Strategies By Herpes Simplex Virus To Evade The Host Immune System
Herpes simplex virus (HSV) is a large, double stranded DNA virus that infects many tissues of the human body (Whitley and Roizman, 2001). Most common are the oral cavity and genital regions, where it causes cold sores and genital lesions, while less common tissues are the eyes and the brain, where it can cause much more serious diseases, like blindness and encephalitis (Whitley and Roizman, 2001; Koyanagi et al, 2017). It is unique in that it infects, replicates, and causes disease in mucosal cells during a lytic cycle before migrating to sensory neurons in the central nervous system to become latent, until periodic reactivation (Paladino and Mossman, 2009).
The virus produces multi-functional proteins, designated infected cell proteins (ICP), that enable it to carry out many functions during infection, latency, and transmission, while maintaining a compact genome (Whitley and Roizman, 2001). These functions include evasion mechanisms that allow HSV to be very widespread across the world. The virus is capable of evading the host immune system so well that it is often asymptomatic, and therefore, easily spread from person to person through close contact with bodily fluids. This review article will touch on some strategies HSV uses to evade the host immune system during both its lytic cycle and latent phase, including disruption of the interferon response, interaction with T cells and dendritic cells, use of vesicles, and epigenetic modulation, as well as vaccines and therapeutic uses for the virus.
Evasion of Interferon Response
The interferon response is one of the first immune responses to a viral infection. It is a rapidly induced pathway that involves the release of interferons (IFNs) from virally infected cells. Although any virally infected cell can produce IFNs, it has been shown that macrophages contribute the majority of IFNs in a viral immune response, due to the fact that they are likely one of the first immune cells that would encounter a virus (Paladino and Mossman, 2009). IFNs are molecules that signal a viral infection and stimulate the transcription of interferon stimulated genes (ISGs) in surrounding non-infected cells, which lead to the production of antiviral proteins and other cytokines. These antiviral proteins and cytokines work to inhibit viral functions, induce apoptosis of infected cells, and activate immune cells, including CD4+ and CD8+ T cells and natural killer (NK) cells (Paladino and Mossman, 2009).
HSV produces many proteins during a lytic cycle that restrict the production, release, and function of IFNs. The viral proteins ICP0, ICP27, ICP34.5, all have a similar early function, albeit through different methods, to suppress IFN regulatory factors (IRFs). Specifically, IRF3 and IRF7 bind together to regulate the transcription of ISGs and production of more IFN. It has been shown that ICP27 works by binding to the TBK1-STING complex, in the TBK1-STING-IRF3 pathway, to inhibit binding of the TBK1-STING complex to IRF3 and activating the transcription of ISGs (Paladino and Mossman, 2009). Other viral proteins, Us11 and vhs, stimulate viral protein synthesis by binding PKR, a host protein kinase, to prevent phosphorylation of eIF-2, a eukaryotic protein initiation factor.
The normal function of PKR is to stop protein synthesis, host and viral, in an infected cell by phosphorylating eIF-2 (Paladino and Mossman, 2009). Binding of PKR to Us11 and vhs allows HSV replication and release of many infectious virions. Another example is a viral protein kinase, UL13, which promotes the expression of cytokine signaling suppressor genes, or SOCS. These SOCS genes do exactly as their name suggests, suppress the production of IFN and cytokines in an infected cell, thereby decreasing the effectiveness of the IFN response (Sato et al, 2017). All these viral proteins are instrumental in HSV evading the interferon response and causing disease within the host.
Evasion of T Cells and Dendritic Cells
Suppress T cell and dendritic cell recruitment
T cells are a major component of the immune response to foreign invaders, like HSV. They activate B cells to make antibodies, destroy infected cells, and release cytokines to recruit other immune cells. HSV has developed many strategies to interfere with immune functions associated with the T cell response during a lytic cycle, including T cell recruitment, activation, receptor signaling, and the interferon response, to evade immune detection. For there to be a meaningful T cell response, T cells must be recruited to the infection site. To combat this in viral encephalitis, HSV produces UL13 kinase which downregulates the expression of the chemokine CXCL9 which, along with CXCL10, attracts CD8+ T cells against HSV specifically to destroy the virally infected cells (Koyanagi et al, 2017).
This allows the virus to continue to reside in the central nervous system and cause encephalitis. HSV also decreases the number of dendritic cells recruited to the infected tissue, thereby decreasing the opportunity for interaction with T cells. This is done by modifying the chemokine receptor CCR7 on the dendritic cell’s surface so it can no longer bind the chemokine sent out to recruit immune cells to the infected cells, resulting in less dendritic cells in the infected tissues and less activation of T cells (Salio et al, 1999).
Inhibit T cell activation
Once a mature dendritic cell has been recruited to the site of infection and is presenting an antigen, it is now able fulfill its main function of activating a T cell. It does this by binding to T cell receptors with a compound ligand, consisting of a major compatibility complex (MHC) molecule and a bound antigenic peptide. This interaction is stabilized and intensified by co-stimulatory molecules such as, CD80, CD83, B7, and others (Murphy and Weaver, 2017). HSV overcomes this interaction in a few ways. One is by simply inhibiting the production and display of co-stimulatory molecules on the cell surface (Salio et al, 1999). ICP22 has been shown to downregulate transcription of the CD80 gene by binding to the CD80 promoter, rendering the promoter incapable of binding to the gene and starting transcription (Matundan and Ghiasi, 2019).
In another study, molecules called L particles were discovered to be able to downregulate the expression of CD83 on the surface of uninfected dendritic cells (Heilingloh et al, 2015). When HSV replicates, it produces whole virions, called H particles, and structures similar to H particles but without any viral DNA or a capsid, called L particles. These L particles cannot infect and replicate like a whole virion but are able to infiltrate uninfected dendritic cells and cause the downregulation of CD83, thereby decreasing the strength and stability of binding to T cell receptors (Heilingloh et al, 2015).
HSV also interferes with T cell activation by simply disrupting the formation of MHC and antigen complexes on the cell surface of antigen presenting cells. HSV viral protein ICP47 binds to TAP, a transporter protein associated with antigen presentation, in the endoplasmic reticulum (ER) membrane to inhibit the transfer of processed peptides into the ER and onto the MHC I molecule (Hill et al, 1995). Without functional TAP, MHC I is not be able to fully fold and be presented on the infected cell surface for recognition by CD8+ T cells (Fruh et al, 1995). This enables the virus to continue to replicate in host cells without be destroyed by cytotoxic T cells. In the formation of MHC II complexes, it has been shown that HSV decreases the expression of invariant chain (Ii), impairing the ability of the MHC II molecule to leave the ER and enter an endosome to bind antigen-specific peptide (Neumann et al, 2003). HSV also produces a glycoprotein (gB) that binds to HLA-DM and HLA-DR dimers to disrupt their binding to MHC II molecules (Neumann et al, 2003). MHC II molecules need to bind HLA-DM/DR dimers to release CLIP, a piece of Ii, and bind a correct peptide. Both strategies inhibit the ability of the MHC II molecule to bind peptide, and if the MHC II molecule cannot bind a correct peptide, then it will not be effective in activating CD4+ T cells.
Interrupt T cell signaling
After a T cell has been activated, a series of phosphorylation events occur that lead to a signaling cascade and the production of cytokines within the cell. HSV protein Us3 decreases the production of interleukin-2 (IL-2) in T cells by inhibiting the phosphorylation and ubiquitination events of host LAT, a linker for activation of T cells (Yang et al, 2015). In a normal immune response, host LAT is phosphorylated by ZAP-70, a protein kinase, after the ITAMs in the T cell have been phosphorylated themselves. This begins the production of IL-2 and other cytokines, to stimulate the differentiation and proliferation of helper T cells (Yang et al, 2015). HSV uses Us3 to inhibit the activation of helper T cells, which, in turn, inhibit the activation of B cells and production of anti-HSV antibodies.
Evasion with Vesicles and Endosomes
Produce microvesicles for protected cell-to-cell movement
Normally during a lytic cycle, HSV enters a host cell, replicates many new virions, and then lyses the cell to release the infectious virions. However, this poses a problem because the virion and its capsid glycoproteins tend to be highly immunogenic. HSV uses a different method of release through exocytosis, which is a slow release of virions from the host cell, in such a way that the virions are taken up by neighboring cells quickly, and spend less time circulating in the body (Bello-Morales et al, 2018). The virus also makes small vesicles called microvesicles, which contain viral proteins, viral DNA or RNA, or infectious virions.
It has been shown that microvesicles allow HSV to evade the immune system by being less immunogenic than free, unprotected virions, and are able to infect cell lines that had previously been immune to free HSV virions. Infection in these previously immune cell lines also occurred after the microvesicles had been mixed with anti-HSV antibodies before introduction into the cells (Bello-Morales et al, 2018). This further confirms that the microvesicles are an effective method to promote cell-to-cell spreading of HSV while not being recognized by the immune system.
Use of host exosomes and lysosomes
When a virus enters a host cell, it takes control of just about every part of the host cell for its own use. Host exosomes are no exception. HSV takes over the exosome pathway and uses it in a complementary fashion to other evasion methods discussed earlier. One example is the STING (stimulator of IFN genes) complex that is involved in the IFN response. Not only does it prevent the binding of STING to IRF3, it can also export STING out of the cell through an exosome to discourage any residual binding (Sadeghipour and Mathis, 2017).
Another example is HLA-DR bound by viral glycoprotein gD to prevent HLA-DR from binding MHC II molecules. HLA-DR binding to MHC II is needed for the proper folding of the molecule around the antigenic peptide, so HLA-DR binding to gD stops MHC II from binding a peptide. After gD binds HLA-DR, it directs it to a host exosome so that it can be exported out of the cell completely, thereby further decreasing the presentation of MHC II on the cell surface (Sadeghipour and Mathis, 2017).
A third example involves the use of a host lysosome to degrade IgG that is bound to the cell surface and endocytosed by an infected cell. During an infection, HSV directs a host cell to produce a transmembrane Fc receptor (gE-gI) which binds IgG that has already bound a viral antigen, specifically the glycoprotein gD. After gE-gI binds IgG: gD complex, the receptor and antibody are both endocytosed into a lysosome where IgG, and possibly gD, are degraded, and gE-gI is recycled back to the cell membrane (Ndjamen et al, 2014). This allows the infected cell to decrease opsonization and recognition by immune cells which would lead to neutralization of the cell. Both the use of microvesicles and exosomes are very clever ways for the virus to use host machinery to evade the immune system and continue to cause disease.
Evasion through Latency
HSV employs a unique mechanism to further evade the immune system after a lytic infection in that it becomes latent in sensory neurons, only to be reactivated later. Little is known about what induces latency, how it is controlled, and how reactivation occurs, making this topic intriguing to researchers. Recent studies have shown that there is an element of epigenetic control in transitioning the viral genome from active to silent (Cliffe and Knipe, 2008). Epigenetic control involves organizing eukaryotic DNA around histones into tightly wound, non-active states called heterochromatin or loosely wound, transcriptionally active states called euchromatin (Cliffe and Knipe, 2008). Once HSV moves to the sensory neurons towards the end of a lytic cycle, it enters the nucleus and begins to associate with the histones present there to modulate its own gene expression. This is due to the absence of viral protein 16 (VP-16) in the sensory neuron nucleus leading to more heterochromatin.
In a mucosal cell nucleus, VP-16 works to stop the accumulation of histones on the viral DNA so more euchromatin is available and more transcription occurs (Herrera and Triezenberg, 2004). During latency, there are few viral genes that are transcribed due to the transition to heterochromatin. These genes, also called LAT genes or latency associated genes, are the only genes that remain as euchromatin and produce any sort of transcript in the sensory neuron’s nucleus until reactivation. LAT genes have been shown to promote latency by silencing lytic genes and preventing apoptosis of infected neuronal cells (Cliffe and Knipe, 2008).
A hallmark of HSV is reactivation into a lytic cycle after becoming latent. One proposed mechanism for how HSV keeps its genome silent while also being open to reactivation is through the binding of cellular CCCTC-binding factor (CCTF) to LAT genes. Binding of CCTF acts as a heterochromatin protector and helps stop the LAT gene from being wound around histones and becoming inactive (Lee et al, 2018). This creates a seemingly perfect balance of “poised latency”, a term coined by Lee et al, the group of researchers that discovered CCTF, to describe the state of HSV in neuronal cells that is both latent and ready to be reactivated at any time (Lee et al, 2018).
Although the exact mechanism of reactivation is unknown, it is widely accepted that there are many actions that can spark reactivation. Most of which are under the umbrella of immune suppression due to stress, other disease, surgery recovery, and menstruation (Suzich and Cliffe, 2018). A mechanism has been proposed that emphasizes a multi-phase pathway to reactivation using immune stress pathways, like the JNK pathway, and interruption of neuronal cell signaling pathways (Cliffe and Wilson, 2017). This mechanism is very intriguing; however, more investigation needs to be done to determine the exact cause for reactivation.
Vaccines and Therapeutic Uses
Preventative and suppressive vaccines
HSV is a widespread and highly ubiquitous disease, and many efforts to produce vaccines to prevent, cure, or suppress the disease have been investigated. As of 2016, there are roughly 15 vaccines contenders, with few in clinical trials and most at a pre-clinical stage (Johnston et al, 2016). HSV vaccines employ a wide range of strategies and antigens to elicit a productive immune response against this elusive virus. One example is a vaccine that uses three viral glycoproteins linked to CpG and alum adjuvant to stimulate more robust antibody production, compared to those produced naturally against the same viral surface glycoproteins (Hook et al, 2018). Another is a vaccine that uses nanoparticles to shuttle a CpG adjuvant linked to two peptides closely resembling the epitopes of HSV glycoproteins (Kopp et al, 2019). The nanoparticle portion comprised of calcium phosphate, which is readily taken up by cells, helps get the vaccine into cells. Once inside, the peptides are processed and displayed to other immune cells. It has been shown that the vaccine is efficient at generating antibodies that inhibit cell-to-cell spreading of the virus (Kopp et al, 2019). A third example is a vaccine, in Phase II of clinical trials, comprised of glycoprotein gD and viral protein ICP4 linked to a Matrix M2 adjuvant. It is one of the furthest along in the process and has been found to be safe and effective at reducing the shedding of the virus and the reoccurrence rates of the lesions in a randomized trial (Bernstein et al, 2017).
Therapeutic use in cancer treatments
The use of viruses as cancer treatment tools, or oncolytic viruses, has been in the works for many years, and just recently has gotten to the point where they are approved for use on patients. Scientists are trying to employ the evasive nature of HSV to help with the delivery of cancer treatments directly to the source without running into the immune system on the way. Interestingly, the first oncolytic virus to be approved by the FDA is a modified HSV used to treat advanced melanoma (Wang et al, 2018). HSV was modified through deletions of the viral proteins ICP34.5 and ICP47 and the addition of GM-CSF, granulocyte-macrophage colony-stimulating factor.
The deletion of ICP34.5 ensures that virus replication will only occur in cancer cells. ICP34.5 is crucial in keeping protein synthesis running in infected cells by inhibiting the host cells response to viral invasion, so the modified virus will be unable to infect healthy cells. However, in cancer cells, that function is not necessary as the viral invasion response is not present. The deletion of ICP47 is needed to keep TAP functional, thereby ensuring the presentation of MHC molecules and the stimulation of an immune response on the cancer cells (Wang et al, 2018; Johnson et al, 2015).
Conclusion and Future Directions
In summary, HSV has evolved many strategies to evade the host immune system and cause disease efficiently and effectively. During a lytic cycle, HSV can interfere with the interferon response, the interaction between T cells and dendritic cells, and the activation of T cells, and can utilize microvesicles and host endosomes to facilitate movement. Its unique characteristic of latency and reactivation using histone modification allows the virus to remain undetected in sensory neurons before reappearing in mucosal cells.
Although more research needs to be done to fully understand how latency works. Some vaccines have been created to prevent new infections and cure or suppress current ones, and there has been an increase in the use of HSV for cancer treatments, including the first FDA approved oncolytic viral treatment. For more vaccines and therapeutic uses to be developed there needs to be continued investigation into the exact mechanisms of the virus latency and reactivation, as well as into the possibilities of combination therapy involving medications and vaccines.
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