HSV antivirals – current and future treatment options Alexander Birkmann and Holger Zimmermann

Herpes simplex virus (HSV) types 1 and 2 can cause infections with clinical manifestations ranging from benign and generally self-limiting blisters or sores as seen in labial and genital herpes through to severe and in rare cases even life-threatening infections. At present, approved treatments for herpes simplex virus are almost all nucleoside analogs. Novel antiviral approaches include therapeutic vaccines, with the most advanced having successfully completed Phase 2 clinical development. Moreover, several small molecules approaches are being developed for the treatment of genital or labial HSV infections. Of particular interest are two novel compounds (amenamevir and pritelivir) belonging to the new class of helicase-primase inhibitors with promising Phase 2 data.

Introduction
Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) are enveloped, double stranded DNA viruses. Like all members of the Herpesviridae, both types persist lifelong in their hosts following infection. Primary locations for latency are the trigeminal nerve (orofacial herpes) and the sacral ganglia (genital herpes), respectively [1]. Both viruses can reactivate at or near the site of the initial infection with results ranging from asymptomatic shed- ding of infectious virus (especially HSV-2) through to painful lesions (as recently reviewed).Clinical manifestations range from the normally benign and generally self-limiting forms such as wide spread labial herpes (mainly caused by HSV-1) and genital herpes (caused by HSV-2 and also increasingly by HSV-1) to the rare but severe and sometimes even life-threatening infections such as herpes encephalitis (HSE). The later mainly affecting either patients be- tween 6 months and 20 years of age or patients that are older than 50 years (summarized in [3]). Though not life threatening, labial and genital herpes can also exert substantial impact on patients’ quality of life as outbreaks can be both painful and stigmatizing, leading to stress and even psychosocial problems [4,5].HSV is transmitted via infectious viral particles derived from either active lesions, or (especially in the case of HSV-2) from asymptomatic shedding [6]. Primary infection typically occurs during childhood in the case of HSV- 1, or later during adolescence in the case of HSV-2, since this virus is mainly transmitted sexually.

Interestingly, the observed increasing proportion of genital herpes cases caused by HSV-1 may be attributed to a decrease in childhood HSV-1 infections and an increase in the prac- tice of oral sex [7,8].Several treatment options are available for herpes simplex infections (see the following chapter for more details). However, in contrast to the closely related varicella zoster virus (VZV), there remains no approved vaccination ap- proach, either prophylactic or therapeutic.With regard to oral antiviral therapy, two major types of treatment can be distinguished [9–11]: First, treatment of an active outbreak (primary infection or recurrence), which is mostly referred to as episodic treatment and is generally of rather short duration, for example, 1–5 days for recurrent genital herpes, 10 days for primary infection and 1 day for labial herpes episodes. Second, suppressive therapy (approved for genital herpes) meaning long-term treatment, which may theoretically require lifelong in- take of antiviral medicine.Antiviral strategiesCurrent HSV drugsThe guanosine analog acyclovir was one of the first antivirals to exhibit both high selectivity and low toxicity [12]. It is active against HSV-1 and HSV-2 as well as against VZV. Once acyclovir has been specifically phos- phorylated by the HSV thymidine kinase (TK) in infected cells, the acyclovir monophosphate is converted by cellular kinases to its active form (triphosphate) (Figure 1). Incorporation of the triphosphate form into the growing DNA chain by the viral polymerase (pol) in place of guanosine triphosphate leads to chain termina- tion and inhibition of viral replication. Acyclovir is used for the systemic treatment of HSV infections including genital and labial herpes as well as HSE and for topical treatment of labial herpes (cold sores). In order to increase oral bioavailability of acyclovir the valine ester, valacy- clovir, was developed [13]. Valacyclovir is indicated as short-term treatment for labial herpes as well as for episodic and suppressive treatment of genital herpes. It Mode of action of nucleoside analogs and helicase-primase inhibitors. Whereas the nucleoside analogs such as acyclovir need to become activated by viral and cellular kinases for inhibiting the HSV DNA polymerase, the helicase-primase inhibitors are active as such and target the viral helicase-primase enzyme complex comprising the HSV UL5, UL8, and UL52 gene products. is also the only drug to have been tested and approved for the reduction of transmission of genital herpes [14].Besides acyclovir and its prodrug valacyclovir two further nucleoside analogs are approved for HSV treatment: pen- ciclovir and trifluridine [15].

Like acyclovir, the guanosine analog penciclovir is specifically phosphorylated by the viral TK. It exhibits slightly lower activity against HSV but has a longer intracellular half-life upon phosphorylation. Since it is not orally bioavailable, penciclovir itself is only used as topical formulation for the treatment of labial herpes. As for acyclovir there is an orally bioavailable prodrug on the market, famciclovir, which is indicated for systemic treatment of labial herpes as well as for episodic and suppressive treatment of genital herpes [16]. Like acyclovir, famciclovir is also active against VZV.Three further treatment options exist for orofacial HSV infections: trifluridine, n-docosanol, and a combination of acyclovir with hydrocortisone [15,17,18]. In contrast to acyclovir and penciclovir, trifluridine and docosanol are not specific to HSV (and VZV). The nucleoside analog trifluridine, a modified form of deoxyuridine, is used to treat herpes keratitis and is also active against other viruses such as vaccinia virus and some adenovirus strains [19]. Docosanol is a saturated fatty alcohol used for topical treatment of recurrent labial herpes; its proposed mode of action is the prevention of viral envelope fusion with the host cell membrane [20].For acyclovir in combination with hydrocortisone it was shown that, in contrast to acyclovir treatment alone, the number of so called prodrome only episodes of recurrent labial herpes are increased, that is the herpes outbreak is prevented [18●]. In addition to these, there is a plethora of nonspecific treatments for labial herpes described on the internet including a range of home remedies such as for example toothpaste (http://www.mothering.com/forum/ 45-women-s-health/1034766-toothpaste-oral-herpes-im-amazed-works.html, visited December 22, 2015).Treatment with nucleoside analogs is effective at reduc- ing the time to healing of recurrent and primary episodes for both genital and labial herpes, when compared to placebo treatment [9–11]. It is important for efficacy that treatment be initiated as soon as possible, especially with regard to treatment of a new recurrence.

Ideally, treat- ment should be started in the prodromal phase as there is only a short window to control the virus while it replicates (e.g. within the first 8 h for labial herpes [21]), that is if started late, treatment has little or no effect on the outbreak [22]. Furthermore, efficacy of nucleoside ana- logs (and also with other therapies such as docosanol) is generally limited and results in only marginal improve- ments in lesion healing time or episode duration [18●].This also holds true for suppressive treatment where viralshedding and outbreaks still occur under treatment, and accordingly transmission is also only reduced by 50% [14]. Taking into account the above mentioned points, there is room for more effective therapy.In immunocompetent patients, the prevalence of nucle- oside analog resistance is low (0.3–0.7%). In contrast, the prevalence of resistant HSV infection in immunocompro- mised patients varies between 2.5% and 25% depending on the underlying illness (e.g. solid organ or hematopoi- etic stem cell transplant) and the degree of immunosup- pression [23]. Immunocompromised patients experience more frequent episodes of reactivation, prolonged dura- tion of symptoms and shedding, increased severity of infection, more extensive lesions, atypical lesions, and a greater potential for dissemination which can even become life-threatening [24]. Because of the mechanism of resistance, that is mutations in the viral TK and pol, approved therapeutic options are very limited. Intrave- nous foscarnet, a phosphonic acid derivative, is the only drug approved for this indication [25]. However, its use is limited by adverse reactions, often including renal im- pairment and necessitating cessation of therapy. Because of foscarnet’s intrinsic toxicity and the need to have patients hospitalized for administration, better tolerated and orally bioavailable therapies for patients with infec- tion resistant to nucleoside analogs are needed.HSV vaccinesIn contrast to the closely related VZV, where both pro- phylactic (chickenpox) and therapeutic (shingles) vac- cines are available, there is no vaccine for HSV infections on the market as of today.

All prophylactic vaccine approaches have failed so far, including a Phase 3 study with a glycoprotein D of HSV-2 (gD2) containing vaccine ‘Herpevac’ (GSK) that enrolled more than 8,000 HSV-1/HSV-2 seronegative women [26]. More promising results have been reported from ongoing therapeutic vaccine programs targeting the reduction of viral shed-ding and clinical lesions of recurrent genital herpes [2●]. At the time of writing this review, 5 candidates werereportedly in Phase 2 clinical development and 1 vaccine candidate in Phase 1 ([27], retrieved November 24, 2015) (Table 1). The most effective therapeutic vaccine candi- date reported is GEN-003 from Genocea Biosciences, Inc., a T-cell directed immunotherapy incorporating the viral antigens ICP4 and gD2 along with an in-licensed adjuvant [28]. The best performing dose showed statisti- cally significant and sustained reduction of HSV-2 shed- ding of 58% versus placebo after 6 months. Furthermore, depending on the dose, the proportion of patients who were lesion-free 6 months after dosing was up to 50 per- cent [29●●].Novel small moleculesSeveral new drugs are currently in development for the treatment of genital or labial HSV infections, but only two compounds comprise novel, specific, small molecule approaches ([27], retrieved November 24, 2015). Both drugs belong to the class of the so-called helicase-primase inhibitors, which were reviewed recently [30] (Figure 1). Briefly, the viral helicase-primase enzyme complex is a heterotrimer consisting of viral UL5 helicase, UL52 pri- mase, and UL8, an accessory protein without enzymatic function. It is required for DNA unwinding at the repli- cation fork and synthesis of primers during virus replica- tion. Since there is no eukaryotic homologue of the helicase-primase complex and since it is essential for viral replication, the helicase-primase complex represents an attractive target for new drug development. Furthermore, helicase-primase inhibitors do not need to become acti- vated by viral enzymes and therefore, can protect both infected and uninfected cells from infection.Amenamevir (or ASP2151), initially discovered and de- veloped by Astellas, is an oxadiazolylphenyl-containing helicase-primase inhibitor with activity against both HSV and VZV [31].

In a Phase 2 study, exploiting a classic episodic study design with time to lesion healing as primary endpoint, 437 of the 695 participants with genital herpes experienced a recurrence and thus received the study drug. The efficacy of once-daily amenamevir was comparable to that of valacyclovir administered twicedaily for 3 days with a time to lesion healing shortened by 1–2 days [32●●]. Adverse findings in a clinical Phase 1 trial led Astellas to suspend the development, though clinical development has meanwhile been resumed by Maruho Co., Ltd. in Japan in HSV (and VZV) patients with multiple orofacial or genital lesions (https://www. clinicaltrials.gov/ct2/results?term=asp2151&Search= Search, visited November 27, 2015). Clinical trials are ongoing at the time of this review.The other helicase-primase inhibitor, pritelivir (AIC316, BAY 57-1293), a thiazolylamide, was discovered by Bayer[33] and is currently in clinical development by AiCuris. To date, two Phase 2 trials have been performed, both using reduction of viral shedding in otherwise healthy people with genital herpes as primary endpoint. The first trial used a parallel group design, and demonstrated a dose dependent and statistically significant reduction of viral shedding, the amount of virus shed, and proportion of days with lesions at the best dose, 75 mg once daily. Shedding was reduced almost 90% compared to placebo [34●●]. In the second study, 100 mg pritelivir once daily was compared with 500 mg once daily valacyclovir in a crossover trial design [35]. A statistically significant re-duction in shedding rate and lesion rate (58% and 60%, respectively), could be shown for pritelivir compared to valacyclovir.

Conclusions
Several therapeutic options are available for HSV infec- tions with the nucleoside analogs acyclovir and penciclo- vir, respectively, and their prodrugs representing the only drugs specifically targeting the virus. The molecules have a common mode of action and therefore also share the same shortcomings such as incomplete suppressive treat- ment, only minimal effect upon acute episodes, and relatively fast emergence of resistance, at least in the immunocompromised host. For HSV-2 patients with genital herpes, a potential alternative may be therapeutic vaccines as data available so far suggest that they may have a sustained impact on viral shedding and, thereby, also on development of outbreaks. However, these vac- cines are specific for HSV-2 and since more new cases are caused by HSV-1, not all genital herpes patients can be protected. Furthermore, therapeutic vaccines are admin- istered iv which may limit patient acceptance and finally, at present they offer a protection of up to only 50% with regard to viral shedding and lesion development. None of these disadvantages apply to the helicase-primase inhi- bitors, currently the only new specific anti-HSV drugs in development. They are equally active against HSV-1 and HSV-2, orally bioavailable and exhibit higher potency than therapeutic vaccines by reducing viral shedding up to 90% compared to placebo. However, both therapeutic vaccines and novel antivirals remain in clinical develop- ment and they still have to successfully complete Phase 3 trials on their way to the Amenamevir market.