labeling experiments performed using markers for neurons (MAP-2), oligodendrocytes (CNPase) and astrocytes (GFAP), most of the new cells were identified as astrocytes, but many expressed neuronal or oligodendrocytic markers. In vivo, vectors have been injected in the rat hippocampus. At 1 month after inoculation, a highly significant increase in BrdU-positive cells was observed in the dentate gyrus of animals injected with the vector expressing FGF-2 and CNTF together, but not in those injected with vectors expressing the single NTFs. Furthermore, double-labeling experiments confirmed in vitro data, that is, most of the new cells identified as astrocytes, some as neurons or oligodendrocytes. These data show the feasibility of the vector approach to induce proliferation and differentiation of neurons and/or oligodendrocytes in vivo.

Replication-competent herpes simplex virus type 1 mutant expressing an autofluorescent glycoprotein H fusion protein.

The heterocomplex of glycoproteins H (gH) and L (gL) of herpes simplex virus type 1 (HSV-1) is essential for viral infectivity and is involved in viral penetration, cell-to-cell spread, and syncytium formation. We constructed an HSV-1 mutant expressing a gH-EGFP (enhanced green fluorescent protein) fusion protein under the control of the gH true late promoter. The EGFP coding sequence was cloned after the gH signal peptide into the HSV-1 genome. Superinfection of transfected, gH-nontranscomplementing cells with gH-negative HSV-1 resulted in a replication-competent recombinant virus. Cells infected with the recombinant virus exhibited strong and stable EGFP-specific fluorescence late in infection, and autofluorescence was detected in purified virions. The recombinant genotype of the mutant was confirmed by PCR. The 140-kD gH-EGFP fusion protein showed an N-linked glycosylation pattern similar to gH-1, was recognized by the conformation-dependent gH-specific monoclonal antibodies 52S and LP11 and formed a heterocomplex with gL which was transported to the cell surface and integrated into the viral envelope. Infectivity of the gH-EGFP mutant was neutralized by antibodies 52S and LP11. To our knowledge, this is the first replication-competent HSV-1 mutant expressing an autofluorescent essential glycoprotein which will be a versatile tool for studies of penetration, late gene expression, transport and tissue spread.

Mice genetic immunization with plasmid DNA encoding a secreted form of HSV-1 gB induces a protective immune response against herpes simplex virus type 1 infection.

Intramuscularly (i.m.) delivered plasmid DNA encoding a secreted form of glycoprotein B of herpes simplex virus type 1 (HSV-1 gB1s) was evaluated for the ability to elicit a protective immune response in Balb/c mice. Animals received three i.m. injections of a gB1s expression plasmid (pRP-RSV-gB1s) or of a wild-type transmembrane gB1 coding plasmid (pRP-RSV-gB1), while control mice were injected with the vector alone (pRP-RSV). A specific antibody response was observed in almost all immunized animals, and in most cases antibodies were also detected after 1 month in the absence of further vaccine boosts. Serum antibodies mostly displayed neutralizing activity against HSV-1. Glycoprotein B1s DNA immunization was also effective in protecting animals against the primary infection induced by a subsequent HSV-1 challenge and limited HSV-1 infection of sensitive ganglia.

Gene transfer into neurones for the molecular analysis of behaviour: focus on herpes simplex vectors.

The use of viral vectors to transfect genes into specific brain-cell populations is a novel approach that can be used to investigate the molecular and cellular basis of brain function. Ideal vectors should be targetable and capable of regulated transgene expression. From the viral vectors developed so far, this article focuses on herpes simplex virus 1 (HSV-1)-based vectors. HSV-1 vectors can be engineered for gene transfer to the brain, which makes them suitable for neuroscience research applications. In particular, genetic manipulations of the virus can almost eliminate toxicity and allow expression of multiple transgenes simultaneously. In some instances, transfection of selected neuronal populations is also possible. Specific alterations in behaviour and in disease models have been described after the viral-vector-mediated expression of specific genes within highly localized brain regions.

The C-terminal cytoplasmic tail of herpes simplex virus type 1 gE protein is phosphorylated in vivo and in vitro by cellular enzymes in the absence of other viral proteins.

Herpes simplex virus 1 glycoprotein E (gE-1) is highly phosphorylated in culture cells during infection. In this report, it is shown that phosphorylation is mediated by host enzymes in human cells stably transfected with gE, in the absence of other herpesvirus products. In contrast, a tailless gE product (C terminus deletion mutant) is not phosphorylated. By using an in vitro kinase assay combined with linker-insertion mutagenesis, it is shown that casein kinase II catalyses the phosphorylation of the C-terminal domain of the protein. Also, it is demonstrated that the serine residues at positions 476 and/or 477 in the cytoplasmic portion of the protein are the major acceptors for the phosphate groups.

Local and systemic inoculation of DNA or protein gB1s-based vaccines induce a protective immunity against rabbit ocular HSV-1 infection.

A secreted form of gB1 (gB1s), previously shown to protect rabbits against HSV-1 ocular infection when inoculated systemically, was delivered to rabbit periocular area to evaluate its vaccine efficacy upon local administration. The efficacy of local or systemic inoculation of a gB1s-DNA-based vaccine in the rabbit model of ocular HSV-1 infection was assessed in parallel flow. Rabbits received four inoculations of the different immunogens, then immune responses and clinical symptoms were evaluated. Both the local protein and the systemic DNA administration elicited a neutralizing antibody response, reduced ocular symptoms with respect to controls (P<0.01), and completely prevented the death of rabbits from encephalitis. Conversely, local DNA vaccination did not induce any detectable antibody response, and could only partially protect rabbits from the development of encephalitis and severe ocular infection.

Membrane protein pattern in hereditary spherocytosis in five subjects from north-east Italy obtained by SDS-PAGE using N,N'-diallyltartardiamide.

In the present study we examined five subjects affected by hereditary spherocytosis (three unsplenectomized and two splenectomized), coming from an area in the north-east of Italy where hereditary spherocytosis is an anaemic disease with very low incidence. All patients showed a low degree of spectrin deficiency (14%), detected with sodium dodecyl sulfate polyacrylamide gel electrophoresis. Moreover, when this analysis was performed with N,N'-diallyltartardiamide as cross-linking agent instead of N,N'-methylenbisacrylamide, some unusual bands appeared in the region between proteins 4.2 and 5, the three unsplenectomized and two splenectomized patients showing different patterns. We hypothesise that some alterations of proteins in this region (e.g. the 4.5 or 4.9 bands), possibly due to proteolysis, must have occurred in relation to the disease.

Replication-defective herpes simplex virus vectors for neurotrophic factor gene transfer in vitro and in vivo.

We report here the construction and the use of two replication-defective herpes simplex virus vectors, SH FGF-2 and TH FGF-2, which efficiently transfer and express the cDNA for fibroblast-growth-factor-2 (FGF-2) in vitro and in vivo. One mutant was deleted in the immediate-early gene encoding ICP4; the other was deleted in ICP4, ICP22 and ICP27. FGF-2--or the control gene lacZ--were inserted in tk, under control of the human cytomegalovirus immediate-early promoter. Infection of Vero cells with SH FGF-2 induced a dramatic increase in FGF-2 protein levels in the first 2 days after infection, with a rapid return to baseline levels within day 4. In contrast, infection of Vero cells with TH FGF-2 displayed FGF-2 levels progressively increasing up to days 4-5, and slowly returning to baseline. Protein extracts of cells infected with TH FGF-2 induced neuronal differentiation of PC12 cells, indicating that the newly synthesized FGF-2 was biologically active. Robust transient transgene expression was also observed in the rat hippocampus after stereotaxical inoculation of TH FGF-2, but not of TH lacZ or of SH vectors. Enhanced gene expression both in vitro and in vivo by the triple immediate-early gene deletion mutant might be attributed to reduced vector cytotoxicity. The present data suggest that TH FGF-2 is suitable for studies of FGF-2 involvement in neurological disorders.

Molecular analysis of behavior by gene transfer into neurons with herpes simplex vectors.

One goal of neuroscience is to define the molecular and cellular basis for behavior and neurological diseases. A novel approach to this problem is based on the use of viral vectors to transfect specific genes into specific brain cell populations. This review focuses on herpes simplex-based vectors. Major advances have recently been made to improve the characteristics of these vectors, in particular to reduce their toxicity, to modulate the greatness and the time-course of transgene expression, to precisely target specific cell populations, and to transfer multiple genes. Much remains to be done to obtain robust and prolonged transgene expression. However, specific alterations in the behavior and in disease models have already been described following the herpes simplex vector-mediated expression of specific genes within highly localized brain areas. Therefore, this research strategy is likely to provide new clues on the cellular and molecular basis of behavior and of neurological diseases.

Heparan sulfate proteoglycan binding by herpes simplex virus type 1 glycoproteins B and C, which differ in their contributions to virus attachment, penetration, and cell-to-cell spread.

Herpes simplex virus type 1 (HSV-1) mutants defective for envelope glycoprotein C (gC) and gB are highly impaired in the ability to attach to cell surface heparan sulfate (HS) moieties of proteoglycans, the initial virus receptor. Here we report studies aimed at defining the HS binding element of HSV-1 (strain KOS) gB and determining whether this structure is functionally independent of gB's role in extracellular virus penetration or intercellular virus spread. A mutant form of gB deleted for a putative HS binding lysine-rich (pK) sequence (residues 68 to 76) was transiently expressed in Vero cells and shown to be processed normally, leading to exposure on the cell surface. Solubilized gBpK- also had substantially lower affinity for heparin-acrylic beads than did wild-type gB, confirming that the HS binding domain had been inactivated. The gBpK- gene was used to rescue a KOS gB null mutant virus to produce the replication-competent mutant KgBpK-. Compared with wild-type virus, KgBpK- showed reduced binding to mouse L cells (ca. 20%), while a gC null mutant virus in which the gC coding sequence was replaced by the lacZ gene (KCZ) was substantially more impaired (ca. 65%-reduced binding), indicating that the contribution of gC to HS binding was greater than that of gB. The effect of combining both mutations into a single virus (KgBpK-gC-) was additive (ca. 80%-reduced binding to HS) and displayed a binding activity similar to that observed for KOS virus attachment to sog9 cells, a glycosaminoglycan-deficient L-cell line. Cell-adsorbed individual and double HS mutant viruses exhibited a lower rate of virus entry following attachment, suggesting that HS binding plays a role in the process of virus penetration. Moreover, the KgBpK- mutant virus produced small plaques on Vero cells in the presence of neutralizing antibody where plaque formation depended on cell-to-cell virus spread. These studies permitted the following conclusions: (i) the pK sequence is not essential for gB processing or function in virus infection, (ii) the lysine-rich sequence of gB is responsible for HS binding, and (iii) binding to HS is cooperatively linked to the process of efficient virus entry and lateral spread but is not absolutely required for virus infectivity.

Red blood cells as delivery system for recombinant HSV-1 glycoprotein B: immunogenicity and protection in mice.

The immunotherapeutic potential of autologous red blood cells (RBC) coupled to the secretory form of herpes simplex virus type 1 (HSV-1) glycoprotein B (gB1s) was examined with a mouse model of HSV-1 infection. C57BL/6 mice were immunized intraperitoneally with gB1s (0.05 microgram per dose) linked to RBC, or mixed with Freund's complete or bound to AlPO4 adjuvants (0.5 microgram per dose). Mice immunized with RBC coupled gB1s were protected against lethal and latent HSV-1 infection, and developed an anti-HSV antibody response, as measured by ELISA and HSV-1 neutralization assays, similar or higher than that elicited by the same antigen in Freund's complete adjuvant, which suggested that autologous RBC coupled to gB1s may provide an effective and safe method of immunization against HSV infection.

Molecular diagnosis of herpes simplex encephalitis.

Herpes simplex virus encephalitis (HSE) is associated with significant morbidity and mortality both in neonates and adults. An early diagnosis can greatly help to reduce mortality in both groups; however, since none of the presenting symptoms is pathognomonic for HSE, a clinical diagnosis is unreliable. On the other hand, the technique of isolating herpes simplex virus (HSV) from brain biopsies, although providing an early and specific diagnosis, has never been widely accepted because of the risk of neurological complications connected with the invasiveness of the method. In addition, several studies reported that the two HSV types appear to be differently associated with specific neurological complications, indicating that it would be important to gain a differential-type diagnosis to guide both prevention strategies and antiviral therapy. Therefore, the need for noninvasive specific and sensitive techniques has given impulse to the development of assays based on the search of either specific anti-HSV antibodies or viral DNA footprints in patient serum or cerebrospinal fluid. While enzyme-linked immunosorbent assays and immunoblot assays, using HSV type-1- and 2-specific viral glycoproteins, are useful for serological typing, the polymerase chain reaction technique has proved highly effective in giving early, precise and specific HSE diagnosis, thus providing a helpful tool for the identification and treatment of this disease.

Expression of the herpes simplex virus type 1 glycoprotein E in human cells and in Escherichia coli: protection studies against lethal viral infection in mice.

The objective of this study was to examine the protective efficacy of purified recombinant herpes simplex virus type 1 (HSV-1) glycoprotein E (gE-1) in the mouse lethal challenge model. A secreted form of gE-1 (hgE-1s) protein, containing amino acids 1-406, was produced in human cells by using the episomal replicating pRP-RSV expression vector. In addition, a portion of the gE-1 (bgE-1t) protein corresponding to amino acids 90-406, was expressed in Escherichia coli as a fusion protein with maltose binding protein using the pMAL-c2 expression vector. Mice vaccinated with hgE-1s developed high serum titres of HSV-1-neutralizing antibodies and were significantly protected from intraperitoneal lethal HSV-1 challenge, whereas mice vaccinated with bgE-1t exhibited only moderate levels of protective immunity. These results demonstrate that the expression of gE-1 in human cells has a strong impact on its protective efficacy and that most importantly the hgE-1s protein could be of values as a component of an HSV multi-subunit vaccine.

Cell-type dependent sensitivity of herpes simplex virus 1 mutants to plaque development inhibition by an anti-gD monoclonal antibody.

Herpes simplex virus 1 (HSV-1) mutants selected in Vero cells either for resistance to plaque development inhibition (PDI) (P1, P2 and P3) or for resistance to neutralization (N1, N2, and N3) against an anti-glycoprotein D (gD) monoclonal antibody (mAb) were characterized both in Vero and BHK cells. In Vero cells P mutants were completely resistant to PDI, while N mutants showed from moderate to good resistance. In BHK cells P mutants lost their resistance to PDI, while N mutants became fully resistant. Cell type influenced the plaque size of the mutants as well. In Vero cells P mutant plaques were larger, and N mutant plaques smaller than wild type virus plaques. In BHK cells all plaques were comparable. With one exception (N2 in BHK) resistance to neutralization could be clearly appreciated at high but not at low mAb:virus particles ratio. For most of the mutants the neutralization values remained approximately the same in Vero and BHK cells. P2 and N2 mutants were more resistant to neutralization in BHK than in Vero cells. However, only for N2 mutant did the change in neutralization resistance go in the same direction as the change in PDI resistance. The results show that it is possible to dissociate the neutralizing and the PDI activities of a mAb and that the sensitivity of a virus to plaque inhibition by an anti-gD mAb is cell-type dependent.