NK cell tumor therapy modulated by UV-inactivated oncolytic herpes simplex virus type 2 and checkpoint inhibitors.

Oncolytic virotherapy is a new and safe therapeutic strategy for cancer treatment. In our previous study, a new type of oncolytic herpes simplex virus type 2 (oHSV2) was constructed. Following the completion of a preclinical study, oHSV2 has now entered into clinical trials for the treatment of melanoma and other solid tumors (NCT03866525). Oncolytic viruses (OVs) are generally able to directly destroy tumor cells and stimulate the immune system to fight tumors. Natural killer (NK) cells are important components of the innate immune system and critical players against tumor cells. But the detailed interactions between oncolytic viruses and NK cells and these interaction effects on the antitumor immune response remain to be elucidated. In particular, the functions of activating surface receptors and checkpoint inhibitors on oHSV2-treated NK cells and tumor cells are still unknown. In this study, we found that UV-oHSV2 potently activates human peripheral blood mononuclear cells, leading to increased antitumor activity in vitro and in vivo. Further investigation indicated that UV-oHSV2-stimulated NK cells release IFN-γ via Toll-like receptor 2 (TLR2)/NF-κB signaling pathway and exert antitumor activity via TLR2. We found for the first time that the expression of a pair of checkpoint molecules, NKG2A (on NK cells) and HLA-E (on tumor cells), is upregulated by UV-oHSV2 stimulation. Anti-NKG2A and anti-HLA-E treatment could further enhance the antitumor effects of UV-oHSV2-stimulated NK92 cells in vitro and in vivo. As our oHSV2 clinical trial is ongoing, we expect that the combination therapy of oncolytic virus oHSV2 and anti-NKG2A/anti-HLA-E antibodies may have synergistic antitumor effects in our future clinical trials.

In vitro studies of the antiherpetic effect of photodynamic therapy.

The number of viral infection cases in the Department of Gynecology and Obstetrics has tended to increase over last few years. Viruses form herpesvirus and cytomegalovirus families are associated with an increased risk for recurrent pregnancy loss. Photodynamic therapy (PDT) is a promising new approach to treat viral infections in which viral particles are inactivated. It exhibits great therapeutic potential, particularly among this group of patients. This study examined the use of PDT to treat herpesvirus infection (HVI) using an in vitro model. In this study, we used the Vero сell lineage as a suitable model of HVI, strains of HSV-1 (strain VR-3) and HSV-2 (strain MS) obtained from The National Virus Collection (London, UK), the photosensitizer Fotoditazine (Veta-Grand, Russia), an AFS physiotherapeutic device (Polironic Corporation, Russia). Laser light irradiation and the photosensitizer had different cytotoxic effects on the Vero cell cultures depending on the doses used. The optimal laser light and photosensitizer doses were determined. PDT had an antiviral effect on an in vitro model of HVI in cell culture. PDT has been shown to be effective treatment for HVI in vitro, leading to a reliable decrease of viral titer.

Herpes simplex virus type 2-infected dendritic cells produce TNF-α, which enhances CCR5 expression and stimulates HIV production from adjacent infected cells.

Prior HSV-2 infection enhances the acquisition of HIV-1 >3-fold. In genital herpes lesions, the superficial layers of stratified squamous epithelium are disrupted, allowing easier access of HIV-1 to Langerhans cells (LC) in the epidermis and perhaps even dendritic cells (DCs) in the outer dermis, as well as to lesion infiltrating activated T lymphocytes and macrophages. Therefore, we examined the effects of coinfection with HIV-1 and HSV-2 on monocyte-derived DCs (MDDC). With simultaneous coinfection, HSV-2 significantly stimulated HIV-1 DNA production 5-fold compared with HIV-1 infection alone. Because <1% of cells were dually infected, this was a field effect. Virus-stripped supernatants from HSV-2-infected MDDCs were shown to enhance HIV-1 infection, as measured by HIV-1-DNA and p24 Ag in MDDCs. Furthermore these supernatants markedly stimulated CCR5 expression on both MDDCs and LCs. TNF-α was by far the most prominent cytokine in the supernatant and also within HSV-2-infected MDDCs. HSV-2 infection of isolated immature epidermal LCs, but not keratinocytes, also produced TNF-α (and low levels of IFN-ß). Neutralizing Ab to TNF-α and its receptor, TNF-R1, on MDDCs markedly inhibited the CCR5-stimulating effect of the supernatant. Therefore, these results suggest that HSV-2 infection of DCs in the skin during primary or recurrent genital herpes may enhance HIV-1 infection of adjacent DCs, thus contributing to acquisition of HIV-1 through herpetic lesions.

[Impact of low-intensity laser on the suppression of infections caused by Herpes simplex viruses 1 and 2: in vitro study]. / Impacto do laser de baixa intensidade na supressão de infecções pelos vírus Herpes simplex 1 e 2: estudo in vitro.

The use of low-level laser to suppress infections caused by Herpes simplex viruses 1 and 2 was evaluated after one to five applications. A gradual reduction in replication of Herpes simplex viruses 1 and 2 was observed, with 68.4% and 57.3% inhibition, respectively, after five applications, thus favoring its clinical use.

Viral activation of macrophages through TLR-dependent and -independent pathways.

Induction of cytokine production is important for activation of an efficient host defense response. Macrophages constitute an important source of cytokines. In this study we have investigated the virus-cell interactions triggering induction of cytokine expression in macrophages during viral infections. We found that viral entry and viral gene products produced inside the cell are responsible for activation of induction pathways leading to IFN-alphabeta expression, indicating that virus-cell interactions on the cell surface are not enough. Moreover, by the use of cell lines expressing dominant negative versions of TLR-associated adaptor proteins we demonstrate that Toll/IL-1 receptor domain-containing adaptor inducing IFN-beta is dispensable for all virus-induced cytokine expression examined. However, a cell line expressing dominant negative MyD88 revealed the existence of distinct induction pathways because virus-induced expression of RANTES and TNF-alpha was totally blocked in this cell line whereas IFN-alphabeta expression was much less affected in the absence of signaling via MyD88. In support of this, we also found that inhibitory CpG motifs, which block TLR9 signaling inhibited early HSV-2-induced TNF-alpha and RANTES expression dramatically whereas IFN-alphabeta induction was only slightly affected. This suggests that virus activates macrophages through distinct pathways, of which some are dependent on TLRs signaling through MyD88, whereas others seem to be independent of TLR signaling. Finally we demonstrate that IFN-alphabeta induction in HSV-2-infected macrophages requires a functional dsRNA-activated protein kinase molecule because cells expressing a dsRNA-dependent protein kinase version unable to bind dsRNA do not express IFN-alphabeta on infection.

CTL are inactivated by herpes simplex virus-infected cells expressing a viral protein kinase.

Numerous cell-to-cell signals tightly regulate CTL function. Human fibroblasts infected with HSV type 1 or 2 can generate such a signal and inactivate human CTL. Inactivated CTL lose their ability to release cytotoxic granules and synthesize cytokines when triggered through the TCR. Inactivation requires cell-to-cell contact between CTL and HSV-infected cells. However, inactivated CTL are not infected with HSV. The inactivation of CTL is sustainable, as CTL function remains impaired when the CTL are removed from the HSV-infected cells. IL-2 treatment does not alter inactivation, and the inactivated phenotype is not transferable between CTL, distinguishing this phenotype from traditional anergy and T regulatory cell models. CTL inactivated by HSV-infected cells are not apoptotic, and the inactivated state can be overcome by phorbol ester stimulation, suggesting that inactivated CTL are viable and that the signaling block is specific to the TCR. HSV-infected cells require the expression of U(S)3, a viral protein kinase, to transmit the inactivating signal. Elucidation of the molecular nature of this signaling pathway may allow targeted manipulation of CTL function.

[EHF-therapy of herpetic keratouveitis]. / KVCh-terapiia gerpeticheskikh keratouveitov.

A device BIK for active suppression of infection has been used against Herpes virus infection type 1 and 2 in 72 patients with keratouveitis. The immunological and genotypic monitoring show that patients with herpetic keratouveitis after BIK treatment have lower titers of antiherpetic antibodies, normal cellular and humoral immunity, weaker viremia and reduced quantity of viral material in corneal tissue. This positive effect persisted for 8-12 months. Thus, BIK device provides a definite, prolonged and stable therapeutic effect in patients with herpetic keratouveitis.

Relationship of the strain and the intraocular amount of herpes simplex virus types 1 and 2 in the induction of anterior-chamber-associated immune deviation.

We investigated the induction of anterior-chamber-associated immune deviation (ACAID) by herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2). Two strains of HSV-1 (VR-3 and F) and four strains of HSV-2 (UW-268, 186, SL, and MS) were inactivated with ultraviolet light, and the anterior chamber of BALB/c mice was inoculated with a viral strain. Immunization against delayed-type hypersensitivity (DTH) was done subcutaneously on day 7 after anterior chamber inoculation. On day 10 after DTH immunization, each virus was inoculated into the foot pad. DTH was evaluated by measuring the foot pad thickness at 24 h after the inoculation into the foot pad. DTH was suppressed by anterior chamber inoculation with more than 10(3) plaque-forming units (PFU) of the VR-3, UW-268, 186, and SL strains, and with 10(5) PFU of the F strain, but not by the MS strain. Until now, HSV-2 was believed not to induce ACAID, however, our results indicate that some HSV-2 strains induce ACAID.ACAID induction by HSV-1 and HSV-2 may depend on both the viral strain and the amount of ocular viral antigen.

Immunotherapy of acute and recurrent herpes simplex virus type 2 infection with an adjuvant-free form of recombinant glycoprotein D-interleukin-2 fusion protein.

Previous studies demonstrated that the adjuvant-free form of a fusion protein consisting of a truncated herpes simplex virus type 1 (HSV-1) glycoprotein D and human interleukin-2 (tgD-IL-2) elicited superior protective immunity in mice. In this study, the immunotherapeutic efficacy of tgD-IL-2 against vaginal HSV-2 infection was investigated using a guinea pig model. Footpad injections of tgD-IL-2 (12.5 micrograms/dose) after the onset of primary lesions strongly suppressed recurrence in the chronic phase of infection; consequently, the number of days with lesions was reduced 65%. Continuous medication with 100 mg/kg/day acyclovir for 5 days failed to suppress recurrent infection. In a UV radiation-induced recurrence model, prophylactic tgD-IL-2 significantly suppressed both duration and severity of disease. A single injection of tgD-IL-2 plus acyclovir produced an additive effect on the suppression of the disease in the acute phase. These results suggest that tgD-IL-2 is a promising immunotherapeutic agent against HSV-2 genital infections.

Herpes simplex virus-induced expression of 70 kDa heat shock protein (HSP70) requires early protein synthesis but not viral DNA replication.

The major 70 kDa heat shock protein (HSP70), which is scarcely expressed in unstressed rodent cells, was apparently induced by infection with herpes simplex virus (HSV). Infection with HSV types 1 and 2 elevated HSP70 mRNA levels within 4 hr post-infection. HSP70 synthesis and accumulation increased in HSV-infected cells. Irradiation of HSV with UV-light abolished the ability to induce HSP70 mRNA. Inhibitors of viral DNA synthesis did not affect the induction of HSP70 in infected cells. Protein synthesis within 2 hr after infection was necessary for HSP70 induction.

Photodynamic inactivation of herpes viruses with phthalocyanine derivatives.

The antiviral photosensitization capacity of 11 different phthalocyanine (Pc) derivatives was examined using herpes simplex virus-1, herpes simplex virus-2 and varicella zoster virus in the search for the most potent sensitizers for viral decontamination of blood. The kinetics of viral photoinactivation were resolved during the stages of viral adsorption and penetration into the host cells. The capacity of Pc in the photodynamic inactivation of viruses was compared with that of merocyanine 540 (MC540), another widely studied photosensitizer. Sensitivity to photoinactivation decreased progressively with time after addition of viruses to their host cells. The viruses were most sensitive to photodynamic inactivation up to 30 min from the initiation of adsorption. Cell-associated viruses, 45-60 min after the onset of adsorption, are highly resistant to photodynamic treatment by most photosensitizers, with the exception of amphiphilic Pc derivatives. Thus the mixed sulfonated Pc-naphthalocyanine derivatives AlNSB3P and AlN2SB2P demonstrated a remarkable decontamination activity even 60 min after the onset of adsorption. Ultrastructural examination of these photosensitized viruses demonstrated damage to the viral envelope which prevented viral adsorption and/or penetration. The non-enveloped adenovirus was found to be resistant to all the dyes tested.