Hepatocyte growth factor incorporated into herpes simplex virus vector accelerates facial nerve regeneration after crush injury.

Hepatocyte growth factor (HGF) promotes regeneration of the central nervous system, but its effects on the peripheral nervous system remain unclear. This study was conducted to elucidate the effect of HGF on regeneration of the murine facial nerve after crush injury. To do so, a replication-defective herpes simplex virus vector that incorporated HGF was prepared (HSV-HGF). The main trunk of the facial nerve was compressed by mosquito hemostats, and HSV-HGF, control vector or medium was then applied to the compressed nerve. We found that mice in the HGF group required significantly fewer days for complete recovery from nerve compression. Furthermore, the amplitude of the evoked buccinator muscle compound action potential increased following HSV-HGF application. HGF expression in and around the compressed nerve was demonstrated by enzyme-linked immunoassay and immunohistochemistry. In addition, HSV-HGF introduction around the damaged nerve significantly accelerated recovery of function of the facial nerve. These data suggest a possible role of HGF in promoting facial nerve regeneration after nerve damage. Furthermore, this viral delivery method may be applied clinically for many types of severe facial palsy during facial nerve decompression surgery.

Oncolytic virotherapy with an HSV amplicon vector expressing granulocyte-macrophage colony-stimulating factor using the replication-competent HSV type 1 mutant HF10 as a helper virus.

Direct viral infection of solid tumors can cause tumor cell death, but these techniques offer the opportunity to express exogenous factors to enhance the antitumor response. We investigated the antitumor effects of a herpes simplex virus (HSV) amplicon expressing mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF) using the replication-competent HSV type 1 mutant HF10 as a helper virus. HF10-packaged mGM-CSF-expressing amplicon (mGM-CSF amplicon) was used to infect subcutaneously inoculated murine colorectal tumor cells (CT26 cells) and the antitumor effects were compared to tumors treated with only HF10. The mGM-CSF amplicon efficiently replicated in CT26 cells with similar oncolytic activity to HF10 in vitro. However, when mice subcutaneously inoculated with CT26 cells were intratumorally injected with HF10 or mGM-CSF amplicon, greater tumor regression was seen in mGM-CSF amplicon-treated animals. Furthermore, mGM-CSF amplicon treatment prolonged mouse survival. Immunohistochemical analysis revealed increased inflammatory cell infiltration in the solid tumor in the mGM-CSF amplicon-treated animals. These results suggest that expression of GM-CSF enhances the antitumor effects of HF10, and HF10-packaged GM-CSF-expressing amplicon is a promising agent for the treatment of subcutaneous tumors.

Suitability of a US3-inactivated HSV mutant (L1BR1) as an oncolytic virus for pancreatic cancer therapy.

Recently, the use of oncolytic viruses against cancer has attracted considerable attention. We studied the potential of the US3 locus-deficient herpes simplex virus (HSV), L1BR1, for oncolytic virus therapy. Its high specificity and potency indicate that L1BR1 is a promising candidate as a new oncolytic virus against pancreatic cancer. Moreover, the virus exhibited the unique characteristic of increasing apoptosis when used in combination with anticancer drugs. We assessed the feasibility of using the US3 locus-deficient HSV named L1BR1 as a new replication-competent oncolytic virus for the treatment of pancreatic cancer. The US3 locus of HSV has been shown to be a key gene in producing a multifunctional protein kinase that inhibits apoptosis induced by viral infections, chemicals and ultraviolet (UV) light. L1BR1 has been reported to be more than 10 000-fold less virulent than the parental virus in mice. In this study, we examined the tumor specificity and oncolytic effect of this attenuated replication-competent virus, L1BR1, in pancreatic cancers derived from SW1990, Capan2 and Bxpc-3cells compared with the parent virus and other well-known oncolytic herpes viruses (R3616 and hrR3). We also studied the efficacy of L1BR1 for the induction of apoptosis as an attribute of this virus in combination with the anticancer drugs 5FU and cisplatin. The combined treatment of the pancreatic cancer cells with L1BR1 and these anticancer drugs enhanced apoptosis significantly. More importantly, L1BR1 showed the lowest replication capacity in normal human hepatocytes, but the highest tumor-reducing effect in vivo among the oncolytic herpes viruses tested. In addition, L1BR1 significantly increased the induction of apoptosis of cancer cells when treated in combination with anticancer drugs although the parental virus inhibited the induction of apoptosis. These results suggest that L1BR1 is promising as a new anticancer oncolytic virus.

Enhanced efficacy of herpes simplex virus mutant HF10 combined with paclitaxel in peritoneal cancer dissemination models.

BACKGROUND/AIMS: Oncolytic viral therapy is used worldwide. Many genetically engineered viruses have been evaluated for their potential as a new therapeutic agent for cancer. HF10, herpes simplex virus (HSV) type-1 clone, has remarkable anti-tumor effects, based on our previous research. In this study, we investigated the ability of HF10 to infect and lyse murine colon cancer cells, CT26, in vitro, and tested its efficacy in an immuno-competent animal model of colorectal cancer. Further, we attempted to evaluate HF10/paclitaxel combination therapy. METHODOLOGY: In vitro, viral replication and cytotoxicity of HF10 against CT26 was observed. In vivo, BALB/c mice harboring carcinomatous peritonitis of CT26 cells were treated with HF10, paclitaxel or HF10 combined with paclitaxel. RESULTS: HF10 is effective for peritoneal dissemination without ascites. The combination of HF10 and paclitaxel prolonged survival of mice bearing carcinomatous dissemination of CT26 compared with the controls, HF10 alone and paclitaxel alone. Paclitaxel did not suppress viral replication and cytotoxicity of HF10. CONCLUSIONS: These results indicate that the combination of HF10 and paclitaxel had a remarkable effect as a cancer therapy and this method is applicable to almost all advanced cancers. This new combination therapy is a potentially epoch-making cancer therapy.

Antiapoptotic activity of herpes simplex virus type 2: the role of US3 protein kinase gene.

In order to determine the ability of herpes simplex virus type 2 (HSV-2) to suppress apoptosis, we examined the effect of HSV-2 infection on apoptosis induced in HEp-2 cells by treatment with 1 M sorbitol. Although a wild-type strain of HSV-2 induced apoptosis in a significant fraction of the infected cells, HSV-2 could suppress sorbitol-induced apoptosis in a manner similar to that of herpes simplex virus type 1 (HSV-1), indicating that HSV-2, like HSV-1, has an antiapoptosis gene. Characterization of the cells infected with a US3-deletion mutant of HSV-2 revealed the necessity of a US3 gene in the antiapoptotic activity of this virus.

Inhibition of influenza A virus replication by a kanamycin derivative.

We studied the antiviral activity and the mechanism of action of a new antiviral agent and kanamycin derivative, 1-N-eicosanoyl-3"-N-trifluoroacetyl kanamycin A (ETKA), against influenza A virus. From yield reduction assays with VERO cells, ETKA showed a significant antiviral activity with negligible cytotoxic effect. In the presence of 20 micrograms/ml of ETKA at which VERO cell growth was not inhibited, virus titer was suppressed to 11.2% of control, and at 100 micrograms/ml virus production was suppressed to more than 99%. ETKA markedly inhibited viral protein synthesis when cells were pretreated with the drug before infection, but there was no inhibition when the drug was added 15 min post-infection. ETKA did not inhibit virus adsorption and penetration. Nor did it affect the activity of viral RNA polymerase in vitro. We found that the drug had a direct inactivating effect on influenza A virus under acidic conditions. These results suggest that ETKA exerts its antiviral action mainly in the early stage, prior to uncoating by direct inactivation of the virus due to the acidic environment of the endocytic vesicle. Aerosol treatment with the drug protected mice against a lethal influenza A virus infection.

US3 protein kinase of herpes simplex virus protects primary afferent neurons from virus-induced apoptosis in ICR mice.

Possible roles of the US3 gene of the herpes simplex virus (HSV) in the interaction between the virus and primary afferent neurons were examined. Neuronal apoptosis was observed in the trigeminal ganglion of mice that were infected with the wild-type (wt) of HSV-2 strain 186 and with US3-deficient mutant virus (L1BR1). In wt virus-infected mice, many HSV-immunoreactive (HSV-ir) cells were seen throughout the trigeminal ganglion, although no apoptotic change was detected. On the other hand, HSV-ir cells in L1BR1-infected mice were found only in the ophthalmic division of the trigeminal ganglions. Examination by HSV-immunohistochemistry combined with the terminal deoxynucleotidal transferase (Tdt)-mediated deoxyuridin 5'-triphosphate (dUTP) nick-end labeling (TUNEL) method showed that DNA fragmentation had occurred in almost all HSV-ir cells in the L1BRI-infected ganglion. Ultrastructurally, many viral particles were detected in apoptotic ganglionic neurons of mice infected with L1BR1. These results indicate that US3 protein kinase (US3pk) played a role in protecting HSV-infected primary afferent neurons from apoptotic cell death. The present study suggests that US3pk plays a role when HSV establishes latent infections in the sensory ganglia.

[Evaluation of vaccination for geriatric patients].

This paper evaluates the immune responses of geriatric patients to three vaccines; Japanese Encephalitis vaccine, inactivated Influenza vaccine and cold-adapted live Influenza vaccine. To evaluate the immune response, serum antibodies and lymphocyte phenotypes were measured of all three vaccines. In addition, nasal specific IgA antibodies were measured of the cold-adapted live Influenza vaccine. In the case of Japanese Encephalitis vaccine and the inactivated Influenza vaccine patients were inoculated subcutaneously with a 0.5 ml dose of the vaccine respectively. While with the cold-adapted vaccine, the doses were administered via spray into the bilateral nasal canals in 0.25 ml doses respectively. Measurements were taken both prior to and four weeks after the inoculations. In all the cases, sera were measured utilizing the hemagglutination inhibition (HI) method. Peripheral lymphocyte phenotypes were also measured utilizing the flow cytometry. In the case of the cold-adapted live influenza vaccine, nasal specific IgA antibodies were measured by the ELISA method. The results were as follows in all three cases: 1. Recipients who showed elevated HI antibody titers of more than 4 folds were 27.7% to 85.0%. 2. Peripheral lymphocyte phenotypes in geriatric patients were the same as the young control group both before and after inoculation. 3. Antibodies to the inactivated Influenza vaccine were maintained at high levels up to three months after inoculation. 4. The significant increased specific IgA antibodies were 50%. In conclusion, this suggests that inoculation of geriatric patients with the above vaccines is both safe and effective.

[Detection of antibody to varicella zoster virus by immune adherence hemagglutination].

Anti varicella-zoster virus (VZV) antibodies were detected by an immune adherence hemagglutination (IAHA) test, and were compared with the CF test, IFA test and ELISA test, respectively. Type O, Rh-positive RBC for IAHA was obtained from five healthy volunteers. All five RBCs had sufficient sensitivity as the indicator cell. Optimum incubation temperature was 37 degrees C in the serum and in the complement. The complement was obtained from guinea pig sera, and the most suitable concentration was 1;100. The convalescent VZV antibody titers were similar to the values obtained from any of the methods previously mentioned. However, mean titers measured by CF were about two-to fourfold lower than in the values of IAHA. Seroconversion rates of the live VZV vaccine, as detected by CF and IFA were relatively low (CF; 76%, IFA; 56%). In contrast, those obtained from ELISA and IAHA showed perfectly (100%). These results indicate that IAHA has sufficient sensitivity and specificity in the detection of VZV antibodies. In addition, the IAHA test is thought to be a rapid and easy test to perform in ordinary laboratories.

[Familial clustering case of hepatitis delta].

We report a familial clustering case of hepatitis delta. All of the members of this family had evidence of past infection of hepatitis B. We investigated the hepatitis delta, three of the members had positive serological hepatitis delta markers. We assayed by polymerase chain reaction the primers corresponding to hepatitis delta antigen. The results of polymerase chain reaction was three positive. The 2nd polymerase chain reaction was used, two geno-type specific primers one was for Japanese S type the other was for Japanese M type7). Three were positive the 2nd polymerase chain reaction for Japanese M, one was negative for all of hepatitis delta polymerase chain reaction.

[Investigation for VP4 region of coxsackie virus A16 RNA sequence from hand-foot-mouth disease patients at eastern district of Shizuoka prefecture in 1995].

In 1995 an investigation was made for VP4 regions of coxsackie virus A16 (CA16) RNA sequence from hand-foot-mouth disease patients in eastern district of Shizuoka Prefecture. Subjects were seven patients who were diagnosed as hand-foot-mouth disease due to CA16 at the Ohashi Pediatric Clinic in Susono City. Throat swabs of patients were extracted to RNA. Extracted RNA were assayed by reverse transcription polymerase chain reaction that primers corresponded to VP4 resion of enteroviruses. PCR products were marked by dye-deoxy terminator methods and assayed by direct sequence methods. RNA sequences were classified into two types. Type 1 were three cases, and type 2 were four. The homology was 90.8% between type 1 and type 2. All cases of sixty-nine amino acids were the same as prototype strain. We concluded that the two type strains of CA16 were prevalented in eastern district of Shizuoka Prefecture in 1995. It was at the same time and was widely noted in the eastern district.

[A case of neonatal herpes infection which proved to be a mixed infection of herpes simplex virus type 1 and type 2].

We encountered a case which proved to be a mixed infection of herpes simplex virus (HSV) type 1 and type 2 in retrospective terms by in situ hybridization (ISH) and polymerase chain reaction (PCR). The case was a male. The gestational age was 39 weeks and 2 days. The birth body weight was 3024 g. A fever developed from the age of 6 days and he was admitted to the neonatal intensive care unit at the age of eight days. AST was 1042 IU/L, and ALT 206 IU/L. In spite of treatment, the patient died at the age of 12 days. Using paraffin embedded tissues, we performed the ISH and PCR on the cerebrum, lungs, liver, spleen, bone marrow, adrenal gland, and kidneys. With the ISH, the lungs, liver, spleen and adrenal gland were both HSV type 1 and type 2. With the PCR, only the liver was positive for type 1, and the lungs, liver, spleen, and adrenal gland were positive for type 2. In the ISH, a probe showing a cross reaction between type 1 and type 2 was used for type 1 probe this time. But a type 2 probe and PCR did not show a cross reaction. We concluded that this case confirmed the presence of mixed infection (HSV type 1 and type 2) in neonatal HSV infection.

Carrier cell-based delivery of replication-competent HSV-1 mutants enhances antitumor effect for ovarian cancer.

Oncolytic viruses capable of tumor-selective replication and cytolysis have shown early promise as cancer therapeutics. We have developed replication-competent attenuated herpes simplex virus type 1 (HSV-1) mutants, named HF10 and Hh101, which have been evaluated for their oncolytic activities. However, the host immune system remains a significant obstacle to effective intraperitoneal administration of these viruses in the clinical setting. In this study, we investigated the use of these HSV-1 mutants as oncolytic agents against ovarian cancer and the use of human peritoneal mesothelial cells (MCs) as carrier cells for intraperitoneal therapy. MCs were efficiently infected with HSV-1 mutants, and MCs loaded with HSV-1 mutants caused cell killing adequately when cocultured with cancer cells in the presence or absence of HSV antibodies. In a mouse xenograft model of ovarian cancer, the injection of infected carrier cells led to a significant reduction of tumor volume and prolonged survival in comparison with the injection of virus alone. Our results indicate that replication-competent attenuated HSV-1 exerts a potent oncolytic effect on ovarian cancer, which may be further enhanced by the utilization of a carrier cell delivery system, based on amplification of viral load and possibly on avoidance of neutralizing antibodies.

Clinical experiment of mutant herpes simplex virus HF10 therapy for cancer.

We reviewed our clinical trial using mutant herpes simplex virus "HF10". We have evaluated the safety and effect of HF10 against recurrent breast cancer since 2003 and also applied HF10 to non-resectable pancreatic cancer since 2005. An oncolytic herpes simplex virus type 1, mutant HF10, has been isolated and evaluated for anti-tumor efficacy in syngeneic immunocompetent mouse models. From long time before clinical trial, we have found that the mutant virus can have remarkable potential to effectively treat cancer in experimental studies using animals, and that all of the surviving mice acquire resistance to rechallenge of the tumor cells. A number of studies have shown that HF10 is effective and safe for use in localized or peritoneally disseminated malignant tumors of non-neuronal origin in animals. Pilot studies using HF10 have been initiated in patients with metastatic breast cancer. For each patient, 0.5 ml HF10 diluents at various doses were injected into test nodule, and 0.5 ml sterile saline was injected into a second nodule. All patients were monitored for local and systemic adverse effects, and the nodules were excised 14 days after viral injection for histopathological studies. All patients tolerated the clinical trial well. While no adverse effects occurred, there was cancer cell death and 30-100% regression histopathologically in recurrent breast cancer. As mentioned above, intratumoral injection of mutant herpes simplex virus HF10 for recurrent metastatic breast cancer was safe and effective. Also a trial for non-resectable pancreatic cancer being carried out on the basis of the above result has proved to be innocuous and has been in progress to assess the clinical benefit and enhance the potentiality of HF10 against cancer.

In vitro and in vivo analysis of human herpesvirus-6 U90 protein expression.

In order to establish a reliable method for the detection of human herpesvirus-6 (HHV-6) B antigens in peripheral blood mononuclear cells (PBMCs) collected from HHV-6 infected patients, we created a polyclonal antibody against the HHV-6 B U90 protein (IEA/ex3) and used it to examine the expression of this protein in virus-infected cells and patients' PBMCs. This antibody reacted with 170 and 195 kDa proteins in HHV-6 B-infected cord blood mononuclear cells. The IEA/ex3 antigen was detected in cord blood mononuclear cells at 6 hr post-infection, and the number of infected cells reached its maximum at 48 hr post-infection. The antigen stained in a punctate pattern and partially localized to the promyelocytic leukemia (PML) protein body. We also examined 60 PBMC samples from 60 febrile children (3-19 months old) and detected IEA/ex3 antigen in the PBMCs by laser-scanning microscopy. HHV-6 was isolated from 31 of the 60 samples. The sensitivity and specificity of the antigen detection were 84% (26/31) and 97% (28/29), respectively, in the samples with virus detected. The mean number of antigen-positive PBMCs was 409/10(6) cells in 20 samples with viral isolation. A significant correlation (r = 0.566; P = 0.008) was observed between the viral load and number of antigen-positive cells. Although IEA/ex3 antigen was detected by laser-scanning microscopy in PBMCs (without cultivation) collected from six patients with isolated virus, it was detected in only one sample by conventional fluorescence microscopy. Increasing the intensity by cultivation (24 hr) resulted in a higher detection rate (5/6) even by conventional fluorescence microscopy, which is available in most hospital laboratories.

Persistent infection of MDCK cells by influenza C virus: initiation and characterization.

Persistent influenza C virus infection was readily initiated in Madin-Darby canine kidney (MDCK) cells at low m.o.i. and has been maintained for over 1 year. The persistently infected (p.i.) cultures were characterized by the following properties: virus infection was limited to a minority of cells, small amounts of infectious virus were produced together with low levels of interferon (IFN) and the cultures were resistant to superinfection by homologous virus and vesicular stomatitis virus, but not by influenza A and B viruses. These properties fluctuated cyclically with passage of the p.i. culture. When p.i. cultures were cured by cultivation in the presence of antiserum, the cultures lost their IFN-producing activity and became as susceptible to homologous virus as normal MDCK cell culture. The results suggest that persistent influenza C virus infection may be regulated by endogenously produced IFN. Under the condition of high m.o.i. a persistent influenza C virus infection could not be initiated in MDCK cells due to the development of cytopathic effects.

Impaired induction of protective immunity by highly virulent herpes simplex virus type 2 in a murine model of genital herpes.

We investigated the immune events in the vagina of mice intravaginally infected with highly virulent herpes simplex virus type 2 (HSV-2) strain 186, and compared them with those induced by HSV type 1 strain KOS, a widely known laboratory strain. Although there was no significant difference between 186 and KOS in the viral replication in the initial stage of infection, inadequate and delayed clearance of virus from the vaginal mucosa was observed in 1 86-challenged mice. The induction of antigen-presenting cells (APC) such as dendritic cells (DC) and macrophages (Mphi) in the vagina was slow in 186-challenged mice, and the number of T cells in the vagina in 186-challenged mice was much lower than that in KOS-challenged mice. Furthermore, the level of IL-12 as well as that of IFN-gamma was significantly lower in 186-challenged mice than in KOS-challenged mice, while the level of IL-4 in 186-challenged mice was higher than that in KOS-challenged mice. On the basis of these observations, we suggest that the weak activation of epithelial cells and the delayed induction of APC by 186-infection may be involved in the inadequate activation of T cells and the ineffective virus clearance from the vaginal mucosa.

Approach to the involvement of influenza B neuraminidase in the cleavage of HA by host cell protease using low pH-induced cell fusion reaction.

ts7, a temperature-sensitive mutant defective in neuraminidase (NA) of influenza B/Kanagawa/73, lacks NA enzymatic activity at the nonpermissive temperature (37.5 C). When MDCK cells were infected with the mutant at the permissive temperature (32 C) and exposed to pH 5.2 medium, extensive cell fusion occurred. In contrast, at the nonpermissive temperature cells did not show cell fusion at all unless they were pretreated with trypsin, suggesting that at 37.5 C the hemagglutinin (HA) of ts7 is expressed at the cell surface in an uncleaved form. It was also found that the replacement of RNA segment 6 of ts7 with that of wild-type B/Lee resulted in the emergence of low pH-induced fusion activity as well as NA enzymatic activity at the incubation temperature of 37.5 C and that the addition of bacterial NA to the cultures infected with ts7 at 37.5 C early in infection brought about low pH-induced cell fusion. We suggest that the removal of neuraminic acid from the carbohydrate moiety of HA by NA is essential for the cleavage of HA by cellular protease.

Herpes simplex virus UL17 protein is associated with B capsids and colocalizes with ICP35 and VP5 in infected cells.

A previous study using a mutant lacking the UL17 gene has suggested that the UL17 protein of herpes simplex virus type 1 (HSV-1) is required for the cleavage/packaging of viral DNA. In this study, we have raised a rabbit polyclonal antiserum which specifically reacted with the UL17 protein which has an apparent molecular mass of 78-kDa in the lysates of HSV types 1- and 2-infected Vero cells. Western blot analysis of intracellular capsids demonstrates that the UL17 protein was associated with B and C capsids. Indirect immunofluorescence studies reveal that it colocalized with the major capsid protein VP5 and the scaffolding protein ICP35 within the nucleus. These results suggest that the association of the UL17 protein with immature B-type capsids is important for its role in cleavage/packaging.

The US2 gene product of herpes simplex virus type 2 interacts with cytokeratin 18.

In order to clarify the biological role of US2 gene product of herpes simplex virus type 2 (HSV-2), a HeLa cDNA library was screened in the yeast two-hybrid system using US2 protein as bait, and several interacting proteins were identified, including cytokeratin 18. US2 protein was co-immunoprecipitated with cytokeratin 18 from HSV-2 infected cell lysates. Analysis of infected orA431 cells by immunofluorescence showed that US2 protein gave filamentous or dot-like cytoplasmic staining pattern, and that it co-localized with cytokeratin 18. When US2 protein was expressed alone, it co-localized with cytokeratin 18. To define the domain interacting with cytokeratin 18, deletion mutant proteins were constructed and cells transfected with mutants were analyzed by indirect immunofluorescence. These results suggest that the N-terminal half of the US2 protein, especially the region containing amino acids 42-77, is important for interaction with cytokeratin 18.