{'fraction_non_alphanumeric': 0.03805021475310599, 'fraction_numerical': 0.02450203222737886, 'mean_word_length': 5.203862660944206, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Combinatorial Herpes Simplex Vaccine Strategies: From Bedside to Bench and Back

The development of vaccines against herpes simplex virus type 1 and type 2 (HSV1 and HSV-2) is an important goal for global health. In this review we reexamined (i) the status of ocular herpes vaccines in clinical trials; and (ii) discusses the recent scientific advances in the understanding of differential immune response between HSV infected asymptomatic and symptomatic individuals that form the basis for the new combinatorial vaccine strategies targeting HSV; and (iii) shed light on our novel “asymptomatic” herpes approach based on protective immune mechanisms in seropositive asymptomatic individuals who are “naturally” protected from recurrent herpetic diseases. We previously reported that phenotypically and functionally distinct HSV-specific memory CD8+ T cell subsets in asymptomatic and symptomatic HSV-infected individuals. Moreover, a better protection induced following a prime/pull vaccine approach that consists of first priming anti-viral effector memory T cells systemically and then pulling them to the sites of virus reactivation (e.g., sensory ganglia) and replication (e.g., eyes and vaginal mucosa), following mucosal administration of vectors expressing T cell-attracting chemokines. In addition, we reported that a combination of prime/pull vaccine approach with approaches to reverse T cell exhaustion led to even better protection against herpes infection and disease. Blocking PD-1, LAG-3, TIGIT and/or TIM-3 immune checkpoint pathways helped in restoring the function of antiviral HSV-specific CD8+ T cells in latently infected ganglia and increased efficacy and longevity of the prime/pull herpes vaccine. We discussed that a prime/pull vaccine strategy that use of asymptomatic epitopes, combined with immune checkpoint blockade would prove to be a successful herpes vaccine approach.


INTRODUCTION
According to the World Health Organization (WHO), over twothirds of the worldwide population in infected with HSV-1 (commonly known to cause oral herpes or cold sores) and HSV-2 (commonly known to cause genital herpes) (1,2). The prevalence of HSV-1 and HSV-2 is 47.8% and 11.9%, respectively, for individuals aged 14 to 49 years according to a 2018 February data brief published by the US Centers for Disease Control and Prevention's National Center for Health Statistics (1,2). In the United States alone, every year, there are 500,000 HSV-1 oral herpes cases; 300,000 HSV-1 and HSV-2 genital herpes cases; 20,000 HSV-1 ocular herpes cases and 1,500 cases of herpes encephalitis (3,4). Apart from being the most prevalent sexually transmitted disease, HSV-1 is the leading cause of infectious blindness in Western countries (5). HSV-1 and HSV-2 are neurotropic viruses that infect the anogenital, oral mucosal lining and the skin and the eyes (6) The immune response to HSV typically controls the acute mucosal infection; however, the virus remains latent in the ganglia, and there is a life-long sporadic low-grade shedding of virus from sensory neurons into the mucosa (6). Thus, while HSV hides for a lifetime in the trigeminal, autonomic, or dorsal root ganglia, it reactivates and sheds asymptomatically making the transmission high. In addition to causing painful blisters, HSV-2 can cause encephalitis and death in newborns from vertical transmission and increases the risk for HIV infection two-three-fold times (7). Antiviral drugs are the only current treatment approved by the Food and Drug Administration (FDA) for treatment of herpetic diseases. Due to the cost, virus resistances and limited effectiveness of antiviral drugs, preventive or therapeutic vaccines are highly desirable to control herpes infection and/or diseases (8). The development of a vaccine that proves effective against one type of the HSV would be helpful for the other type due to the genetic similarity between HSV-1 and HSV-2. However, due to virus latency and HSV immune evasion, immunotherapy and vaccine development against the virus have become a real challenge. As of 2018, a number of different HSV vaccine candidates were at different stages of clinical trials (9, 10) ( Table 1).
One common denominator in these vaccines is the use of the whole virus or whole virus proteins, which contain both protective "asymptomatic" epitopes and pathogenic "symptomatic" epitopes. Our developed "asymptomatic" herpes vaccine approach which is based on understanding the immune mechanisms by which seropositive asymptomatic individuals are "naturally" protected from recurrent herpes disease throughout their lives. Clinical and pre-clinical studies have proved that the T cell-based immune system in the mucosa lining of the genital tract plays a crucial role in the prevention of HSV acquisition. A better mucosal vaccine approach to boost effector memory T cell responses will serve instrumental in developing an effective HSV vaccine (45). Our latest approach of using adenoviral vectors delivering chemokines and asymptomatic dominant epitopes to induce and pull antiviral CD4 + and CD8 + T cells to the site of reactivation (i.e., ganglia) and replication (i.e., epithelia) would be an effective combinatorial herpes simplex vaccine strategy. Moreover, another combinatorial herpes simplex vaccine strategy that consists of reversing T cell exhaustion by immune checkpoint blockade would be a successful strategy to clear herpes infection (46). In this review, we highlight the current clinical trials in herpes vaccine development and emphasize the significance of using the asymptomatic epitope approach in a combinatorial vaccine strategy.

HSV VACCINES: FROM PAST TO PRESENT
The success of vaccines against other alpha herpes, like the chicken-pox and shingles vaccine, has given hope for the development of a vaccine against HSV (47) ( Table 1). Four main vaccine approaches have been designed and tested in the past four decades to fight off herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) infections and diseases (48): (1) Inactivated "killed" HSV vaccines; (2) Live-attenuated HSV vaccines; (3) Replication-defective HSV vaccines; and (4) Subunit HSV vaccines (9,(49)(50)(51)(52)(53)(54). Each of these types of vaccine approaches has its pros and cons when it comes to safety, immunogenicity, and protective efficacy.

Inactivated "Killed" HSV Vaccines
HSV is a highly successful neurotropic virus that resides in the nervous system and therefore presents the risk of developing neuro-pathogenesis and life-threatening Herpes Simplex Encephalitis (HSE). Thus, back in the 70s and 80s, the first whole inactivated HSV vaccine approach used "kill" the whole virus after exposure to heat, UV-light (55) or chemicals (56,57). These whole inactivated HSV vaccines induced antibodies, but not T cells, and as such have not been successful in the protection against recurrent HSV-1 or HSV-2 infections and diseases (58)(59)(60). Therefore, the live-attenuated HSV vaccines (61-66) and replication-defective HSV vaccines were introduced (51, 58-60, 67-71).

Live-Attenuated HSV Vaccines
Live-attenuated HSV vaccines contrast inactivated HSV vaccines produced by "killing" the virus and reducing the neurovirulence of HSV-1 or HSV-2, while keeping them viable. In the past 24 years, many live-attenuated HSV vaccines have been introduced and tested in both the mouse and guinea pig models mainly in a prophylactic setting (instead of a therapeutic setting). However, due mostly to safety concerns, only a few of these live vaccines have progressed into clinical trials (63). Live-attenuated HSV vaccines include:

Replication-Defective HSV Vaccines
Replication-defective virus vaccines, also called DISC (Disabled Infectious Single Cycle) virus vaccines, are defective for one or more genes that are essential for viral genome replication or synthesis and assembly of viral particles. In normal cells, they express viral gene products but do not replicate to form progeny virions. Replication-defective HSV vaccines can stimulate immune responses but produce no progeny viral particles. However, because they do not replicate and spread in the host, replication-defective virus vaccines may be less immunogenic, specifically less T cell stimulators because they have a relatively limited capacity to solicit professional antigen presenting cells  (87). The efficacy of the HSV-2 DgD vaccine in prophylactic and therapeutic settings has yet to be evaluated in the guinea pig model of primary and recurrent genital herpes. Compared to clinical trials using adjuvanted subunit vaccines (e.g., the adjuvanted gD/gB vaccine trials), many live attenuated/replication defective vaccines-based Phase 1 trial trials, were either terminated or did not progress to Phase II, because of: (i) A lack of immunogenicity; and/or (ii) Concerns related to safety of using a live virus as vaccine, as detailed above.

Subunit HSV Vaccines
A variety of subunit HSV vaccine approaches have been developed including proteins, DNA and peptide epitope-based vaccines (88,89). Traditional protein-based vaccines are safe compared to live-attenuated and replication-defective HSV vaccines. Recombinant soluble HSV-2 glycoprotein D (gD) has been the most promising subunit vaccine that went into extensive clinical evaluation. Over the past 25 years, there has been one Phase II therapeutic genital herpes vaccine and three Phase III clinical trials of prophylactic subunit vaccines, all using the HSV-2 gD (or mixed with gB in one trial) (90)(91)(92)(93)(94)(95). Back in 1994, the first therapeutic vaccine trial delivered the gD with aluminum salt (i.e. Alum) adjuvant in 98 symptomatic genital herpes patients who reported 4 to 14 recurrences per year (96). Unfortunately, this vaccine reduced the frequency of recurrences by only 24% despite that the vaccine boosted neutralizing antibodies to HSV-2 four-fold over baseline levels (96). These disappointing results from the first therapeutic gD/Alum vaccine trial suggested that for therapeutic protection; a vaccine must: (1) Induce CD4 + and CD8 + T cell responses, in addition to neutralizing antibodies, (2) Incorporate HSV-2 antigens other than gD; and (3) Must test different adjuvants, other than Alum. Three years later in 1997, the Chiron vaccine trial used a combination of gD and gB delivered together with the MF59 Novartis' adjuvant, an oil-in-water emulsion of squalene oil, using the same target population of genital herpes patients as in the 1994 trial. This gB/gD/MF59 vaccine did not elicit T cell responses, produced high levels of neutralizing antibody to HSV-2, yet had only a 9% efficacy (94). This trial suggested that: (1) besides neutralizing antibodies, a protective vaccine must induce antiviral CD4 + and CD8 + T cell responses; (2) a therapeutic vaccine must incorporate HSV-2 antigens other than gB and gD; and (3) must test different adjuvants, other than Alum and MF59. Later, two GlaxoSmithKline (GSK) vaccine trials (one reported in 2004 and the other in 2012), used the gD protein delivered together with a different adjuvant, the 3-0-deacylated monophosphoryl lipid A (MPL), a TLR4 agonist (93) together with Alum (gD/MPL/Alum vaccine). The first trial enrolled discordant couples, who have regular partners with genital herpes, while the second trial enrolled HSV seronegative women who have multiple and random partners (93). The first trial, reported in 2004, showed a 74% efficacy against genital herpes disease caused by HSV-2 (93). Unfortunately, later, results using the same gD/MPL/Alum vaccine reported in 2012, showed only 58% efficacy against genital HSV-2 disease (13). The apparent contradictions in efficacy against genital HSV-2 disease, of the two GSK trials that used the same gD/ MPL/Alum vaccine, is puzzling. The difference in efficacy in the two clinical trials attributed to different populations enrolled in each trial (i.e. discordant couples vs. random seropositive women with multiple partners) (13). In the first clinical trial, the distinguishing feature of discordant couples was that they were a highly selected group in which the uninfected partner is potentially repeatedly exposed to HSV by the infected partner. This likely increased risk of infection and disease, hence lowering the threshold of seeing a significant effect of the therapeutic vaccine. In other words, the attack rates of HSV-2 genital disease were high among discordant couples making easy to see a significant reduction following therapeutic vaccination. In contrast, the second clinical trial that enrolled random seropositive women, with multiple lifetime sexual partners, in which the attack rate and the risk of infection and disease was much lower and hence likely raised the threshold of seeing a significant effect of the therapeutic vaccine. Regardless of the targeted population, the first GSK vaccine trial that produced 74% protective efficacy also stimulated both T cells and neutralizing antibodies (13). In 2016-2018, a Genocea vaccine trial (designated as Gen-003) used a combination of ICP4 and gD2 truncated proteins with a novel adjuvant, named Matrix M-2 (MM-2) (89). Matrix M is a saponin-based adjuvant that has a balanced B and T cell immuno-stimulatory profile. This trial reported a significant reduction of recurrent herpes lesions and genital viral shedding (90)(91)(92). This protection appeared to correlate with blood-derived antiviral CD4 + and CD8 + T cell responses (90)(91)(92). Due to ethical and practical limitations, none of the vaccine clinical trials have investigated the local tissue resident CD4 + and CD8 + T cells in dorsal root ganglia (DRG) and vaginal mucosal tissues.

MODIFIED RNA (MRNA) VACCINE PLATFORMS AGAINST HSV-1 AND HSV-2
RNA vaccines, during the current pandemic, have emerged as a versatile approach against emerging viral infections to overcome the challenges confronted with the conventional vaccine strategies [1][2][3][4][5][6][7] . mRNA is the carrier of the genetic information necessary for the endogenous proteins synthesis, it does not integrate into the genome and safely metabolized and eliminated by the cells [8][9][10] . RNA-based vaccines have been shown safe in animal models and in human clinical trials and trigger a strong innate immune response. Many strategies have been used to increase the delivery and immunogenicity of mRNA while diminishing innate immune sensing 11 . Free and protaminecomplexed mRNA were among the first approaches to provide robust antigen expression and immune-stimulation [12][13][14] . This vaccine set-up showed the ability to induce strong immunity and protective efficacy against lethal influenza or rabies viral infections in many animal models 4,15 . The first ever prophylactic mRNA-based vaccine (CV7201) in healthy human volunteers was made against rabies. This vaccine was generally safe and led to the induction of neutralizing antibody that waned one year after the first vaccination 8 . The success of mRNA vaccines has greatly benefited from the development of lipid-and polymer-based nanoparticles that protect RNA from degradation, enhanced cell uptake and improve delivery to the translational machinery. Currently, lipid nanoparticles (LNPs) are the most frequently used and effective agents for in vivo delivery of mRNA vaccines 9,16,17 18 showed that nucleosidemodified mRNA in lipid nanoparticle vaccine encoding for glycoproteins gC, gD, and gE induced strong and protective immunity against acute and latent herpes simplex virus type 2 infection in mice. Indeed, and in a side-by-side experiment they compared two vaccine platforms: (1) Trivalent gC2/gD2/gE purified glycoproteins were given with adjuvants (CpG and Alum) 19 and (2)

LESSONS LEARNED FROM PAST HSV VACCINE CLINICAL TRIALS
The vaccine clinical trials produced valuable lessons that should help improve future herpes subunit vaccines. Specifically, these trials emphasize four major gaps in our current knowledge: (1) The need to incorporate protective herpes protein Ags, other than gB and gD, in the development of a future herpes therapeutic vaccine (3); (2) The need to design a vaccine strategy that induces anti-viral CD4 + and CD8 + T cellmediated immunity (in addition to HSV-specific neutralizing antibodies) for a better protection against recurrent herpes (3). This includes exploring new adjuvants and antigen delivery systems, and (3) The need to develop a mucosal vaccine strategy that would induce strong tissue resident CD4 + and CD8 + T RM cells (beside mucosal antibodies such as IgA) that would reduce virus reactivation from latently infected dorsal root ganglia (DRG) and subsequent virus shedding in the genital tract and recurrent herpetic disease. This is because of the failure of past parenteral subunit vaccines that elicit systemic immune responses against HSV-2. Although most of these vaccine research trials have not been promising, we have gained a better understanding of the correlates of protective immunity for a therapeutic HSV vaccine, forming the platform for novel combinatorial vaccine strategies against HSV.

Phenotypic and Functionally Differential HSV-Specific Memory CD8 + T Cell Subsets in Asymptomatic and Symptomatic HSV Infected Individuals
Understanding the immune mechanisms by which seropositive asymptomatic individuals are protected from recurrent herpes disease is significantly important as exploiting it can elicit a T cellbased immune response in the mucosa lining the genital tract to prevent HSV acquisition. Recurrent genital herpes disease occurs following periodic reactivation of the virus that travels the axons of DRG neurons to re-infect the genital tract (GT), where lytic replication leads to herpetic lesions and transmission (15). In asymptomatic individuals (ASYMP) HSV reactivation never causes recurrent disease (16)(17)(18)20). In symptomatic individuals (SYMP), HSV reactivation often causes painful recurrent genital disease (17,19,21,22). Reports on HSV therapeutic vaccine trials have shown that both innate and adaptive immunity play an equal role in directing the right immune response to prevent disease by causing a low to noshedding of the virus. Our research group has explored the differential immune scenarios present in asymptomatic protected individuals that gives them the natural immunity to contain recurrence of herpes. The asymptomatic and symptomatic individuals are strikingly different in their HSVspecific CD8 T memory cell immune-profile. After resolution of primary genital herpes infection, a heterogeneous pool (in terms of anatomic distribution, phenotype and fu) of HSV-specific memory CD8 + T cells develops (23) and can be divided into three major subsets: (1) effector memory CD8 + Figure 1. Regarding anatomic distribution, effector memory CD8 + T EM cells and central memory CD8 + T CM cells circulate between lymphoid and non-lymphoid tissues, such as the DRG and GT (24). The third subset does not enter circulation, but is instead selectively retained in infected tissues, such as DRG (25)(26)(27) and GT (25,28), as a tissue-resident memory CD8 + T RM cells. These CD8 + T RM cells are poised for immediate response to reactivation from DRG (25,29) and inhibit virus replication at GT (25). T RM cells have altered T cell trafficking patterns due to the down-regulation of T cell homing molecules CD62L and CCR7 (30)(31)(32)(33)(34). The phenotypic profile of T CM cells is CD8CD103 low CD62L high CCR7 high . T EM cells are CD8 + CD103 low CD62L low CCR7 low . T RM cells are CD8 + CD103 high CD62L low CCR7 low CD11a high CD69 high (24,35,36). T CM and T EM cells, but not T RM cells, express CD103. T CM cells must proliferate and undergo differentiation for effector function (37)(38)(39)(40). In contrast, T EM and T RM cells are already differentiated and poised for immediate effector function (41). We recently discovered that most HSV-specific CD8 T cells from ASYMP individuals expressed low levels of lymphoid homing markers (CD62L low CCR7 low ), suggesting that these T cells are predominantly of a CD8 + T EM cell subset. In contrast, most HSV-specific CD8 + T cells from SYMP individuals are predominantly of T CM cell subset (42). Moreover, a decline in the number and function of memory CD8 + T cells positively correlated with severe recurrent genital disease in SYMP individuals. The critical role of antigen-specific CD8 T cells has been demonstrated in studies using various animal models (43,44). We are now beginning to appreciate the differences observed in CD8 T cell memory population in symptomatic and asymptomatic HSV infected individuals, and understand the importance of stimulating tissue-resident memory T cells for prevention of HSV infection in the mouse model (44). T cellbased immunotherapeutic strategies to treat recurrent herpes infection and disease are emerging for HSV, and our laboratory has contributed significantly towards developing human asymptomatic CD8 T cell epitopes for HSV immunotherapy (20,44,97,98). In the last fifteen years of vaccine development, we have succeeded in identifying new HLA-A2*01 restricted "asymptomatic" human CD4 + and CD8 + T cell epitopes from HSV-1 gB and gD glycoproteins and from HSV-1 VP11/12 and VP13/14 tegument proteins. Ocular herpes models using HLA-A2*01 restricted transgenic mouse and rabbits have shown that these asymptomatic human epitopes stimulated protective CD8 T cell responses (21,99,100). Presently, we are making significant headway with novel combinatorial approaches to use these epitopes as a SAPN (self-assembling protein nanoparticle) with built-in flagellin domains as a therapeutic HSV vaccine.

PRIME AND PULL VACCINES USING ADENOVIRAL VECTORS DELIVERING EPITOPES TOGETHER WITH T-CELL CHEMOKINES INTO HSV INFECTED TISSUES
Chemokines are naturally produced by our immune system and could serve as safer and reliable adjuvants (101). Memory CD8 + T cells specific for HSV play an important role in inhibiting HSV-1 reactivation from TG and subsequent viral shedding in tears that trigger the recurrent corneal herpetic disease. The CXC chemokine ligand 10 (CXCL10)/CXC chemokine receptor 3 (CXCR3) pathways are critical in promoting T cell immunity against many viral infections (102). In a "prime and pull" strategy, a topical chemokine was applied to the genital mucosa after subcutaneous vaccination to pull HSV-specific CD8 T cells and was shown to be associated with decreased  disease upon challenge with HSV-2 (103). The CXCL10/CXCR3 pathway also affects TG-and cornea-resident CD8 + T cell responses to recurrent ocular herpes virus infection and disease (104). Chemokines can also be co-delivered in a DNA vaccine for immunomodulation. Adenovirus-CCL21 transduced class I peptide-pulsed DC, and autologous DC-adenovirus CCL21 vaccines are currently in Phase I clinical trials for the treatment of malignant melanoma and stage IIIB-IV or recurrent non-small lung cancer respectively while XCL1 along with the IL-2 gene (CHESAT tumor vaccine) is in a clinical trial for neuroblastoma (101). Pre-clinical studies in HSV have shown immuno-potentiation of DNA vaccines by co-delivery of chemokines such as CCR7 ligands and IL-8, RANTES delivered to the mucosa (105,106). We are in the advent of testing multi-epitope vaccine that co-delivers chemokines using adenovirus vectors. A "Prime-Pull-Keep" Therapeutic Vaccine (PPK Vaccine) is being designed to boost Neutralizing IgG/IgA antibodies and boost the number and function of antiviral CD4 + and CD8 + T RM cells within the cervico genital muco-cutaneous (CGMC) and DRG tissues. The PPK vaccine is expected to help STOP the virus reactivation from latently infected DRG, virus shedding and virus replication in CGMC, thus curing or reducing recurrent genital herpes disease ( Figure 1).

Laser Adjuvants
As an alternative to currently used conventional adjuvants, the chemical-and biological-free laser-adjuvant offers a welltolerated, simple to produce method to enhance mass vaccination for widespread viral infections (107). Studies from our laboratory have reported that skin exposure of B6 mice with the FDA approved non-ablative fractional diode laser (PaloVia Laser), followed by an intradermal delivery of a HSV peptide vaccine, safely induced potent and sustained HSV-specific CD8 + T cells, detected in both the draining lymph nodes (DLN) and in the vaginal mucosa (VM) (108). In the vaginal mucosa of lasertreated and peptide vaccinated mice, we observed more HSVspecific effector memory CD8 T cells. Following an intravaginal HSV-2 challenge, we found decreased genital herpes lesions and increased DC infiltrates around the laser-treated skin area. These findings have important implications for the development of efficient vaccine immunization strategies against HSV-1 and HSV-2.

IMMUNE CHECKPOINT BLOCKADE COMBINED WITH THERAPEUTIC HERPES VACCINE
Total or partial loss of T cell function (dysfunction) occurs following repetitive HSV latent/reactivation cycles (109)(110)(111) and exposure to antigens is termed exhaustion (112) and is usually linked with expression of T cell co-inhibitory receptors: PD-1, TIM-3, LAG3 (CD223), TIGIT, PSGL-1, 2B4 (CD244), GITR, CTLA-4 (CD152), CD160, and BTLA (CD272) (113,114). T cell dysfunction requires two signals: (1) T cell receptor (TCR) engaged by MHC presenting an HSV epitope (113); and a (2) T cell co-inhibitory receptor (e.g., PD-1) engaged by ligand (i.e., PDL-1). In humans, latent HSV in sensory ganglia is accompanied by chronic CD8 T cell infiltrates (115). A portion of viral reactivation in sensory ganglia appears to be controlled by CD8 T cell-mediated mechanisms (111,116,117). Recently, we compared the expression levels of eight known T cell coinhibitory receptors on blood-derived HSV-specific CD8 T cells from symptomatic and asymptomatic HSV infected individuals and discovered that, HSV-specific CD8 T cells from symptomatic individuals expressed significantly higher levels of T cell co-inhibitory receptors like PD-1, LAG-3, TIM-3 and TIGIT ( Figure 1). This phenotype correlated with functional exhaustion of HSV-specific CD8 T cells in symptomatic individuals with increased virus titers and severe disease. In mice, like humans, HSV-1 latently infected sensory ganglia have chronic CD8 T cell infiltrates (118). HSV-specific CD8 T cells producing IFN-g and Granzyme B appear to suppress (or abort) induced viral reactivation in explanted mouse sensory ganglia (118,119) and may similarly reduce detectable HSV-1 and HSV-2 reactivation in vivo (120)(121)(122)(123). During acute (11 days) and latent (30 days) post-infection HSV-1infection of mice, most effector CD8 T cells from sensory ganglia simultaneously express high levels of 2 to 3 immune checkpoint receptors (e.g. PD-1 and LAG-3) (39,111,116,117). This phenotype correlated with functional exhaustion of sensory ganglia-derived CD8 T cells and increased virus reactivation from infected sensory ganglia explants (39,111,116,117). Pembrolizumab and nivolumab are the first of the anti-PD-1 pathway family of checkpoint inhibitors to obtain FDA approval for the treatment of melanoma. The FDA has also granted approval of nivolumab for squamous cell lung cancer and Hodgkin lymphoma (HL), and MPDL-3280A, for bladder cancer and non-small cell lung cancer (124). From 2014-2017, the FDA approved several different anti-PD-1 mAbs opening the field of next vogues of so-called "immune checkpoint therapy mAbs" (125)(126)(127). Blocking the PD-1/PD-L1 (128-135) pathway in animal models demonstrated an improvement in CD8 + T cell effector function against persistent viral infections (136). Recent reports show that the natural constitutive PD-L1 expression on corneal cells impacts the HSV-1 infection of corneas. Genetic deficiency in PD-L1 using B7-H12/2 mice and the use of anti-PD-L1 blocking Ab significantly enhanced HSV-1 clearance from corneas of C57BL/6 mice mediated mainly by monocytes/macrophages (137). Based on our preliminary data of PD-L1 and GAL-9 blockade, we hypothesized that blocking PD-1, LAG-3, TIGIT and/or TIM-3 immune checkpoint pathways will help in restoring the function of HSV-specific CD8 + T cells in latently infected DRG and increasing efficacy and longevity of a therapeutic herpes vaccine.

HERPES VACCINE-SAFETY EVALUATION
Safety concerns for vaccines include: (i) the potential inherent toxicities of the antigen and the adjuvants, as well as potential toxicities due to interactions of the components present in the final formulation; and (ii) the possibility that the vaccine induces inflammatory responses that may lead to undesired toxic side effects. Some adjuvants may elicit elevated levels of proinflammatory cytokines and other mediators of toxicity, irrespective of the immune response against the antigen. Preclinical standard repeated-dose toxicology studies performed in animals will identify whether intrinsic toxicity and immunotoxicity are: (i) confined primarily to the sites of injection; (ii) caused by the delivery method (i.e., the side effects are seen in both control and vaccinated animals) or (iii) caused by the intended immune responses to the vaccine (i.e., side effects occur with greater frequency and severity in vaccinated animals compared to controls). (1) Parameters for monitoring of systemic toxicity: Toxicity studies, repeated-dose toxicity studies, address the potential for systemic toxicity including, but not limited to, the systemic effects on the immune system. A broad spectrum of information should be obtained from the toxicity study, and both in-life and postmortem data should be collected. This routinely includes careful monitoring of body weight and food consumption, body temperature, histopathology, clinical chemistry, hematology, coagulation parameters and acute phase reactants. (2) Parameters for monitoring of local reactogenicity: Local toxicity studies of intramuscularly administered vaccines should preferably be conducted in animals with sufficient muscle mass, (such as rabbits) to test the full human dose of the final vaccine formulation.

CONCLUSIONS
Since most of the current HSV vaccine candidates were not promising individually in clinical trials, combinatorial vaccine approach seems to be the most appropriate in the present scenario to further advance HSV vaccine trials. Combinatorial application practically poses many problems and hence requires optimization in animal models. For example, one such approach optimized in the guinea pig model in our laboratory, is illustrated in Figure 1.
Results from clinical trials of the HSV vaccine indicate that it is essential to explore combinatorial approaches in the discovery of an effective therapeutic vaccine. Our long-term goal is to develop a long-lasting immunotherapeutic vaccine against genital herpes. HSV-specific CD8 + T cells are critical in preventing HSV reactivations from neurons of DRG and in limiting the severity of GT inflammatory lesions by reducing HSV replication (138)(139)(140)(141)(142). By harnessing the immune mechanisms active in seropositive asymptomatic individuals that make them "naturally" protected from recurrent herpes disease, we came up with a multiple-asymptomatic/protective epitope-based vaccine strategy, a promising HSV vaccine candidate when combined with other T cell-based immunotherapies like immune-checkpoint blockade or immunomodulation using various chemokines.

EXPERT REVIEW
▪ The latest failures of most of the clinical herpes vaccines indicate that immunotherapeutic vaccine against HSV should be efficient in eliciting antigen-specific immune responses that contain reactivation of the virus, to control both recurrent lesions and viral shedding. Our vaccine research approach is based on the understanding and harnessing of immune strategies that make the seropositive asymptomatic individuals "naturally" protected from recurrent herpes disease throughout their life. We realized that the best strategy for an effective HSV vaccine would be to elicit a T cell-based immune response that boosts HSV specific effector memory T cell functionalities in the mucosal lining to prevent HSV-1/HSV-2 acquisition/reactivation. ▪ Much remains unknown about the protective immune effector of herpes, however, improved knowledge of HSV immunoepidemiology, and immunopathology should help guide new vaccine strategies for HSV. In the last fifteen years of vaccine development, we have succeeded in identifying many protective "asymptomatic" human CD4 + and CD8 + T cell epitopes from HSV-1 gB and gD glycoproteins and from HSV-1 VP11/12 and VP13/14 tegument proteins. We are currently progressing with novel combinatorial approaches to use these epitopes as a SAPN with built-in flagellin domains as therapeutic HSV vaccine. A Prime-Pull-Keep Therapeutic Vaccine (PPK Vaccine) is designed to boost Neutralizing IgG/ IgA antibodies (Abs) and boost the number and function of antiviral CD4 + and CD8 + T RM cells within the cervico genital muco-cutaneous (CGMC) and dorsal root ganglia (DRG) tissues. PPK vaccine is expected to help STOP the virus reactivation from latently infected DRG, virus shedding and virus replication in CGMC, thus curing or reducing recurrent genital herpes disease. ▪ Since most of the current HSV vaccine candidates were not promising individually in clinical trials, a combinatorial vaccine approach seems to be the most appropriate in the present scenario to further advance HSV vaccine trials. Combinatorial application practically poses many problems and hence requires optimization. We are currently optimizing these combinatorial approaches in animal models. We came up with multiple-asymptomatic/protective epitope-based vaccine strategy which will be a promising HSV vaccine candidate when combined with other T cell-based immunotherapy-like immune-checkpoint blockade or immunomodulation using various chemokines.

AUTHOR CONTRIBUTIONS
AC, ND, RS, SP, P-GC, and LB: conceived and designed the experiments, performed the experiments, contributed reagents, materials, and analysis tools. AC, ND, RS, SP, P-GC, LZ, HV, HC, KH-C, and LB wrote the paper. All authors contributed to the article and approved the submitted version.

FUNDING
This work is supported by Public Health Service Research R01 Grants EY026103, EY019896 and EY024618 from National Eye Institute (NEI) and R21 Grant AI158060, AI150091, AI143348, AI147499, AI143326, AI138764, AI124911 and AI110902 from National Institutes of allergy and Infectious Diseases (NIAID) (to LB), and in part by The Discovery Center for Eye Research (DCER) and the Research to Prevent Blindness (RPB) grant.