Evolution of rational vaccine designs for genital herpes immunotherapy

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ScienceDirect Evolution of rational vaccine designs for genital herpes immunotherapy Johanna Katharina Kaufmann and Jessica Baker Flechtner Immunotherapeutic vaccines have emerged as a novel treatment modality for genital herpes, a sexually transmitted disease mainly caused by herpes simplex virus type 2. The approaches to identify potential vaccine antigens have evolved from classic virus attenuation and characterization of antibody and T cell responses in exposed, but seronegative individuals, to systematic screens for novel T cell antigens. Combined with implementation of novel vaccine concepts revolving around immune evasion and local recruitment of immune effectors, the development of a safe and effective therapeutic vaccine is within reach. Here, we describe the vaccine approaches that currently show promise at clinical and pre-clinical stages and link them to the evolving scientific strategies that led to their identification. Address Genocea Biosciences Inc., Cambridge Discovery Park, 100 Acorn Park Drive, Cambridge, MA 02140, USA Corresponding author: Kaufmann, Johanna Katharina ([email protected])

Current Opinion in Virology 2016, 17:80–86 This review comes from a themed issue on Preventive and therapeutic vaccines Edited by Mark J Mulligan and Harriet L Robinson

These significantly reduce the frequency and quantity of viral shedding [7,8] and control symptomatic disease in both recently and long-term infected individuals [9,10]. However, efficacy is dependent on compliance and the reduction of symptoms and shedding is incomplete, as outbreaks still occur even when patients are on chronic therapy. In fact, antiviral therapy reduces transmission rates by only about 50% [11]. Consequently, alternative treatment options, such as immunotherapeutic vaccines that effectively prevent lesions and viral spread, are needed. Of note, this will likely require a stronger induction of effective immune responses than those elicited naturally [12]. Here, we summarize recent or ongoing approaches to develop therapeutic genital herpes vaccines by linking strategies for antigen discovery to rational vaccine design (Figure 1, Table 1).

Exploiting the proven concept of virus attenuation Live-attenuated or replication-incompetent viruses have been used historically as an effective vaccine strategy in prophylactic and some therapeutic settings. Examples include polio, chickenpox or rabies, among others (reviewed in [13]). The vaccine viruses do not cause disease, but contain the majority of viral antigenic epitopes and therefore are potent inducers of humoral and cellular immune responses.

http://dx.doi.org/10.1016/j.coviro.2016.01.021 1879-6257/# 2016 Elsevier B.V. All rights reserved.

Immunotherapy for genital herpes is an alternative to antivirals Genital herpes is a sexually transmitted disease, primarily caused by herpes simplex virus type 2 (HSV-2) [1] and increasingly by the closely-related HSV type 1 (HSV-1) [2]. More than 500 million people are infected worldwide and an estimated 23 million new infections add to the disease burden each year [3]. Most patients are not aware of their infection and are therefore prone to viral transmission during asymptomatic viral shedding [4]. In addition, genital herpes has been associated with serious disease in immunodeficient individuals and with increased susceptibility to HIV infection [5,6]. Currently, treatment of genital herpes relies on suppressive antiviral therapy with nucleoside analogs, like acyclovir. Current Opinion in Virology 2016, 17:80–86

An early example of the virus attenuation approach, that followed a proof-of-concept trial for herpes immunotherapy using subviral particles [14], is the AuRx Inc. live-attenuated ICP10DPK virus with a deletion of an enzymatic domain of the HSV-2 ribonucleotide reductase, which severely impairs viral growth. In animal models of HSV-2 infection, ICP10DPK prevented close to 90% of recurrences [15,16] and in humans with documented genital herpes, it significantly reduced the frequency of recurrences [17]. A second example is the gH-deleted, disabled infectious single cycle (DISC) vaccine. Although this vaccine induced immune responses in seronegative individuals, it failed to boost immunity and protect against disease or viral shedding in seropositive subjects in a phase 2 clinical trial [18]. Additional replication-defective mutants of HSV-2 are currently in clinical development. Of note, dl5-29 and its derivatives, deleted in the UL5 and UL29 genes, have been extensively characterized in animal models of HSV1 and HSV-2 to prepare for human trials [19,20]. Although originally developed by Sanofi Pasteur as a prophylactic www.sciencedirect.com

Vaccines for genital herpes immunotherapy Kaufmann and Flechtner 81

Figure 1

1990-1992 Chiron gD2 Phase 2

1990

1997 Skinner Vaccine Phase 2

1995

1997 Chiron gD2/gB2 Phase 2

2003-2009 GSK Herpevac Prophylaxis1 Phase 3

2000-2002 AuRx ICP10ΔPK Phase 1/2a

2000

2013-2015 Vical VCL-HB01 Phase 1/2a

2005

2001-2003 3M Pharmaceuticals Resiquimod - Topical Therapy Phase 2

2004-2005 PowderMed2 pPJV7630 Phase 1

2010

2006 GSK DISC Phase 2

2013-2017 Sanofi HSV529 Phase 1/2a

2015

2012-2015 Agenus HerpV Phase 1/2a

2015-2017 Admedus Phase 2

2014-2016 Genocea GEN-003 Phase 2 1. The only preventative vaccine included on this timeline. 2. Not included in review, no published data.

Adjuvant

Glycoprotein

Peptides

DNA

Protein

Attenuated Virus

Antibody

T cell

Current Opinion in Virology

Chronology of the progress in therapeutic vaccine development for genital herpes. The therapeutic genital herpes vaccines discussed in this review that were or are in clinical development are depicted chronologically. For each vaccine, the sponsor, phase and duration of the trial are indicated. As a reference, the phase 3 Herpevac trial is also included, although it was directed toward prophylaxis of genital herpes. Symbols indicate the composition of the vaccine (attenuated virus, entry glycoproteins, proteins, peptides, DNA, and adjuvants) and what type of immune mediators were intended to be primarily induced (antibodies and T cell responses). End dates of ongoing trials are estimated.

vaccine candidate [21], studies in HSV-1-infected guinea pigs suggest that this virus may also have therapeutic benefit in humans [19]. This candidate is currently being evaluated as both a prophylactic and therapeutic vaccine in a Phase 1 clinical trial (NCT0195212). Attenuated virus vaccines have shown promise for immunotherapy and, to date, no serious side effects have been observed in humans. However, their safe and consistent manufacturing is complex. Additional conceptual safety risks, such as mutation to a pathogenic strain or recombination of a replication-defective mutant with wild-type strains as observed with some prophylactic vaccines [13], lead the push toward vaccine alternatives with fewer inherent risks that may be simpler to produce.

Natural antibody responses uncover viral entry mediators as vaccine antigens Safety concerns and challenges associated with manufacturing whole virus vaccines have fueled efforts to identify individual viral proteins capable of inducing protective or therapeutic immune responses. Rationally, www.sciencedirect.com

surface proteins involved in viral entry and cell-to-cell spread are attractive candidates, since antibodies directed against these proteins might inhibit spread of reactivated virus, thereby reducing symptoms, or prevent primary infection and establishment of latency. Neutralizing antibody responses to natural HSV-2 infection in humans are mainly directed against glycoproteins gB, gD, and to a lesser extent to gH/gL, all key players in the viral entry process [22]. Chiron Corp. tested several vaccine candidates following this subunit strategy: the initial vaccine was comprised of a truncated gD protein adjuvanted with alum. In a clinical study, it significantly reduced the frequency of symptomatic recurrences and viral shedding by approximately a third and was the first proof-of-concept for immunotherapy of a chronic viral disease in humans [23]. The results argue that the induction of humoral immune responses, either systemically and/or locally in the genital mucosa, can mediate effective immunotherapy. An alternative therapeutic vaccine contained both gD and gB together with the squalene emulsion MF59 as adjuvant. While four Current Opinion in Virology 2016, 17:80–86

82 Preventive and therapeutic vaccines

Table 1 The status of immunotherapies in clinical testing for genital herpes Vaccine type Live-attenuated vaccines

Subunit vaccines

DNA vaccines

Topical therapy

Developer

Vaccine

Current phase a

Skinner

HSV-1 subviral particles

–b

AuRx

ICP10DPK

–

GSK c

–

Sanofi

DISC (disabled infectious single cycle) HSV529

Chiron

gD2 plus alum

–

Chiron

gG2 and gB2 plus MF59

–

Agenus

HerpV: 32 peptides + HSP70 + QS-21

–

Genocea

GEN-003: gD2DTMR, ICP4.2 + Matrix-M2

2

PowderMed Admedus

pPJV7630 ubiquinated and unmodified gD2

– 2

Vical

VCL-HB01: (gD2, VP11/12, and VP13/14 plus + Vaxfectin1)

1/2a

3M Pharmaceuticals

Resiquimod1

–

1/2a

Comments

Ref.

Reduced severity of recurrences in Phase 2 Reduced number of recurrences and illness days in Phase 1/2a Safe in Phase 1/2a, no effect on viral shedding or lesions Primarily developed as prophylactic vaccine; theoretical safety concerns; trial ongoing (NCT02571166) First proof-of-concept for viral immunotherapy in humans; induced antibodies and reduced viral shedding and lesions in Phase 2 Induced antibodies but no reduction in overall frequency of recurrences in Phase 2 Induced T cell responses and durable reduction in viral shedding rate (up to 15%) in a Phase 1/2a triald Phase 2 trial ongoing (NCT02114060); induced T cell and antibody responses; durable reduction of viral shedding (up to 58%) and lesion rates (up to 69%) e

[14] [15–17] [18] [19–21]

[23]

[24,25]

[37,38]

[39,40]

No published data Phase 2 trial ongoing (ACTRN12615000094572); induced T cell responses in Phase 1 Phase 1/2a trial ongoing (NCT02030301); no effect on viral shedding or lesion rates compared with placebo f

– [43]

Recruitment of T cells to genital mucosa; prolongation of time to first recurrence

[45–48]

[44]

a

As of January 2016. Discontinued or on hold. c GlaxoSmithKline. d Unpublished; http://www.agenusbio.com/docs/press-releases/2014/agenus-vaccine-shows-significant-reduction-in-viral-burden-after-herpvgenerated-immune-activation.php. e Unpublished; http://ir.genocea.com/releasedetail.cfm?ReleaseID=935492. f Unpublished; http://www.vical.com/investors/news-releases/News-Release-Details/2015/Vical-Reports-Top-Line-Results-From-Phase12-Trial-of-Therapeutic-Genital-Herpes-Vaccine/default.aspx. b

injections were safe, immunogenic and able to mitigate the duration and severity of the first genital recurrence post-vaccination, absolute recurrence frequency did not differ from placebo treatment [24]. Furthermore, the vaccine failed to meet clinical endpoints in two prophylactic studies [25]. A growing body of evidence suggests that cellular immune responses are an essential component of successful herpes immunotherapy. Not only are herpes virus recurrences more frequent in immunosuppressed individuals, but virus-specific CD8+ T lymphocytes, Current Opinion in Virology 2016, 17:80–86

persisting in the peripheral mucosa and genital skin after the previous herpetic lesion, reduce the frequency and clinical severity of HSV-2 reactivation and facilitate viral clearance [26,27]. Moreover, differences in pre-existing T cell responses may have contributed to conflicting outcomes of the promising phase 2, but subsequent unsuccessful phase 3 prophylactic studies with the Herpevac vaccine developed by GlaxoSmithKline, containing gD, alum and monophosphoryl lipid A [28,29]. Thus, more recent vaccine development aims at the identification of T cell antigens with therapeutic potential. www.sciencedirect.com

Vaccines for genital herpes immunotherapy Kaufmann and Flechtner 83

Systematic antigen screens identify multiple T cell antigens Immunotherapeutic T cell antigens, which do not need to be restricted to surface-exposed envelope proteins, were initially identified by assessing the interferon-g (IFN-g) responses to HSV-2 immediate early gene products in symptomatic patients [30] and to a set of HSV-2 proteins in exposed seronegative people [31]. Comparing the specificity profile of these responses may predict T cell antigens potentially protective against recurrences or even primary infection. A study by Posavad et al. identified ICP4, ICP0 as well as ICP10 as vaccine candidates [31], and a follow-up study corroborated these targets not only in peripheral blood, but also in the genital mucosa of HSV-2-infected individuals [32]. While these and other studies provide important insights into the diversity and breadth of T cell responses [33,34] and hypotheses for their application, these low-throughput assays are limited to a preselected set of test antigens. A less biased approach was employed in antigen screens performed by Corixa, which analyzed the specificity of lesion-infiltrating lymphocytes from HSV-2-seropositive subjects by IFN-g quantification, using a preselected set of 48 proteins for restimulation [35]. IFN-g responses in CD4+, but not CD8+ T cells correlated with viral shedding and recurrence rates, emphasizing the need for substantial Th1-type responses in therapeutic herpes vaccines [36]. Similarly, Agenus Inc. is developing HerpV, a vaccine comprised of 32 HSV-2 peptides, complexed with an HSP70 chaperone and QS-21 adjuvant; peptides were selected based on algorithms predicting HLA binding, synthesis feasibility, and proteasomal processing. Although the selected peptide epitopes originate from proteins spanning all classes of herpes proteins (immediate early to late and capsid to envelope proteins), the algorithms were not entirely unbiased, since 32 proteins were rationally chosen as input for the in silico analysis [37]. HerpV significantly induced CD4+, and at a lesser frequency, CD8+ T cell responses to HSV-2 antigens in humans, validating the computational approach [38]. In a recent clinical trial, HerpV was reported to have reduced viral shedding in HSV-2-infected subjects by approximately 15% after boost immunization (Agenus; URL: http://www.agenusbio.com/docs/ press-releases/2014/agenus-vaccine-shows-significantreduction-in-viral-burden-after-herpv-generatedimmune-activation.php). The most comprehensive T cell antigen screen performed to date, unlimited by HLA haplotype and with comprehensive coverage of the entire HSV-2 proteome, was developed at Genocea Biosciences by combining (i) bacterial libraries of all proteins predicted to be expressed by HSV-2 with or without listeriolysin O to facilitate antigen presentation to CD4+ and CD8+ T cells, respectively, with (ii) an in vitro screening platform that utilizes www.sciencedirect.com

autologous antigen-presenting cells and T lymphocytes [39]. Comparisons of antigen responses between symptomatic and asymptomatic or exposed seronegative subjects helped identify and prioritize both previously identified and novel T cell antigens. Protein subunits of two of these were formulated with the adjuvant MatrixM2 to form the vaccine candidate GEN-003 [40]. In therapeutic guinea pig models, GEN-003 significantly reduced both lesions and viral shedding [40]. At the optimal dose in a phase 1/2a trial (NCT01667341), viral shedding and symptoms in HSV-2-infected subjects were reduced by 52% and 65%, respectively. Furthermore, all measures of immunity increased, including CD4+ and CD8+ T cell and IgG responses to both antigens, as well as virus-neutralizing titers (JB Flechtner et al.., in review). A second phase 2 dose optimization study is in progress (NCT02114060). While several of the whole virus and subunit vaccines show promise as immunotherapies, thus far, patients’ symptoms have not been completely suppressed. Immune responses elicited by a therapeutic vaccine likely need to exceed a threshold well above responses normally occurring in infected subjects to substantially impact disease [12]. To this end, classic vaccine approaches need to be combined with novel, rationally designed strategies to significantly boost immune responses.

Novel strategies may boost immune responses One approach to enhance the effectiveness of a therapeutic vaccine for genital herpes is to prevent immune evasion from complement neutralization and antibodydependent cytotoxicity. By adding the respective immune evasion proteins gC and gE to other antigens, an antibody response elicited against these glycoproteins may (i) prevent gC shielding of complement factor C3b, disinhibiting the complement cascade, and (ii) sterically hinder gE from binding IgG Fc domains that recognize other surface proteins like gD [41,42]. A proof-of-concept study in mice has fueled further exploitation of this approach [42]. Alternatively, DNA rather than protein can be used to boost immune responses. The combination of codonoptimized DNA constructs encoding both ubiquinated and unmodified gD aims at a balanced induction of antibody and T cell responses [43]. After providing a high level of protection against establishment of ganglionic latency in mice, this candidate is now being clinically investigated as a therapeutic vaccine by Admedus Ltd. (ACTRN12615000094572). Similarly, the therapeutic potential of a plasmid DNA vaccine encoding gD and the tegument proteins VP11/12 and VP13/14 is being evaluated; the latter are both inducers of robust CD8+ T cell responses [44]. Formulated with the cationic Current Opinion in Virology 2016, 17:80–86

84 Preventive and therapeutic vaccines

lipid-based adjuvant Vaxfectin1, the vaccine reduced viral shedding and symptomatic recurrences in guinea pigs [44]. However, a safety and efficacy trial in HSV-2seropositive subjects, sponsored by Vical, failed to meet its primary endpoint (Vical; URL: http://www.vical.com/ investors/news-releases/News-Release-Details/2015/ Vical-Reports-Top-Line-Results-From-Phase-12Trial-of-Therapeutic-Genital-Herpes-Vaccine/default. aspx).

School, Boston, MA) for their helpful discussion of the manuscript. Thank you to Jessica Klepac (Spectrum Science, Washington, DC) for help with the figure. We apologize to all colleagues whose work could not be cited owing to space and reference limitations.

Lastly, efforts to induce or enhance local T cell responses in the genital mucosa are being revived. The immunostimulatory compounds Resiquimod and CpG motif-containing oligonucleotides successfully reduced recurrent lesions when applied topically in mice and guinea pigs [45,46]. Resiquimod also prolonged the time to first recurrence in infected patients [47]. Currently, a ‘prime and pull’ strategy is being optimized in animal models, in which topical application of chemokines recruits T cells to the genital tract, that have been stimulated previously through a conventional immunization [48]. Although regular topical administration of an immunotherapeutic may be difficult to implement in humans, the concept of enhancing local immunity is important to consider.

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Remaining hurdles for effective immunotherapy of genital herpes A therapeutic vaccine for genital herpes would improve millions of lives. We have described several promising approaches directed toward this goal and likely, combinatorial targeting of both B and T cells is paramount. Further characterization of local immune responses and the identification of correlates of protection that are translatable from animals to humans will greatly facilitate improved vaccine design. Recently, a workshop held at the National Institute of Allergy and Infectious Diseases noted that only a combined effort of academic, industrial, and governmental collaborations can enhance the development of effective herpes vaccines; the group agreed upon several recommendations with respect to improved animal models, vaccine manufacturing, and human trial design [5]. Together with the establishment of appropriate objective measures of clinical benefit, their implementation will accelerate the successful development of safe and effective genital herpes vaccines.

Conflict of interest

References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as:  of special interest  of outstanding interest

5. 

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7.

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Drs. Flechtner and Kaufmann are paid employees of Genocea Biosciences with equity ownership. Dr. Flechtner is an inventor on multiple issued and pending patents.

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Acknowledgements

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We would like to thank Emilio Flano, Sybil Tasker (both at Genocea Biosciences, Cambridge, MA) and Darren Higgins (Harvard Medical Current Opinion in Virology 2016, 17:80–86

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Vaccines for genital herpes immunotherapy Kaufmann and Flechtner 85

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47. Spruance SL, Tyring SK, Smith MH, Meng TC: Application of a topical immune response modifier, resiquimod gel, to modify the recurrence rate of recurrent genital herpes: a pilot study. J Infect Dis 2001, 184:196-200. 48. Shin H, Iwasaki A: A vaccine strategy that protects against genital herpes by establishing local memory T cells. Nature 2012, 491:463-467.

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