Understanding the interplay between oHSV and the host immune system: Implications for therapeutic oncolytic virus development

Abstract

Oncolytic herpes simplex viruses (oHSV) preferentially replicate in cancer cells while inducing antitumor immunity, and thus, they are often referred to as in situ cancer vaccines. OHSV infection of tumors elicits diverse host immune responses comprising both innate and adaptive components. Although the innate and adaptive immune responses primarily target the tumor, they also contribute to antiviral immunity, limiting viral replication/oncolysis. OHSV-encoded proteins use various mechanisms to evade host antiviral pathways and immune recognition, favoring oHSV replication, oncolysis, and spread. In general, oHSV infection and replication within tumors results in a series of sequential events, such as oncolysis and release of tumor and viral antigens, dendritic cell-mediated antigen presentation, T cell priming and activation, T cell trafficking and infiltration to tumors, and T cell recognition of cancer cells, leading to tumor (and viral) clearance. These sequential events align with all steps of the cancer-immunity cycle. However, a comprehensive understanding of the interplay between oHSV and host immune responses is crucial to optimize oHSV-induced antitumor immunity and efficacy. Therefore, this review aims to elucidate oHSV’s communication with innate and adaptive immune systems and use such interactions to improve oHSV’s potential as a potent immunovirotherapeutic agent against cancer.

Graphical abstract

Saha and colleagues explored oHSV interactions with the host immune system, highlighting strategies to enhance oHSV’s potential as an immunotherapeutic agent. OHSV induces oncolysis while simultaneously inducing antiviral and antitumor immunity, which limits oHSV activity. OHSV uses various mechanisms to escape host immunity, favoring its replication and oncolysis.

Introduction

Oncolytic viruses (OVs), including oncolytic herpes simplex virus (oHSV), represent a unique class of antitumor agents capable of preferentially targeting and killing cancer cells while sparing normal cells and tissues,^1,2,3 and inducing antitumor immunity^4,5 (Figure 1). Due to these unique properties (tumor-selective replication and induction of antitumor immunity), oHSVs are often referred to as in situ cancer vaccine.⁶ The in situ vaccine effect is more pronounced with armed oHSV expressing immunostimulatory cytokines.^7,8,9 The oHSV-induced vaccine effect and immunostimulation convert immunologically desert or cold tumors into inflamed or hot tumors (Figure 1),^4,10 making them susceptible to other forms of immunotherapy, such as immune checkpoint blockade.^7,11,12,13 Among OVs, HSV type 1 (HSV-1) was one of the first genetically modified OVs¹⁴ to achieve regulatory approval for the treatment of cancer.⁶ Talimogene laherparepvec (T-VEC or Imlygic), a type I oHSV, obtained approval from the U.S. Food and Drug Administration (FDA) in 2015 for treating advanced melanoma, representing a significant milestone in the field of oncolytic immunovirotherapy.^15,16

Figure 1.

The complex relationship between oHSV, TME, and the host immune system

Intratumoral injection of oHSV leads to oncolysis and converts an immunologically desert or cold TME (indicated in blue) into an inflamed or hot TME (indicated in red) through a series of coordinated immunological effects. These include (1) recruitment of innate immune cells from the draining lymph node to the TME, such as NK cells, macrophages, and immature DCs (IDCs); (2) IDC-mediated uptake of released soluble viral/tumor antigens, and IDC maturation enhanced by the release of danger signals from oHSV-infected tumor cells; (3) the mature DCs migrate to draining lymphoid organs where antigen presentation to T cells (CD4⁺ and CD8⁺ T cells) results in their priming and activation; and (4) activated T cells (notably effector CD8⁺ T) traffic from the lymphoid organs to the TME, whereupon recognition of cognate antigen on tumor cell MHC class I induces cytotoxic effects against the antigen-bearing tumor cells. Antigen or MHC class I-negative cells may also be lysed due to local bystander effects. During oHSV-host immune interactions in the TME, oHSV uses various immune evasion mechanisms (also depicted in Figures 2, 4, and 5), inhibiting the antiviral activity of DCs, NK, and T cells. This figure was generated with BioRender.com.

OHSV replication and oncolysis trigger diverse host innate and adaptive immune responses (Figure 1), characterized by a series of sequential events, which include (A and B) oHSV infection, oncolysis, and the release of tumor- and viral-associated antigens,⁴ (C) antigen uptake and maturation of antigen-presenting cells (APCs) such as dendritic cells (DCs), (D) migration of DCs from the tumor site to the nearest lymphoid organ, regional antigen presentation, and T cell activation, (E) T cell migration and infiltration into the tumor microenvironment (TME), (F) recognition of tumor and/or viral antigens, and (G) killing of oHSV-infected and non-infected tumor cells and virus clearance (Figure 2).^6,17 This phenomenon aligns with the cancer-immunity cycle,^18,19,20 a concept that describes seven sequential steps that are thought to mediate tumor clearance by the immune system.^18,19,20 While these responses are generally directed against the tumor, they also contribute to antiviral immunity²¹ (Figure 3), and interestingly, antiviral immunity helps in establishing antitumor immunity.²² However, HSV-1 has evolved mechanisms (which also apply to oHSVs) to counteract host antiviral pathways (Figure 4) and to evade immune cell recognition (Figure 5), thus promoting its replication and spread within the TME.^17,23,24,25 Understanding the interplay between oHSV and host immune responses is essential, as it can help to improve the therapeutic potential and safety of oHSV. Hence, the goal of this review is to provide a more comprehensive understanding of oHSV’s relationship with the host’s immune system and how this relationship can be used to improve oHSV-induced antitumor immunity.

Figure 2.

OHSV infection induces sequential immunological events, mimicking the cancer-immunity cycle

(A) OHSV infection of the TME leads to the induction of innate immune responses, including infiltration of IDCs, macrophages (Mϕ), and NK cells. (B) OHSV infection results in lysis of tumor cells, leading to the release of viral and tumor antigens. (C) The soluble antigens are then engulfed and processed by IDCs, leading to DC maturation. (D) The mature DCs then migrate to the nearest lymphoid organ for the presentation of tumor/viral antigens to T cells, resulting in T cell activation and clonal expansion, and generation of oHSV and/or tumor-specific T cells. (E) The virus and/or tumor-specific T cells migrate out from the lymphoid organ and infiltrate into the TME. (F) The accumulated T cells in the TME recognize tumor and/or viral antigens, resulting in the (G) release of killer cytokines, such as IFN-γ, perforin, and granzyme B, which all contribute to the lysis of tumor cells or oHSV-infected tumor cells. This results in the further release of tumor and viral antigens and oHSV virions. The newly released oHSV virions further infect the surrounding cancer cells and initiate step B, resulting in the continuation of the aforementioned immunological events. This cyclic process continues until the oHSV, tumor cells, and/or oHSV-infected tumor cells are eliminated. OHSV uses various inhibitory mechanisms to escape from the inhibitory effect of the host immune system (as depicted above); for instance, HSV γ34.5 and ICP47 inhibit DC maturation and antigen presentation (depicted in step D), which can eventually disrupt the sequential immunological events, leading to oHSV escape from T cell immune surveillance. This figure was generated with BioRender.com.

Figure 3.

Innate and adaptive antiviral immune responses against HSV

Once oHSV is injected into the tumor lesions, it interacts with various components of the innate and adaptive immune system. Specific anti-tumor mechanisms are depicted and include activation of the (1) serum complement/immunoglobulin cascade, (2) cytokine/chemokine signaling pathway, (3) innate immune cells, and adaptive immune cells, such as (4) T and (5) B cells. These systems contribute to oHSV clearance and indirectly to antitumor immunity. This figure was generated with BioRender.com.

Figure 4.

HSV-1 uses a multitude of mechanisms to escape the host immune response by expressing genes that impair the host’s antiviral activities

Production of type I IFN and signaling pathways are considered crucial for antiviral innate immunity. Various HSV-1 genes can counter signaling molecules within antiviral signaling pathways (such as the TLR signaling pathway, RIG-I-like receptor or RLR signaling pathway, DNA sensor signaling pathway, and IFN-stimulated genes), which promotes HSV replication in cancer cells. Defined signaling pathways induced following HSV infection include the following. (I) The TLR signaling pathway: TLR2 and TLR3 signaling to TRAF6 is inhibited by Us3, TRAF3 is inhibited by UL36 deubiquitinate, MyD88 and NF-KB subunits p50 and P65 are inhibited by ICP0, nuclear translocation of NF-KB is inhibited by UL42 and Us3. (II) RIG-I-like receptor (RLR) signaling pathways: Us11 binds to PACT, RIG-1, and MDA-5 to inhibit interaction with MAVS; ICP34.5 binds to TBK1 and inhibits IRF3 phosphorylation, as does VP24; Us3 hyperphosphorylation of IRF3 blocks its activation; ICP0 inhibits IRF3 translocation to the nucleus. (III) DNA-sensor signaling pathway: Upon detecting the presence of cytosolic DNA, cyclic GMP-AMP synthase (cGAS) synthesizes cyclic GMP-AMP (cGAMP). cGAMP binds to and activates STING, which in turn stimulates IRF3, leading to type I IFN production for antiviral activity. The oHSV US3 protein antagonizes the cGAS/STING signaling pathway, resulting in hyperphosphorylation of IRF3, a substrate of TBK1, inhibiting the activity of IFN-β, leading to immune evasion. (IV) IFN-stimulated genes: UL41 inhibits viperin, ZAP, and tetherin through mRNA degradation; Us11 inhibits OAS and PKR; PKR phosphorylates eIF2α, which shuts down protein synthesis; ICP27 prevents STAT-1 phosphorylation and activation. This figure was generated with BioRender.com.

Figure 5.

Immune evasion mechanisms of oHSV

Mechanisms associated with HSV-mediated immune escape are shown. Escape from NK cells: OHSV leverages TGF-β to suppress NK cell activity. HSV-1 gD glycoprotein downregulates CD112, a ligand for the DNAM-1 receptor of activating NK cells. HSV’s gD-induced downregulation of CD112 ligand reduces the ability of NK’s DNAM-1 receptor to bind to the target cell surface (i.e., HSV-infected cells). The reduced interaction between DNAM-1 (from NK) and CD112 (HSV-infected cells) results in the inhibition of NK cell degranulation, rendering them unable to bind to and kill target cells (HSV-infected). HSV also inhibits the activity of NKT cells through reduced surface expression of CD1d molecule on APCs, leading to APC’s inability to stimulate NKT cells. Escape from DC antigen presentation: HSV ICP0 degrades CD83, a DC maturation marker, and ICP34.5 inhibits DC maturation. HSV-1 also induces apoptosis in DC via activation of IRE-1α and prevents DC migration via activation of the LFA-1 adhesion molecule. Escape from signaling pathways: OHSV interferes with apoptotic pathways involving caspase 3, Bid, cytochrome c, and programmed cell death protein 4 (PDCD4). The viral protein Us3 plays a pivotal role in inhibiting these pathways. By disrupting these signaling cascades, oHSV subverts host defense, enabling its survival and replication in infected cells. Escape from T cell: HSV-1 infection of T cells initiates a cascade of events, including T cell receptor (TCR) remodeling, leading to the production of IL-10, an immunosuppressive cytokine that not only aids HSV-1 replication but also remodels the T cell response. HSV ICP47 targets the TAP, a crucial component in the presentation of MHC class I restricted antigens to cytotoxic T cells. HSV ICP22 promotes proteasomal degradation of MHC-I-related (MR1) protein, whereas HSV’s virion host shutoff (vhs) RNase protein downregulates MHC-I-related (MR1) antigen presentation through degradation of MR1 transcripts. Consequently, HSV-1-infected cells, including cancer cells, experience downregulation of MHC-I peptide presentation to CD8⁺ T cells, hindering their immune recognition. Moreover, HSV-1 infection also blocks CD4⁺ T cells by interfering with the MHC class II processing pathway. Escape from immunogenic cell death mechanisms: Viral protein Us11 inhibits autophagy. Additionally, several HSV proteins, including ICP6, ICP10, Us3, Us5, and Us6, inhibit apoptosis pathways, collectively enhancing the virus’ ability to survive and replicate within host cells. Escape from other early host defense mechanisms: HSV gE/gI enhances FcγR binding to IgG, thus inhibiting antibody-mediated complement activation. Additionally, HSV gC inhibits components of the complement system, including C5a, C3b, and properdin. These actions collectively help HSV to evade host immune responses. This figure was generated with BioRender.com.

Interplay between oHSV and the innate immune system

OHSV interaction with serum proteins

Serum lipoproteins, complement, immunoglobulins, and fatty acids bind to HSV, thus reducing viral infectivity.^24,26 In vivo complement depletion by cobra venom factor (CVF) treatment enhances the transduction of oHSV to intracerebral tumors after intravascular oHSV administration, confirming oHSV interactions with complement.²⁶ HSV’s interaction with the innate serum immunoglobulins was confirmed when anti-IgG or -IgM-treated sera from humans and rats (not mice) significantly lost their capacity to inactivate oHSV.²⁷ Additionally, cyclophosphamide (CPA) treatment, which partially suppresses IgM activity in rodents and humans and significantly increases oHSV propagation in orthotopic brain tumors in rats, resulting in tumor regression.²⁸ OHSV propagation was further improved when CPA was given in combination with CVF.²⁶

HSV-1 uses envelope glycoproteins for entry into host cells, and several studies show that viral glycoproteins also contribute to HSV-mediated immune escape mechanisms (Figure 5). Although two domains of HSV’s gC are involved in modulating the complement pathway, other domains of HSV’s gC evade antiviral host complement proteins.^29,30 For example, several domains of viral gC bind to and inactivate C3b complement protein, and one domain of gC binds to and inactivates C5a complement protein, inhibiting C3-C5 and C3-properdin interactions (Table 1).²⁹ These interactions reduce complement-mediated neutralization of cell-free HSV^29,30,31,32 and complement-mediated lysis of virus-infected cells.²⁹ However, these immune evasion mechanisms from the complement system may vary between virulent clinical isolates and laboratory HSV strains and may be more pronounced in clinical isolates.²⁷ HSV-1 encodes two Fc receptors (FcRs): (1) viral glycoprotein E (gE) and glycoprotein I form a complex that acts as an FcR to bind to the Fc domain of the monomeric IgG, inhibiting its function potentially through antibody bipolar bridging^24,26; and (2) gE alone acts as another FcR that binds to IgG complexes.³³ The HSV-1 FcR-IgG interaction blocks IgG-mediated Fc-mediated complement activation and prevents IgG from targeting HSV and HSV-infected cells, promoting immune evasion (Figure 5).³⁴

Table 1.

List of HSV-1 genes or proteins and their interactions with various host factors

HSV-1 gene or protein	Function	Reference
Glycoprotein B	forms complexes with HLA-DR or HLA-DM and inhibits MHC class II presentation	Neumann et al.87
Glycoprotein C	inactivates C3b and C5a complement proteins; inhibits C3-C5 and C3-properdin interactions	Lubinski et al.29
Glycoprotein D (Us6)	causes degradation/downregulation of CD112 (a ligand for activated NK cell receptor) and inhibits NK cell degranulation and NK-mediated cytotoxicity	Grauwet et al.62
	causes degradation of CD83 in mature DCs	Heilingloh et al.77
	prevents staurosporine- or Fas-induced apoptosis of the HSV-1-infected monocytic cells via activation of NF-kB	Sciortino et al.151 and Marino-Merlo et al.152
Glycoproteins E and I	binds to the Fc domain and blocks IgG-mediated complement activation and prevents IgG from targeting HSV-infected cells	Ma et al.,24 Ikeda et al.,26 Dubin et al.,33 and Lubinski et al.34
Glycoprotein H/L	activates IRF and NF-κB pathways, leading to the production of antiviral type I IFN	Danastas et al.36
Glycoprotein J (Us5)	inhibits CTL-induced apoptosis through the inhibition of FasL- and granzyme B-mediated pathways	Jerome et al.150
UL12.5	induces production of antiviral type I IFN response via the RNA polymerase III/RIG-I pathway	Berry et al.40
UL31	antagonizes IFN-β responses through the inactivation of IKKi and IRF3	Gong et al.48
UL41 (vhs)	downregulates MR1 through the degradation of MR1 transcripts	Samer et al.86
US3	reduces production of antiviral type I IFN via phosphorylation of RIG-I	van Gent et al.42
	antagonizes the antiviral cGAS/STING signaling pathway	You et al.43
	hyperphosphorylates IRF3 and inhibits the activity of IFN-β	Wang et al.44
	blocks TCR signaling in T cells by impairing the TRAF6-mediated ubiquitination of the linker for activation of T cells	Yang et al.112
	inhibits apoptosis via inhibition of programmed cell death protein 4, caspase-3 activation, and granzyme B-mediated cleavage of the proapoptotic protein Bid	Wang et al.147 and Cartier et al.148
US11	suppresses the RLR signaling pathway via direct interaction with RIG-I and MDA5	Xing et al.45
	inhibits autophagy through the disassembly of the TRIM23-TBK1 complex or via interaction with EIF2AK2 (PKR)	Liu et al.129 and Lussignol et al.130
	inhibits autophagy through cleavage of caspase-8-p18 and Atg3	Musarra-Pizzo et al.133
	inhibits the activity of TBK1 via the formation of the Us11-HSP90 complex	Liu et al.46
VP24	inhibits the DNA-sensing signal pathway through the inactivation of IRF3	Zhang et al.49
ICP0	enhances NK recognition and lysis of HSV-infected cells via upregulation of ligands for NCRs, such as NKp30, NKp44, and NKp46	Barrow et al.54
ICP6	blocks caspase-8, leading to inhibition of death receptor (DR) ligand (such as TNF-α and Fas ligand)-mediated apoptosis	Dufour et al.144
	inhibits TNF-induced necroptosis by preventing RIP1 from interacting with RIP3	Guo et al.145
ICP22	promotes proteasomal degradation of MR1 and MHC class II proteins	Samer et al.86
ICP34.5	controls IRF3 activation	Manivanh et al.47
	blocks autophagy through interaction with phosphorylated host protein PPP1CA/PP1α	Ripa et al.127
	inhibits DC maturation through IKK and protein phosphatase 1 alpha	Jin et al.80
ICP47	inhibits the TAP and MHC class I presentation	Oldham et al.83

Several strategic adjustments can be made to counter serum protein-mediated oHSV inactivation, first by developing oHSV variants that are less susceptible to neutralization by serum proteins (complement and immunoglobulins) and second by delivering oHSV loaded in mesenchymal stem cells, which can prevent oHSV neutralization by host serum proteins. This strategy could be specifically applicable to intravenous oHSV therapy.³⁵ Moreover, combining complement depletion agents with immunomodulatory treatments (e.g., CPA) may further enhance oHSV propagation within tumors.^26,28

OHSV interaction with innate immune signaling pathways

OHSV infection of tumor cells can trigger various innate cellular immune signaling pathways (Figure 4).^25,36,37,38 HSV-induced activation of these pathways results in the production of cytokines and chemokines within the TME.³⁸ For instance, the HSV glycoprotein H/L complex binds to αvβ3-integrins, activating the interferon regulatory factors (IRF) and nuclear factor (NF)-κB pathways, leading to the production of antiviral type I interferons (IFNs).³⁶ HSV-1 infection-mediated production of cytokines/chemokines is crucial for the induction of subsequent steps of antitumor immunity (Figure 2), but they also contribute to antiviral immunity,²² limiting oHSV replication/spread within the TME. However, generally, the antiviral type I IFNs and their associated signaling pathways (Figure 4) are defective in transformed cells (but not in healthy cells), thus allowing oHSV replication in tumor cells, and subsequent oncolysis.^17,38,39 Furthermore, various HSV genes can counter many signaling molecules of these antiviral signaling pathways, thus further favoring oHSV replication in cancer cells (Figure 4).

HSV’s UL12.5 impairs the mitochondrial network in infected cells, resulting in the production of the antiviral type I IFN response via the RNA polymerase III/RIG-I pathway⁴⁰; however, Us3 protein can reverse this anti-HSV effect.^41,42 Us3 protein binds to and phosphorylates RIG-I. RIG-I phosphorylation inhibits TRIM25-induced RIG-1 ubiquitination and reduces RIG-1 binding to MAVS, resulting in reduced production of anti-viral type I IFNs,⁴² suggesting that US3-mediated immune evasion may favor oHSV replication and oncolysis. The US3 protein also antagonizes the cGAS/STING signaling pathway⁴³ (Figure 4) and hyperphosphorylates IRF3, a substrate of TANK-binding kinase 1 (TBK1), inhibiting the activity of IFN-β, leading to immune evasion.⁴⁴ Like Us3, Us11 protein directly interacts with RIG-I and MDA5, leading to suppression of the RLR signaling pathway.⁴⁵ The Us11 protein also inhibits the activity of TBK1 via the formation of the Us11-HSP90 complex, promoting HSV replication.⁴⁶ Following HSV infection, the pattern recognition receptors (PRRs) recruit TBK1, which activates IRF3, resulting in the production of antiviral type I IFNs.⁴⁷ However, the TBK1-binding domain (TBD; amino acids 87–106) of ICP34.5 controls IRF3 activation (Figure 4), although the TBD itself does not have any impact on IRF3 activation or on HSV replication.⁴⁷ UL31 antagonizes IFN-β responses through the inactivation of IKKi and IRF3,⁴⁸ and VP24 inhibits the DNA-sensing signal pathway through the inactivation of IRF3⁴⁹ (Table 1; Figure 4). A list of HSV-1 genes (or proteins) and their interactions with the different host factors is provided in Table 1.

In summary, the interactions between HSV and host antiviral signaling pathways pose challenges and opportunities for cancer therapy. On the one hand, the HSV-encoded proteins suppress innate immune responses, promoting viral replication and oncolysis (Figures 4 and 5). On the other hand, HSV infection and replication also triggers antiviral signaling pathways, limiting viral replication and oncolysis (Figure 4). Studies suggest that transient inhibition of innate antiviral signaling may help initial oHSV replication, spread within the TME, and oncolysis, thus enhancing oHSV efficacy against cancer.^50,51 Additional studies underscore the importance of anti-HSV immunity in generating clinical antitumor immunity.²²

OHSV-induced activation or inhibition of host natural killer cells

Intratumoral administration of 1716 (an oHSV devoid of ICP34.5) triggered monocytes and DCs to produce CXCL9 (MIG) and CXCL10 (IP-10), associated with a significant increase in the infiltration of natural killer (NK) and CD8⁺ T cells into the tumor, resulting in control of ovarian cancer growth.⁵² HSV-1 infection of peripheral blood mononuclear cells (PBMCs) in vitro leads to increased production of interleukin (IL)-15, enhancing the cytotoxic ability of NK cells, thereby inhibiting HSV replication. Neutralization of IL-15 abrogates HSV-induced NK activity, resulting in increased HSV replication.⁵³

HSV ICP0 (an immediate-early gene) enhances the upregulation of ligands for natural cytotoxicity receptors (NCRs), such as NKp30, NKp44, and NKp46,⁵⁴ thereby enhancing NK recognition and lysis of HSV-infected cells (Figure 3),⁵⁵ eventually decreasing the efficacy of oHSV therapy. Conversely, increased oHSV replication and anti-glioma efficacy were observed in mice devoid of NCRs, further demonstrating the inhibitory effect of NK cells on oHSV therapy.⁵⁶ In a recent study, NK cell depletion enhanced the efficacy of an oHSV-expressing IL-2 cytokine in a murine model of glioblastoma multiforme (GBM).⁸ Transforming growth factor (TGF)-β treatment of NK cells makes NK cells less cytotoxic to oHSV-infected GBM or GBM stem cells, and co-culture of TGF-β-treated NK cells, macrophages, or microglia with oHSV-infected GBM cells results in a higher oHSV yield than co-culture of untreated innate immune cells with oHSV-infected GBM cells.⁵⁷ These in vitro findings are replicated in animal models of GBM, as TGF-β pretreatment, which decreases intratumoral infiltration of NK cells and macrophages and microglia, before oHSV therapy, resulted in enhanced oHSV efficacy, such as inhibition of GBM growth and prolongation of survival of mice bearing xenograft or syngeneic tumors.⁵⁷ These studies indicate that innate cellular responses are inhibitory to oHSV, and reversing these responses enhances oHSV efficacy. Co-administration of CPA, which inhibits innate immune responses, with oHSV enhanced tumor cell-specific CTL activity and antitumor efficacy in the Lewis lung carcinoma model.⁵⁸

NK cells degranulate in response to stimulation by virus-infected target cells and cause cytotoxicity.⁵⁹ However, HSV infection reduces degranulation in NK cells, rendering them unresponsive to target (K562) cell stimulation. This suggests that HSV-induced functional paralysis of NK cells⁶⁰ is not related to defects in the recognition of target cells.⁶¹ HSV gD glycoprotein expression during HSV infection or gD transfection causes degradation and downregulation of CD112, a ligand for the DNAX accessory molecule 1 (DNAM-1) receptor of activating NK cells.⁶² HSV gD-induced downregulation of CD112 ligand reduced the ability of NK’s DNAM-1 receptor to bind to the target cell surface (i.e., HSV-infected or gD-transfected cells).⁶² The reduced interaction between DNAM from NK cells and CD112 (gD-expressing or HSV-infected cells) results in the inhibition of NK cell degranulation, rendering them unable to bind to and kill target cells (HSV-infected or gD-transfected) (Table 1).⁶² HSV also inhibits the activity of NK T (NKT) cells through reduced surface expression of the CD1d molecule on APCs, leading to APC’s inability to stimulate NKT cells (Figure 5).^63,64

In summary, the role of NK cells in oHSV-induced antitumor immunity is associated with counter-regulatory responses that can lead to paradoxical responses. While some studies highlight the importance of NK cell infiltration for controlling tumor growth after oHSV infection,^52,65 others demonstrate that NK cells can inhibit oHSV replication and reduce oHSV-mediated antitumor efficacy.^8,56,57 To address this complexity, transiently suppressing NK cell function (e.g., with TGF-β or CPA) before oHSV infection^26,28,57 may allow oHSV-infected cancer cells to temporarily evade NK cell recognition, facilitating the completion of the viral replication cycle, resulting in oncolysis and the initiation of subsequent immunological events (Figure 2).

OHSV interaction with the host DCs activates other innate immune cells

OHSV infection leads to infiltration of plasmacytoid DCs (pDCs) and monocytes,^66,67 which are among the first cells, besides NK cells, recruited to the site of infection.⁶⁷ HSV gD interacts with HVEM for entry into epithelial DCs, followed by HSV replication within DCs, resulting in DC maturation and enhanced expression of antiviral innate cytokines, such as IFN-α, IFN-β, and IFN-γ.⁶⁸ HSV has been shown to interact with conventional DCs via activating PRRs, such as toll-like receptor (TLR) 2 and TLR9.^69,70 HSV-PRR interaction activates DCs, resulting in the secretion of immunomodulatory cytokines such as IL-6 and IL-12⁷¹ (Figure 3). HSV-TLR2 interaction is also required for the activation of IL-15 gene expression in monocytic cells, and this HSV-TLR2-induced IL-15 expression helps with the translocation of NF-κB to the nucleus via recruitment of TIRAP/Mal/MyD88 and activation of TRAF6/IRAK1,⁷² all of which are known to induce antiviral activity. Another study also confirmed that HSV interaction with TLR2/9 results in the recruitment and activation of NK cells and monocytes to the site of infection, thus restricting HSV spread.⁷³

Specifically, TLR2/9 recognition of HSV infection results in functional maturation of monocyte-derived tumor necrosis factor (TNF)-α and inducible nitric oxide synthase (iNOS)-producing DCs (Tip-DCs), and activation of NK cells. These Tip-DCs present cognate antigens, leading to enhanced effector activity of HSV antigen-specific CD4⁺ and CD8⁺ T cells at the site of infection and the nearest draining lymph nodes.⁷³ Interestingly, TLR2/9 recognition of HSV causes direct activation of NK cells (without the help of DCs) via activation of the p38 MAPK pathway.⁷³ DCs and monocytes/macrophages produce IL-1β and iNOS (Figure 3), which, in conjunction with type I IFNs, control HSV infection.⁷⁴ Vogel et al.⁶⁷ demonstrated that UV-inactivated HSV-1 induces the upregulation of CD69, an activation marker, on NK cells, and the pDC-derived IFN-α is responsible for the activation of NK cells. Similar to the pDC-derived IFN-α, TNF-α secreted from HSV-1-stimulated monocytes also contributes to the effector function of NK cells (Figure 3), since neutralization of TNF-α significantly diminishes CD69 upregulation induced by HSV-1 on NK cells.⁶⁷

OHSV can preferentially influence DC maturation and migration

For effective and potent antitumor immune responses, DCs must be activated and mature. However, oHSV can inhibit DC maturation through the degradation of CD83, a DC maturation marker.^75,76 Heilingloh et al.⁷⁷ demonstrated that HSV ICP0 causes degradation of CD83 in mature DCs (Figure 2), which is independent of its E3 ubiquitin ligase function. HSV infection of monocyte-derived DCs induces rapid degradation of CYTIP (a cytohesin-interacting protein that is upregulated on mature DCs and helps in DC motility), resulting in activation of β2 integrin adhesion molecules, mainly LFA-1^78,79 (Figure 5), and thus, impairing the migration of DCs from the site of infection to draining lymph nodes.^78,79

The HSV neurovirulence factor γ34.5 inhibits DC maturation through I_κB kinase (IKK) and protein phosphatase 1 (Table 1),⁸⁰ while the beclin-binding domain (BBD) inhibits autophagosome maturation, which is critical for host defense and antigen presentation in DCs.⁸¹ HSV infection causes apoptosis in DCs via activation of the inositol-requiring enzyme 1 alpha (IRE-1α), an unfolded protein response factor, impairing their migration and antigen presentation (Figure 5).⁸² Pharmacological inhibition of the endonuclease activity of IRE-1α by MKC-3946 significantly reduced apoptosis in HSV-infected DCs and increased their migration, resulting in increased activation of HSV-specific CD4⁺ and CD8⁺ T cells.⁸² Overall, HSV-mediated inhibition of DC maturation and migration results in reduced T cell activation and proliferation, leading to HSV escape from antiviral T cell responses.^75,76

OHSV influences host major histocompatibility complex expression and antigen presentation

HSV uses several distinct mechanisms to escape immune surveillance by T cells. HSV ICP47 inhibits the transporter associated with antigen processing (TAP)⁸³ (Figure 5) in HSV-infected cells, including cancer cells. TAP inhibition results in the downregulation of major histocompatibility complex (MHC) class I-peptide complex presentation on target cells, allowing HSV-infected cells to escape immune recognition by CD8⁺ T cells.⁸⁴ An oHSV armed with a TAP inhibitor, bovine herpesvirus UL49.5 (which inhibits both murine and human TAP, in contrast with human HSV ICP47, which does not inhibit mouse TAP), significantly reduced tumor burden in syngeneic murine tumor models compared with an oHSV armed with a non-functional UL49.5 or mock, eliciting both local and systemic antitumor immunity. This improved efficacy is CD8⁺ T cell-dependent and associated with increased oHSV replication within the TME.⁸⁵

HSV ICP22 (a US1 regulatory protein) promotes proteasomal degradation of MR1, whereas HSV’s virion host shutoff (UL41, vhs) RNase protein downregulates MR1 through the degradation of MR1 transcripts (Table 1).⁸⁶ In addition to evading CD8⁺ T cells, HSV infection also evades CD4⁺ T cells by targeting MHC class II processing pathway factors, such as invariant chain (li), human leukocyte antigen (HLA)-DR, and HLA-DM. HSV infection reduces the expression of li, and HSV gB forms complexes with HLA-DR or HLA-DM (Figure 5).⁸⁷ HSV infection of GBM cells decreased cell surface expression of MHC class II proteins, resulting in immune escape from CD4⁺ T cell recognition, while infection with HSV mutants in UL41 or γ34.5 led to increased cell surface expression of MHC II proteins.⁸⁸

Understanding the complex oHSV-DC interactions can help to harness the potential of DCs to improve oHSV-based immunotherapies and effective antitumor immunity. Indeed, several strategies to improve DC antigen presentation after oHSV virotherapy have been suggested: (1) Since ICP34.5 impairs DC maturation, deletion of the γ34.5 gene can enhance DC maturation and migration. In fact, many oHSVs in clinical development, including T-VEC, are ICP34.5 deleted.⁸⁹ (2) Modulation of intracellular pathways involved in HSV-induced DC apoptosis could be beneficial, e.g., pharmacological inhibition of the IRE-1α could reduce DC apoptosis, thereby preserving DC’s function. (3) Selecting an HSV candidate for enhanced antigen presentation, e.g., deleting HSV ICP47, which inhibits TAP and MHC class I presentation,⁸³ or ICP22 and vhs RNase, which downregulate MR1 and MHC class II proteins,⁸⁶ could improve viral recognition by CD8⁺ and CD4⁺ T cell responses, respectively. Interestingly, several oHSVs, including T-VEC and G47Δ (approved in Japan for GBM), lack ICP47.^89,90 In contrast, Pourchet et al.⁸⁵ showed that oHSV expression of bovine herpesvirus UL49.5 (a TAP inhibitor for both human and mouse TAP, whereas HSV-1 ICP47 is inactive in rodents) elicits potent local and systemic antitumor immunity, although UL49.5-expressing oHSV evades CD8⁺ T cell recognition. Finally, (4) OHSVs expressing various immunomodulatory agents, such as IL-12^39,91, CD40 agonist monoclonal antibodies,⁹² and others, may enhance DC maturation and activation, resulting in effector T cell responses with improved antitumor immunity. Further research is needed to validate these approaches and optimize their translation into effective cancer immunotherapies.

OHSV interaction with host macrophages can be beneficial or inhibitory to oHSV activity

Macrophages play a dual role in the context of oncolytic virotherapy, showing both antiviral and antitumor activity.⁹³ Macrophage depletion increases oHSV titers in the TME, suggesting anti-HSV activity of macrophages.⁹⁴ OHSV injection into GBM tumors leads to robust infiltration of macrophages into the tumors and polarization of macrophages/microglia toward an M1-like proinflammatory phenotype, which in turn produces a significant level of TNF-α.⁹⁵ This induces the apoptosis of HSV-infected GBM cells. The use of TNF-α-neutralizing antibodies reverses the anti-HSV effect of TNF-α, showing increased oHSV replication and leading to improved survival of mice bearing orthotopic GBM tumors.⁹⁵ The inhibition of intratumoral infiltration of macrophages and other monocytic lineages into the GBM TME by liposomal clodronate⁹⁴ or CPA^96,97 resulted in enhanced oHSV propagation with improved therapeutic efficacy.

Both M1- and M2-like macrophages can be critical in controlling HSV infection, with M2 polarization via HSV expression of IL-4 being more effective than M1 polarization via HSV expression of IFN-γ against HSV latency, reactivation, and disease.⁹⁸ Monocytes and macrophages produce IL-1β and iNOS, which, in conjunction with type I IFNs, control HSV infection (Figure 3).⁷⁴ In contrast, Kwan et al.⁹⁹ demonstrated that human macrophages support oHSV 1716 replication, and macrophage depletion leads to reduced antitumor activity of oHSV 1716. Macrophage-derived IL-6 may play a dual role in interacting with HSV since one study showed that IL-6 controls HSV infection,¹⁰⁰ whereas another group showed that an increased IL-6 level enhanced oHSV replication in glioma cells via STAT3 activation, which inhibits the antiviral type I IFN pathway in oHSV-infected cancer cells.¹⁰¹ Inhibition of STAT3 activation by a STAT3 inhibitor (LLL12) resulted in a significant reduction of oHSV replication.¹⁰¹ As opposed to the outcome of STAT3 inhibition (which reduces oHSV replication), the inhibition of STAT1 activation by the JAK1/2 inhibitor ruxolitinib increases productive oHSV (R3616) replication in malignant peripheral nerve sheath tumor (MPNST) cells via reduced expression of IFN-stimulated genes.¹⁰² Ruxolitinib has also been shown to increase oHSV susceptibility in melanoma cells.¹⁰³

In summary, macrophages have a dual role, both antiviral and antitumor immunity, which underscores the complex interplay between oHSV, macrophages, and the TME in shaping therapeutic outcomes. Further investigations into targeted modulation of macrophages and their associated immune responses (antiviral and antitumoral) are needed to refine oHSV-based immunotherapies and maximize their clinical impact in treating cancer.

Interplay between oHSV and the adaptive immune system

OHSV induces antiviral and antitumor T cell responses

OHSV infection and oncolysis result in immunogenic cell death¹⁰⁴ and the release of tumor-associated (and viral) antigens, which are taken up by DCs, leading to their activation^17,105 and generation of both oHSV antigen- and tumor antigen-specific T cell responses (Figure 2) in vivo and control of tumor growth.^106,107 OHSV replication within mouse tumors reached a peak on day 2 post infection and then gradually declined,¹⁰⁸ likely due to the antiviral action of innate immune and HSV-specific T cells, leading to HSV clearance.^106,109 In a recent phase 1 clinical trial, intratumoral oHSV CAN-3110 treatment of GBM resulted in somewhat improved survival, associated with enhanced infiltration of CD4⁺ and CD8⁺ T cells into tumors and positive HSV-1 serology with clearance of CAN-3110 virus from the injected tumor, suggesting that CAN-3110 induced antitumor as well as antiviral immunity.⁴ Further evaluation of GBM tumors shows a time-dependent non-significant decrease in the number of CD8⁺ T cells over a period of several months in HSV-1 seronegative patients and more than in the HSV-1 seropositive patients, without much alteration of CD4⁺ T (and CD20⁺ B cells).⁴ The longer presence of CD8⁺ T cells in the tumors of HSV-1 seropositive patients possibly contributed to their longer survival time compared with HSV-1 seronegative patients.⁴ While many published studies have identified defined tumor antigens, further work is needed to determine whether tumor neoantigen responses occur and contribute to favorable patient outcomes.

HSV uses various mechanisms to evade T cell responses (Figure 5). For example, HSV infection of T cells causes TCR remodeling, resulting in p38-dependent synthesis of IL-10, an immunosuppressive and HSV replication-friendly cytokine.¹¹⁰ HSV can induce T cell apoptosis via direct or indirect mechanisms.¹¹¹ Direct HSV infection of Jurkat cells (a T cell leukemia cell line) or human CD4⁺ T cells (obtained from human PBMCs) results in their apoptosis. Indirectly, T cells undergo apoptosis when they are co-cultured with HSV-infected fibroblasts.¹¹¹ HSV tegument protein Us3 blocks TCR signaling in T cells (Figure 2) by impairing the TRAF6-mediated ubiquitination of the linker for activation of T cells (Table 1), resulting in reduced T cell activation and IL-2 production, which may eventually enhance HSV replication.¹¹²

Although HSV evasion of T cell activity may lead to improved HSV replication, HSV-induced modulation of T cells or T cell infiltration is critical for therapeutic antitumor effects.^7,8,108,113 For instance, intratumoral application of oHSV G47Δ or G47Δ expression of IL-12 immunostimulatory cytokine enhanced median survival of mice bearing orthotopic brain tumors, which was associated with reduced infiltration of immunosuppressive regulatory T cells (Tregs) and an increased T effector to Treg ratio, a hallmark of immunotherapy success in the clinic.^7,108 Similar to G47Δ-IL12, T-VEC treatment reduces Tregs in human melanoma responders.¹¹⁴ G47Δ expression of IL-2 (another immunostimulatory cytokine), similar to IL-12 expression, also results in increased infiltration of CD8⁺ T cells into the tumor, resulting in improved survival compared with control-treated mice.⁸ Systemic T cell modulators, such as immune checkpoint blockade, were also shown to enhance antitumor efficacy of oHSV expressing IL-12, which was associated with infiltration of T cells (CD4⁺ and CD8⁺) and M1-like macrophages into the TME.⁷ These studies demonstrate that oHSV infection beneficially modulates T cells, including T cell infiltration, for improved antitumor immunity; however, none of these studies establishes a direct link to whether enhanced T cell infiltration has any negative impact on oHSV replication or spread within the TME.

In conclusion, the above studies indicate that oHSV closely interacts with T cells, and such interactions play a critical role in shaping the immune response against both the virus and tumor cells. Although oHSV-induced T cell infiltration to the TME could be inhibitory to oHSV replication,^106,109,115 robust T cell infiltration is an integral, necessary step for an improved oHSV-mediated antitumor immunity (Figure 2). Thus, to further optimize the oHSV-induced antitumor immunity, future strategies should focus on enhancing T cell responses, which could involve the use of those therapeutics that beneficially modulate T cell activation and infiltration within the TME, such as oHSV-expressing T cell modulators, e.g., IL-12, 4-1BBL, OX40L, and checkpoint inhibitors.^{116,117,118,119}

OHSV interaction with B cells is inhibitory to oHSV activity

B cells can also interact with HSV and may limit HSV infection (Figure 3).^{120,121,122,123} B cells initially generate anti-HSV IgM antibodies that react with HSV and also present antigens to CD4⁺ T cells, leading to the generation of HSV-specific CD4⁺ T cells and the production of antiviral IFN-γ.¹²⁴ Loss of B cells increases HSV dissemination to the brain, leading to encephalomyelitis and mortality. B cell-deficient mice have a lower lethal dose 50 (2.2 × 10⁷ plaque-forming units [PFU]) than wild-type C57BL/6 mice (2.3 × 10⁸ PFU), indicating that B cells play a role in controlling HSV infection.¹²⁵ B cell interactions with DCs contribute to anti-HSV protection by recalling CD4⁺Th1⁺ memory T cells that secrete noncytolytic IFN-γ; such that in the absence of both DCs plus B cells, anti-HSV protection is lost, resulting in HSV infection and death.¹²⁶ The administration of hyperimmune sera prevents HSV encephalomyelitis-induced mortality in B cell-deficient mice, further confirming the role of HSV-B cell interactions in controlling HSV infection.¹²⁵ In response to HSV reactivation, CD20⁺ B and antibody-secreting cells infiltrate and co-localize with T cells in the HSV-infected tissues, suggesting a role of B cells in limiting HSV reactivation.¹²¹ Similarly, in a recent clinical study, oHSV CAN-3110 treatment of GBM resulted in a non-significant increase in intratumoral infiltration of CD20⁺ B cells, alongside a significant increase in infiltration of CD4⁺ and CD8⁺ T cells.⁴

Interplay between oHSV and the cell death mechanisms

Autophagy is a host defense mechanism that participates in the degradation and clearance of HSV-infected cells¹²⁷ and also contributes to viral antigen processing and presentation, leading to the generation of adaptive anti-HSV immunity.¹²⁸ Several HSV proteins contribute to escape mechanisms from the effects of autophagy. HSV Us11 inhibits autophagy through the disassembly of the TRIM23-TBK1 complex¹²⁹ or via interaction with EIF2AK2 (PKR), resulting in the inhibition of EIF2AK2-mediated EIF2S1 (eIF2α) phosphorylation,¹³⁰ and thus enhancing HSV replication.^131,132 Musarra-Pizzo et al.¹³³ demonstrated that Us11 interacts with caspase 8, leading to the induction of caspase-8-p18 cleavage, and the activated-caspase-8 further cleaves Atg3 protein, resulting in inhibition of autophagy and thus favoring HSV replication.

HSV ICP34.5 blocks autophagy in infected cells¹²⁷ through interaction with phosphorylated host protein PPP1CA/PP1α, which dephosphorylates eIF2α (Table 1),¹³⁴ and HSV inhibition of autophagy in M1, not M2, macrophages enhances HSV replication.¹³⁵ The BBD of ICP34.5 contributes to HSV-mediated autophagy inhibition,^127,136 since increased autophagy is observed in cell culture infected with HSV-1 with BBD-deleted ICP34.5.¹³⁷ Switching the ICP34.5 with its human ortholog GADD34 did not influence viral replication, e.g., NG34, an oHSV devoid of ICP34.5 and ICP6 but expressing GADD34, demonstrated similar replication efficiency as rQnestin34.5 (an oHSV expressing ICP34.5 from the nestin promoter but devoid of ICP6) in glioma cells but higher than that of rHSVQ1 (an oHSV devoid of ICP34.5).¹³⁸ Δ68H-6 (an oHSV devoid of BBD in ICP34.5) efficiently replicates in GBM stem-like cells compared with ICP34.5-deleted oHSV 1716-6, which barely replicates in GSCs.¹³⁷ However, there is a contradictory study reporting that virus-induced autophagy has no effect on HSV-1 replication, and even the absence of autophagy does not produce any major effect on the replication of ICP34.5 BBD HSV-1 mutant.¹³⁹

Cell death, either by apoptosis or necroptosis, is a critical host defense mechanism that contributes to the clearance of virus-infected cells.^140,141 HSV counteracts these cell death processes via different mechanisms. Us11 interacts with caspase 8 and promotes the accumulation of caspase-8-p18 in HSV-infected cells, and the accumulation of p18 does not induce signaling pathways associated with apoptosis (Table 1).¹³³ Likewise, pharmacological inhibition of autophagy with autophagy inhibitors, such as 3-methyladenine or bafilomycin A1, inhibits oHSV RH2 (lacking ICP34.5)-induced cell death of squamous cell carcinoma cells without affecting oHSV replication, allowing the infected cells to survive longer.^142,143

Other HSV proteins such as ICP6, ICP10, Us3, Us5 (also called gJ), and Us6 (also called gD) also inhibit apoptosis of HSV-infected cells (Table 1; Figure 5). The HSV-1 R1 subunit of ICP6 or HSV-2 R1 subunit of ICP10 binds to and blocks caspase-8, leading to inhibition of death receptor (DR) ligand (such as TNF-α and Fas ligand)-mediated apoptosis.¹⁴⁴ Both R1 subunits (ICP6 and ICP10) also inhibit TNF-induced necroptosis by preventing receptor-interacting protein kinase 1 (RIP1) from interacting with RIP3.¹⁴⁵ However, Huang et al.¹⁴⁶ reported a contradictory observation, i.e., ICP6 interaction with RIP1/RIP3 results in the initiation of necroptosis, thus restricting virus replication (Table 1; Figure 5).

The Us3 is an anti-apoptotic protein that inhibits apoptosis of HSV-infected cells via inhibition of programmed cell death protein 4 (PDCD4),¹⁴⁷ blockade of caspase-3 activation, and inhibition of granzyme B-mediated cleavage of the proapoptotic protein Bid, preventing HSV-infected cells from recognition and lysis by the MHC class I-restricted CD8⁺ T cells.¹⁴⁸ Thus, Us3 deletion promotes apoptosis and oncolysis of the HSV-infected cells.¹⁴⁹ The Us5 (gJ) protein inhibits CTL-induced apoptosis of HSV-1-infected cells through the inhibition of FasL- and granzyme B-mediated pathways.¹⁵⁰ Like Us5, Us6 (gD) also prevents staurosporine- or Fas-induced apoptosis of the HSV-1-infected monocytic cells via activation of NF-kB (Table 1).^151,152

In summary, autophagy and other cell death mechanisms are generally inhibitory to HSV-1 infection and replication. However, HSV-1 uses various mechanisms to inhibit/escape cell death mechanisms (which can also be achieved by pharmacological inhibition) (Figure 5), allowing HSV-1-infected cells to survive longer, leading to the completion of the viral replication cycle and release of more viral particles to infect the surrounding cancer cells for an enhanced antitumor effect. Additionally, in HSV-1-infected cells, the use of strategies that activate apoptosis or necroptosis pathways, which are inhibited by various HSV proteins (ICP6, ICP10, Us3, Us5, Us6, and Us11), can promote oncolysis, leading to the release of tumor and viral antigens, and the initiation of the subsequent immunological events (depicted in Figure 2).

OHSV and the cancer-immunity cycle

OHSV infection of tumor cells, oncolysis, and subsequent steps involved in the induction of antitumor immunity (Figure 2) follow the seven steps of the cancer-immunity cycle,^18,19,20 which represents a series of immunological events that occur following the application of various immunotherapies, including OVs such as oHSV.¹⁹ Understanding oHSV interaction in regards to inducing antitumor (antiviral) immunity may help improve strategies to promote oHSV-induced antitumor efficacy. A brief discussion of how oHSV drives the antitumor/anti-HSV immunity follows:

Steps A and B: OHSV infection, oncolysis, and release of tumor and viral antigens

Once oHSV is injected into tumors, it must first overcome the neutralizing effect of complement/immunoglobulins before entering cancer cells (Figure 3). Once oHSV enters the host cancer cells, it encounters various obstacles, including upregulation of innate immune signaling pathways (Figure 4), leading to the production of cytokines and chemokines that recruit innate immune cells such as NK cells, NKT cells, DCs, and macrophages (Figure 1).¹⁷ This intracellular immune response contributes to both direct anti-HSV clearance and induction of systemic immune effects (Figures 1 and 2). Although HSV infection induces antiviral immunity, studies indicate that OV-induced antiviral immunity is necessary to boost antitumor immunity.^21,22 Likewise, prior studies demonstrated that pre-existing anti-HSV immunity enhances the antitumor effects of oHSV therapy, although it is unclear whether pre-existing antiviral T cells or pre-existing humoral immunity (or both) plays a role in controlling tumor burden.^153,154 This is an interesting topic that requires further research. OHSV uses various immune evasion mechanisms to overcome the antiviral effects of the innate immune system, allowing it to complete the full replication cycle and resulting in oncolysis (Figures 4 and 5). In fact, the intratumoral administration of T-VEC results in enhanced apoptosis in the TME and a significant reduction in tumor growth, suggestive of oncolysis. The detection of gB (HSV-1 antigen)- and gp100 (tumor antigen)-specific CD8⁺ T cells indicates the release of both tumor and viral antigens following oHSV-induced oncolysis.¹⁰⁶ However, the process of oncolysis and the eventual release of tumor/viral antigens can be counteracted by several HSV proteins, such as ICP6, ICP10, ICP34.5, Us3, Us5, Us6, and Us11, which inhibit various cell death mechanisms in HSV-infected cells (Figure 2).

Step C: Antigen uptake and DC maturation

APCs, such as DCs, take up and process soluble tumor-associated (and viral) antigens, leading to DC maturation. In response to oHSV infection, mature DCs produce immunomodulatory cytokines such as IL-12 and IFN-γ, among others, further orchestrating immune effects within the TME.¹⁷ Intratumoral T-VEC treatment leads to the generation of tumor- and/or viral antigen-specific T cells, specifically cytotoxic CD8⁺ T cells,¹⁰⁶ suggestive of APC-mediated processing and presentation of both viral and tumor antigens. T-VEC also encodes granulocyte-macrophage colony-stimulating factor, which helps to recruit and mature DCs. In another study, intratumoral injection of an oHSV expressing IL-12 led to increased expression of genes associated with antigen processing and presentation within tumors and an increased number of mature DCs and CD8⁺ T cells in the spleens of virus-treated mice, suggesting that oHSV-IL12 treatment induced DC maturation and migration to lymphoid organs for antigen presentation to T cells, such as CD8⁺ T cells.¹¹³ However, HSV proteins, such as ICP34.5 (which inhibits DC maturation) and ICP47 (which downregulates MHC class I presentation), inhibit APC-mediated antigen presentation and processing, countering the sequential events of the oHSV-induced antitumor immunity (Figure 2). HSV can also induce apoptosis in DCs via activation of IRE-1α and prevent DC migration via activation of LFA-1 (Figure 2).

Step D: DC migration, antigen presentation, and T cell activation

Intratumoral oHSV treatment leads to the expression of genes associated with DC maturation and activation and CD8⁺ T cell activation, demonstrating HSV involvement in activating APCs and T cells.¹¹³ This study also suggests that the mature DCs migrated from the TME to the nearest lymphoid organ for antigen presentation to T cells, leading to T cell activation and clonal expansion and generation of HSV- and tumor-specific T cells.¹⁰⁶ However, HSV ICP0 degrades CD83,⁷⁷ and HSV ICP34.5 inhibits DC maturation through IKK and PP1α,⁸⁰ which can lead to reduced priming and activation of DCs and T cells (Figure 2).

Step E: T cell migration and infiltration to tumors

The activated tumor- and/or virus-specific CD8⁺ T cells traffic from the lymphoid organs to the tumor site,¹⁰⁶ as evidenced by an increased number of accumulated viral gB antigen-specific and tumor gp100 antigen-specific CD8⁺ T cells in the virus-treated TME compared with controls.¹⁰⁶ This is likely mediated by the induction of local chemokine gradients following oHSV infection.

HSV Us3 inhibits T cell activation by blocking TCR signaling in T cells (Table 1),¹¹² while the accumulated T cells can inhibit oHSV activity (Figure 2). The efficiency of T cell recruitment to the tumor site is more pronounced when tumors are treated with oHSV armed with a potent immunomodulatory cytokine. For instance, oHSV expression of IL-12¹⁰⁸ or IL-2⁸ leads to significantly enhanced accumulation of CD8⁺ T cells in the TME; however, neither IL-12 nor IL-2 expression showed robust efficacy.^8,108 IL-12 expression, when combined with immune checkpoint blockades (anti-PD-1 plus anti-CTLA-4), leads to robust efficacy and significant survival benefits,⁷ suggestive of oHSV-induced infiltrated T cells being counterbalanced by their expression of immunosuppressive molecules such as PD-1 and CTLA-4. Thus, to improve the oHSV efficacy and maintain the effector state of the virus-induced infiltrated T cells in the tumor site, a second line of immunotherapies may be required,⁷ as also indicated in the recently updated version of the cancer-immunity cycle.¹⁸ While this has been shown in murine models, a combination of oHSV and checkpoint blockade has not been validated in the clinic to date.

Steps F and G: T cell recognition and killing of tumor cells

Since both virus- and tumor-specific T cells are present in the TME,¹⁰⁶ it suggests that these T cells recognize viral and tumor antigens within the TME, release killer cytokines (such as IFN-γ, granzyme B, and perforin), and kill tumor cells, including oHSV-infected ones. This leads to oncolysis and subsequent release of antigens (Figure 2). This allows for antigen spread, potentially to tumor-associated neoantigens, with the cycle continuing until oHSV, tumor cells, and/or oHSV-infected tumor cells are eliminated.

In summary, by strategically manipulating or optimizing each step of the oHSV-induced antitumor immune responses, including antigen release, presentation, T cell activation, trafficking, and infiltration, we can use various strategies to enhance the therapeutic potential of oHSV. For instance, (1) Antigen release can be used to develop strategies to enhance oHSV propagation within the TME,^26,28 leading to enhanced oncolysis and release of antigens; (2) Antigen presentation enhances APC-mediated antigen presentation by incorporating immunomodulatory molecules into oHSV, such as IL-12^39,91 or CD40 agonist monoclonal antibody;⁹² (3) T cell activation, trafficking, and infiltration enhance CD8⁺ T cell activation by applying oHSV-expressing immunomodulators (such as IL-12, IL-2, and among others);^8,108 and finally, (4) T cell recognition and killing is important. HSV ICP47 protein downregulates MHC class I presentation and thereby diminishes immune recognition.⁸⁶ However, oHSVs that have deleted ICP47, such as T-VEC, can promote T cell recognition of virus-infected cancer cells.

Concluding remarks

OHSV-based immunotherapy, immunovirotherapy, is a rapidly evolving field that holds great promise to treat cancer due to its unique ability to preferentially target and destroy cancer cells¹ and synergize with other forms of immunotherapies for enhanced antitumor efficacy.⁷ The interaction between oHSV and the host immune system is complex, and a comprehensive understanding of this interaction, such as how oHSV enhances the cellular immune response in the TME and how immune cell-mediated immune effects work against tumors (and HSV), is essential to improve the therapeutic outcomes of oHSV immunovirotherapy for cancer. Thus, future studies should focus on defining oHSV-host immune interactions to maximize their therapeutic efficacy. This includes but is not limited to developing new genetically modified oHSVs to evade neutralization by host serum factors, optimizing oHSV delivery methods to bypass neutralization by pre-existing anti-HSV-1 antibodies, and using oHSV armed with immunomodulatory agents or tumor antigens¹⁵⁵ to modulate the host immune system for sustained antitumor immunity. The former two strategies are relevant to systemic oHSV administration.

Another important strategy is to promote tumor-specific virus replication.⁴ It is generally accepted that ICP34.5, although it endows robust HSV replication, induces neuropathogenicity,¹⁵⁶ and thus, many oHSVs in clinical development, including T-VEC, are ICP34.5 deleted.⁶ However, Chiocca et al. developed a unique oHSV expressing ICP34.5 under the control of the nestin promoter (CAN-3110) and tested it in GBM,⁴ a high nestin-expressing¹⁵⁷ and immunologically cold tumor.^119,158 Nestin promoter-driven ICP34.5 expression restricts oHSV CAN-3110 replication/pathogenicity to GBM cells¹⁵⁹; thus, intratumoral application of CAN-3110 resulted in improved survival without encountering serious dose-limiting toxicities in the treated patients.⁴ This nestin-driven tumor targeting is limited to nestin-expressing tumors; however, this strategy, transcriptionally targeted oHSV, is applicable to any cancer-selective transcriptional regulatory sequence.¹⁶⁰ Likewise, several studies have reported the development of receptor-targeted oHSVs, lacking any mutations except those in HSV glycoproteins necessary for targeting, thus, containing intact ICP34.5.^161,162,163 ICP34.5 deletion promotes oHSV attenuation and often reduces its replication and spread in tumor cells.¹³⁷ In contrast, oHSV with intact ICP34.5^161,162,163 may enable rapid viral replication and provoke a robust immune response, leading to early viral clearance and diminished therapeutic efficacy, as well as decreased safety. To address these challenges, repeated dosing of oHSV can be used as a treatment strategy since it has shown promise in the treatment of brain tumors.¹⁶⁴

OHSV infection and oHSV-mediated oncolysis generate both antitumor and antiviral immunity (Figure 2). Although antiviral immunity prevents oHSV replication and spread within the TME, studies suggest that oHSV-induced antiviral immunity helps to elicit clinical antitumor immunity.²² However, transient suppression of antiviral innate immune responses, such as suppression of NK cell activity^8,57 or pharmacological inhibition of innate immunoglobulins with CPA,²⁸ may help to improve oHSV-mediated treatment outcomes. Other strategies like enhancing antigen presentation and DC maturation could be crucial steps toward improving the overall efficacy of oHSV-based immunotherapies. Moreover, introducing genetic modifications in oHSV to enhance antigen presentation and T cell activation within the TME could significantly improve therapeutic outcomes.

Another strategy in which oHSV may be useful is pre-conditioning the TME before adoptive T cell or CAR-T cell therapy.^165,166 The local changes induced by oHSV and establishing chemokine gradients may enhance T cell recruitment and retention. This is an interesting strategy for improving CAR-T cell approaches in patients with solid tumors. Further, oHSV may be used to express microRNAs that modulate immune responses.¹⁶⁷ Local expression of specific microRNAs can regulate immune responses in the context of virus-induced immunogenic cell death and is another concept that awaits translational investigation. Like replicating oHSV vectors, non-replicating HSV vectors (one of which was approved by the FDA for gene therapy of skin diseases) are safe but may induce limited immune responses and result in persistent expression of therapeutic transgenes.¹⁶⁸ These features may allow non-replicating HSVs to be used for cancer therapy in a non-oncolytic mechanism; however, the optimal role of non-replicating HSV in cancer treatment and its interactions with the host immune system have yet to be determined.

The interplay between oHSV and the TME (and the immune cells) and the induction of robust antitumor immune responses underscores its potential as an in situ cancer vaccine. Overall, continued research efforts should be directed toward unraveling the complex interaction between oHSV and the host immune system. By strategically manipulating each of the immunological events after oHSV infection (Figure 2) and by mechanistically exploring the HSV-host immune cell interactions, we can further exploit the therapeutic potential of oHSV for effective tumor eradication and improved patient outcomes. This requires a multidisciplinary approach integrating virology, immunology, and clinical oncology to move the field forward in understanding the full therapeutic potential of oHSV as an effective immunotherapeutic agent against cancer.

Acknowledgments

D.S. was supported in part by a fund from the DOD (W81XWH-20-1-0702), and S.D.R. was supported in part by a grant from the NIH (R01 CA160762) and the Thomas A. Pappas chair in Neurosciences. All figures, including the graphical abstract, were generated with BioRender.com.

Author contributions

K.A. Prepared figures with BioRender.com; H.W., H.L.K., H.J.N., and S.D.R. edited the manuscript and provided relevant expertise and critical inputs; D.S. conceptualization, wrote the manuscript, edited figures, funding acquisition. All authors contributed to the article and approved the submitted version.

Declaration of interests

S.D.R. is a co-inventor on patents relating to oHSVs, owned, and managed by Georgetown University and Massachusetts General Hospital, which have received royalties from Amgen and Acti\Vec Inc., and acted as a consultant and received honoraria from Replimune, Cellinta, and Greenfire Bio, and honoraria and equity from EG 427. H.L.K. is an employee of Ankyra Therapeutics and has received honoraria for participating on advisory boards for Castle Biosciences, Midatech Pharma, Marengo Therapeutics, and Virogin.