Vaccine prospect of Kaposi sarcoma-associated herpesvirus

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Vaccine prospect of Kaposi sarcoma-associated herpesvirus Ting-Ting Wu1,2,3, Jing Qian1,3, Jian Ang1,3 and Ren Sun1,2,3 Infection of Kaposi sarcoma-associated herpesvirus (KSHV) or human herpesvirus-8 (HHV-8) is estimated to account for 34,000 new cancer cases globally. Unlike other herpesviruses, KSHV is not ubiquitous but is highly prevalent in some areas, such as sub-Saharan Africa where Kaposi sarcoma is the leading cancer among adults. While latent infection of KSHV plays a major and direct role in tumorigenesis, viral lytic replication also makes significant contributions to this process. Efforts to develop a KSHV vaccine are limited, but studies with EBV have provided important lessons. Informative vaccine research has been conducted in the mouse infection model of a closely related rodent virus, murine gammaherpesvirus-68 (MHV-68 or gHV-68). This mouse model has generated fundamental principles for an effective vaccination strategy. KSHV vaccines designed to prevent a naı¨ve host from infection and to boost the immune control of KSHV in persistently infected people will have major impact on individuals who are at a high risk of developing KSHV-associated diseases. Addresses 1 Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA 90095, United States 2 Dental Research Institute, University of California at Los Angeles, Los Angeles, CA 90095, United States 3 ZJU-UCLA Joint Center for Medical Education and Research, The Second Affiliated Hospital, College of Medicine, Zhejiang University, China Corresponding author: Wu, Ting-Ting ([email protected]) Current Opinion in Virology 2012, 2:482–488 This review comes from a themed issue on Human tumour viruses (old and new)

led to extensive epidemiological studies that strongly support KSHV as the likely cause of KS, PEL, and MCD. Here we will briefly summarize several basic aspects of KSHV and then discuss the prospect of KSHV vaccines as preventive and therapeutic strategies.

KSHV virology Like all herpesviruses, infection of KSHV has two distinct phases, known as lytic replication and latency. Latency is the hallmark of herpesviruses, characterized by limited gene expression without virion production, and is the fundamental strategy utilized by the virus to escape or evade host immune control while maintaining its genome in infected cells. Periodically, the latent virus reactivates to enter lytic replication, during which the viral genes are fully expressed in a cascade manner (immediate early, early, and late genes), leading to the production of infectious particles, often resulting in the lysis of infected cells. Shuffling between two life cycle phases allows herpesviruses to efficiently establish life-long persistent infections in hosts. Genetic analysis of the KSHV genome assigns the virus to the gamma-2 herpesvirus, which also includes herpesvirus saimiri (HVS) and murine gammaherpesvirus-68 (MHV-68 or gHV-68). A majority (>75%) of the predicted open reading frames from the KSHV sequence are homologous to those of HVS and MHV-68. These three viruses together with another human virus, Epstein Barr virus (EBV), belong to the gamma subfamily of herpesviruses, characterized by their ability to establish latent infection in lymphocytes.

Edited by Janet S Butel and Hung Fan For a complete overview see the Issue and the Editorial Available online 12th July 2012 1879-6257/$ – see front matter, # 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.coviro.2012.06.005

Introduction Kaposi sarcoma-associated herpesvirus (KSHV), also referred to as human herpesvirus-8 (HHV-8), is the most recently identified human herpesvirus. The discovery of KSHV in 1994 was made by Chang et al. [1], who found unique herpesvirus-like DNA sequences present in most Kaposi sarcoma (KS) lesions from acquired immunodeficiency syndrome (AIDS) patients. Subsequent findings indicate that the KSHV genome is also strongly associated with two types of B-cell tumors that frequently develop in AIDS patients: primary effusion lymphoma (PEL) [2] and multicentric Castleman disease (MCD) [3]. These results Current Opinion in Virology 2012, 2:482–488

During latency, KSHV expresses only a subset of genes to maintain its genome and to preserve the latently infected cells. LANA/ORF73 mediates the distribution of viral genomes into daughter cells following cell division [4] and is absolutely essential for the persistence of KSHV genome in cells [5]. Moreover, the majority of KSHVpositive cells in tumors express a latent gene program. Therefore, latency is generally thought to play a major and direct role in viral tumorigenesis. One major function for the viral genes expressed during latency is believed to enhance the growth and survival of latently infected cells, which can increase transformation potential. For example, LANA, v-cyclin, vFLIP, Kaposin A, vIRF3, and viral microRNAs, when individually expressing, are able to deregulate the control of cell proliferation and death (for review see [6,7]). LANA can interrupt several pathways that regulate cell proliferation and apoptosis [8–10], the cellular processes that are targeted by vcyclin and vFLIP as well [11–13]. vcyclin promotes cell-cycle progression of quiescent cells [11], but it also induces DNA damage www.sciencedirect.com

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responses, senescence and growth arrest [14,15] or apoptosis [16], depending upon cell types. The vcyclin-induced senescence and growth arrest can be relieved by inactivating p53 or autophagy [14,17]. vFLIP activates NF-kB signaling [18,19] and suppresses autophagy [13], both of which lead to protection against cell death [12,13]. Interestingly, the anti-autophagy function of vFLIP can rescue cells from senescence induced by vcyclin, demonstrating a coordinate regulation exploited by KSHV to ensure the survival and growth of latently infected cells [17]. The most abundant latent transcripts, T0.7 [20], encode three kaposin proteins [21], of which kaposin A is potentially oncogenic [22] and Kaposin B stabilizes mRNAs that encode a variety of proteins involved in many biological processes, such as cell growth and inflammatory responses [23–25]. The viral miRNAs are clustered in the major latency locus and from the potential targets revealed so far, they appear to regulate the host immune responses, cellular proliferation and apoptosis (for review see [26,27]). For KSHV to enter lytic replication, expression of a viral immediate early gene product RTA is sufficient [28,29]. While lytic replication eventually leads to cell death, it can contribute to tumorigenesis via indirect mechanisms [6,7]. Lytic replication with virion production can generate new infections and replenish the pool of latently infected cells, which may subsequently develop into transformed cells. In addition, expression of viral lytic genes that are capable of inducing paracrine signaling can induce angiogenesis, protect latently infected cells from apoptosis, and promote their growth [30].

Prevalence and transmission of KSHV Unlike other herpesviruses, KSHV infection does not occur ubiquitously in the general population. KSHV prevalence is low (<10%) in most areas, but is high in some Mediterranean countries (4–35%) and in Africa (30–60%) [31–33]. In endemic areas, the prevalence increases with age in children and it does not correlate with sexual behaviors in adults, clearly indicating that KSHV is mainly transmitted by non-sexual contacts [34]. However, in non-endemic areas, such as the United States, KSHV prevalence is much higher among MSM (men who have sex with men) than in the general population [35], suggesting KSHV transmission through sexual interactions or certain behaviors associated with sexual activity [36]. The exact mechanism of transmission remains to be elucidated. Saliva seems to be the major source of transmission because saliva contains KSHV more frequently and at a higher level than other body fluids [36,37]. A much less frequent route of transmission is through organ transplants [38,39]. Transmission via blood transfusion is rare but may also occur [40].

rare neoplasm affecting elderly men in certain Mediterranean courtiers, people in endemic sub-Saharan Africa, and in transplant recipients undergoing immunosuppressive therapy. In early 1980s, KS began to rise in people infected with human immunodeficiency virus-1 (HIV-1) and is one of the most frequent malignancies developed in AIDS patients. While the incidence of KS has significantly declined since the introduction of highly active antiretroviral therapy (HARRT), there are still approximately 34,000 new cases worldwide [41] and 90% of KS occurs in KSHV highly prevalent sub-Saharan Africa. Unlike most other tumors, KS is comprised of various cell types and the cells infected with KSHV are spindle cells that display endothelial markers [6,7]. In addition to proliferation, the pathogenesis of KS involves inflammation and angiogenesis, mediated by both viral and cellular proteins that are capable of inducing autocrine and paracrine signaling [6,7]. While the majority of KSHV-associated cells in KS are latently infected, viral lytic gene products can be detected in a small fraction of infected cells [42]. Furthermore, treatment by ganciclovir, an anti-viral drug that blocks KSHV lytic replication, markedly reduced the risk of KS in AIDS patients [43], supporting the role of lytic replication in KS pathogenesis. B-cell lymphoproliferative diseases

KSHV is linked to two B-cell lymphoproliferative diseases, PEL and MCD. Unlike KS and PEL where most cells in the tumor lesion are infected with KSHV, only some cells in MCD are positive for KSHV genes [44]. Among KSHV-associated lesions, MCD has the largest fraction of KSHV-infected cells that express viral lytic genes. Moreover, symptoms of MCD are associated with a high KSHV viral load in the blood [45]. Indeed, targeting KSHV lytic replication has shown some promising efficacy in treating MCD [46,47].

KSHV vaccine Interests and efforts to develop a KSHV vaccine are limited. It is often argued that there is no unmet demand for a KSHV vaccine because disease incidence in the majority of KSHV-infected people is very low. However, a KSHV vaccine will have a major impact on people that have staggering tumor risk, such as those with a high risk of HIV-1 infection, under immunosuppression, or living in endemic African areas. Moreover, while some therapeutic approaches are available to treat KSHV-associated malignancies [48,49], they are costly and not readily accessible in resource-limited countries, especially in sub-Saharan Africa where KS continues to be one of the most common malignancies among adults [50,51] and is an eminent problem for children [52].

KSHV-associated diseases

KSHV-specific immune responses

Kaposi sarcoma

Most KSHV-infected healthy individuals are able to control persistent infections of KSHV without developing diseases. An understanding of this exquisite immune

KS is the most frequent malignancy associated with KSHV infection. Before the AIDS epidemic, KS was a www.sciencedirect.com

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control may be helpful for the development of vaccines. However, the immune responses against KSHV are not extensively studied. KSHV-specific CD8 T-cell responses are found to target both early and late lytic proteins as well as two KSHV latent proteins, LANA and K12/Kaposin [53,54]. In addition, studies have shown that individuals with asymptomatic KSHV persistent infection have better CD8 T-cell responses than KS patients [55–57]. Much less is known about KSHV-specific CD4 T-cell responses. Neutralizing antibodies against KSHV infection are also induced [58]. However, the target of neutralizing antibodies has not been elucidated. The importance of neutralizing antibodies in controlling KS development is indicated by the fact that HIV-positive individuals without KS had higher neutralizing antibody responses than HIV-positive KS patients [58].

Challenges to herpesvirus vaccine development

Despite many efforts and diverse approaches, the only licensed herpesvirus vaccine is a live-attenuated virus, the Oka vaccine against varicella-zoster virus. Major challenges to vaccine development for herpesviruses include the immunologically silent nature of latency and the large collections of immune evasion genes encoded by the viruses. A live vaccine that can induce a broad spectrum of immune responses against a full repertoire of viral antigens is more likely to be effective than killed or subunit vaccine. However, safety concerns about the potential of establishing latent infection have hampered the development of a live herpesvirus for vaccination, especially as latency of EBV and KSHV is directly associated with tumorigenicity. Another major obstacle to vaccine research on EBV and KSHV is the lack of an amenable animal model for their infection. So far, EBV and KSHV can only infect primates other than humans. A New World primate species, common marmosets, was recently described to support KSHV infection. These KSHV-infected marmosets developed B-cell hyperplasia, with one of the orally infected common marmosets developing a KS-like lesion [62]. Although this KSHV primate model undoubtedly provides an excellent opportunity for vaccine research, it is not readily accessible or manipulatable, and thus it will be more likely to be used for validation rather than exploratory studies.

Lessons from EBV studies

Significant efforts have been made to develop vaccines against the other tumor-associated human gamma-herpesvirus, EBV. The ultimate prophylactic EBV vaccine is to induce sterilizing immunity that prevents EBV infection. However, as reduced EBV latent infection may lower cancer incidence and viral shedding for subsequent transmission, an EBV vaccine that can reduce the latent viral load might still be beneficial. Current EBV vaccine development is focused on the major and most abundant envelope protein, gp350, which is the primary target of the neutralizing antibodies in human sera [59]. In a phase two trial, a gp350-based vaccine significantly reduced the incidence of infectious mononucleosis (symptomatic primary EBV infection) but failed to decrease the overall infection rate [60]. It is not surprising that subunit vaccines targeting individual proteins are unable to prevent infection of complex viruses, like EBV or other herpesviruses. Nevertheless, it will be imperative to determine whether this gp350-based vaccine modifies the EBV load in the blood of individuals that became infected after immunization. A recent meeting report highlights the need, opportunities, and strategies to develop EBV vaccines [61], which can provide some important guidance to future KSHV vaccine development.

Vaccine studies in the MHV-68 mouse infection model

Mouse infection with MHV-68, a rodent gamma-herpesvirus closely related to KSHV, has been exploited as an experimental model to explore proof of principle vaccination strategies [63,64,65,66,67,68,69,70,71,72,73,74] (Table 1). The goal of these studies is to pursue a vaccine strategy that can prevent or reduce long-term viral latency (>28 days post-infection) and hence lower the tumor risk. Strategies such as subunit vaccines targeting lytic and latency-associated viral proteins, heat-inactivated virions and replication-deficient viruses reduce the level of acute infection, but have little impact on long-term latency. These results are consistent with the notion that MHV-68 is able to establish a constant latent reservoir regardless of

Table 1 Vaccination strategies tested in the MHV-68 mouse infection model. Effects Strategy

Antigen

Epitope Protein

Lytic (gp150, ORF6, ORF61, gB) Lytic (gp150, M3) Latent (M2)

Heat-inactivated virus Replication-deficient virus Replication-competent virus a

Lytic replication

Acutea latency

Long-terma latency

Reference

Reduced Reduced No effect Reduced Reduced Prevented

Reduced Reduced Reduced Reduced Reduced Prevented

No effect No effect No effect No effect No effect Prevented

[63,64] [65,66] [67] [68] [69] [70–74]

Acute latency is measured at 14 days after challenge infection and long-term latency is measured after 28 days post-challenge.

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the initial viral load [75]. The only effective vaccination strategies that prevent long-term MHV-68 latency are based on replication-competent viruses, which also protect against acute infection. These studies in the MHV-68 model have profound implications for development of KSHV and EBV vaccines: a vaccine that can reduce acute infection may not have any effect on the steady-state level of latent infection and it may be necessary to consider the vaccine approach of a live virus. In response to the safety concern about the potentially oncogenic latency of a live virus, some of the replication-competent MHV-68 viruses were engineered to be deficient in latency. Importantly, these latency-deficient viruses still afford protection [71,72,73,74], providing a novel paradigm for a live gamma-herpesvirus vaccine. One of the latency-deficient MHV-68 viruses was constructed by a dual strategy (deletion of ORF73 and constitutive expression of RTA) [74]. Like in KSHV, MHV-68 ORF73 is required for genome maintenance during latency [76] and RTA is a viral immediate early protein that is sufficient to disrupt viral latency and drive the virus to lytic replication [77]. Therefore, we constructed a latency-deficient MHV-68 virus by replacing the latency locus, including ORF73, with a constitutive overexpression cassette of RTA that ensures lytic replication. A similar dual strategy can be used to generate a latency-deficient KSHV or EBV virus for vaccination [78]. Removal of latent infection is a major, but also just the first, step towards a safe live vaccine. The next challenge will be to attenuate viral lytic replication without losing immunogenicity. A rational approach is to eliminate viral immune evasion genes. Excellent candidate viral genes for targeted inactivation include ORFs that block type I interferon responses (e.g. ORF36, ORF54) and MHC class I presentation (e.g. K3), which are required for efficient MHV-68 infection in vivo, but not in vitro [79–81]. While a primate infection model is available for KSHV, mouse infection of MHV-68 provides a cost-effective and tractable experimental system for developing proof of concept vaccination strategies.

derived from latent proteins, such as LANA/ORF73 and K12/Kapsosin, will likely increase its efficacy.

Conclusions Since the discovery of KSHV in 1994, research has been focused on the molecular virology and potential mechanisms of viral oncogenesis. Much less attention is given to development of a KSHV vaccine. Yet, vaccines against KSHV provide an affordable opportunity to prevent and treat virus-associated diseases, especially in resource-limited areas. Important information has been obtained from studies of EBV and a murine gamma-herpesvirus, but many critical scientific questions remain to be addressed. With the ongoing HIV-1 epidemic in Africa where the prevalence of KSHV is high and HARRT coverage is limited, efforts need to be ramped up to develop safe and effective vaccines that are urgently needed to protect people against KSHV-associated diseases.

Acknowledgements We apologize that we could not include many important references due to space limitations. We thank Ronika Sitapara Leang, Yoon Hoon Kim, and Jun Feng for their comments and editing. This work was supported in part by grants from National Institute of Health (CA91791, DE14153, and DE15752), UCLA Center for AIDS Research (CFAR) NIH/NIAID AI028697, UCLA Jonsson Comprehensive Cancer Center (JCCC) NIH/ NCA P30 CA016042, and from National Basic Research Program of China (2011CB504803, 2011CB504305).

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Therapeutic vaccine

Developing a therapeutic vaccine to boost pre-existing anti-KSHV immunity should be considered as a parallel goal. Several lines of evidence support the hypothesis that increased KSHV lytic replication contributes to KS development [37,82–84]. Moreover, individuals with asymptomatic KSHV persistent infection have better CD8 T-cell and neutralizing antibody responses than KS patients do [55,56,58]. Thus, a vaccine to increase the immune control of KSHV lytic replication and to decrease the KSHV viral load in people already infected may reduce the risk of KS and even reduce shedding of viruses for transmission. For a therapeutic vaccine, incorporation of epitopes www.sciencedirect.com

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