According to the World Health Organization (WHO), over two-thirds 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.

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, 49–54). Each of these types of vaccine approaches has its pros and cons when it comes to safety, immunogenicity, and protective efficacy.

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 ^1–7. 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 ^8–10. 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 sensing¹¹. Free and protamine-complexed mRNA were among the first approaches to provide robust antigen expression and immune-stimulation ^12–14. 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. Recently, the Food and Drug Administration (FDA) issued an Emergency Use Authorization (EUA) for the Pfizer-BioNTech COVID-19 (BNT162b2) vaccine (Pfizer, Inc; Philadelphia, Pennsylvania), nucleoside-modified mRNA vaccine formulated lipid nanoparticle- encoding the spike glycoprotein of SARS-CoV-2, the virus that causes coronavirus disease 2019 (COVID-19)⁷. This technology has encouraged other groups working on vaccines against cancer and viral pathogens to use the NLP-formulated mRNA platform. Recently, the Friedman group¹⁸ showed that nucleoside-modified 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) ¹⁹and (2) modified mRNA encoding the 3 glycoproteins formulated in lipid nanoparticles (LNP) ²⁰. The RNA was modified to increase the cellular uptake and prevent the innate immunity sensors from inhibiting the translation machinery ²¹. The mRNA-LPN vaccine demonstrated to induce effective T-follicular helper and germinal center B cell responses translated into high titers and durable antibodies responses ²² that outperform the glycoproteins-based vaccine in preventing HSV-1 and HSV-2 genital infection and in protecting mice and guinea pigs against intravaginal HSV-2 infection ²⁰.

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 cell-mediated 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.

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).

Total or partial loss of T cell function (dysfunction) occurs following repetitive HSV latent/reactivation cycles (109–111) 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 co-inhibitory 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-γ 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 (120–123). 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” (125–127). 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.

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.

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 (138–142). 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.

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.

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.

The authors alone are responsible for the views expressed in this review article, and they do not necessarily represent the decisions, policy, or views of the institutions, with which they are affiliated.

Author HV was employed by TechImmune, LLC.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.