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Arvin A, Campadelli-Fiume G, Mocarski E, et al., editors. Human Herpesviruses: Biology, Therapy, and Immunoprophylaxis. Cambridge: Cambridge University Press; 2007.

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Human Herpesviruses: Biology, Therapy, and Immunoprophylaxis.

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Chapter 72Epstein–barr virus vaccines

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1 and 2.

1 Division of Virology, Department of Cellular and Molecular Medicine, School of Medical Sciences, University of Bristol, UK
2 Tumour Immunology Laboratory, Division of Infectious Diseases and Immunology, Queensland Institute for Medical Research, Herston, Australia

Introduction

Primates and their γ-herpesviruses enjoy a largely peaceful coexistence where a balance of power has been reached over evolutionary time. Coevolution probably began before primate speciation and has allowed these viruses to develop sophisticated systems for the evasion of host immune responses. As a consequence, herpesvirus vaccines have been especially difficult to design because of viral latency, persistence, and immune modulation. Epstein–Barr virus (EBV) persists for the life of the individual in the face of a range of antibody responses, some of which are virus-neutralizing in vitro and a multitude of cell-mediated responses, including viral-specific CD8+ T-cells, CD4+ T-cells and NK cells. At least 95% of the adult population is infected with EBV and, for the vast majority, there are no clinical consequences whatsoever and an asymptomatic carrier state is maintained. It is not clear whether advantages are conferred to humans by lifelong EBV infection, but it is possible that some immunological effects, such as bias of the T-cell receptor repertoire are provided on a population-wide basis. Whether unselective mass vaccination of healthy individuals to prevent or modify EBV infection may cause more problems than it would solve must be considered.

M. A. Epstein first put forward ideas on the development of EBV vaccines in 1976. These original proposals were based on the notion that vaccination might prevent EBV infection and break the link in the complex chains of events that lead to EBV-associated disease. Since that time, a better understanding of EBV biology has led to the elaboration of more sophisticated vaccination strategies. Presently, it seems most unlikely that vaccination of any kind will achieve sterilizing immunity against herpesviruses. The murine γ-herpesvirus, MHV68, establishes the same steady-state levels of lytic and latent infection whatever the route of infection or dose. It may be that a single virus particle successfully infecting a single target cell will be enough to establish persistent infection in a susceptible subject (Tibbetts, 2003). The goal of EBV vaccination is the prevention of disease and not of infection. Vaccination that could modify infection, or at least the subsequent immunological status of the infected person with respect to EBV, may prevent or minimize disease. It should be noted that EBV will not have evolved to evade vaccine-induced immune responses where they are qualitatively and/or quantitatively different from naturally occurring immune responses. An important precedent for herpesvirus vaccination is the attenuated varicella zoster virus (VZV) Oka strain vaccine that may not prevent infection but is able to prevent disease. More recently, the concept of therapeutic vaccination to treat EBV-associated tumors themselves has begun to emerge (Khanna et al., 2001; Ong et al., 2003; Khanna et al., 2005).

An understanding of the life cycle and cellular habitats of EBV should be an essential prerequisite in the rational design of EBV vaccines. Unfortunately, the biology of EBV in vivo remains poorly understood and the various approaches to EBV vaccine design discussed below are, of necessity, based on a number of unproven assumptions. EBV is an orally transmitted infection and is able to infect B-cells travelling in the circulation in the oropharynx or resident in lymphoid tissue in this region. The point of infection is presumed to be the oropharyngeal epithelium but the identity of the primary target cell is not clear. It has not been possible to convincingly demonstrate the presence of EBV in oropharyngeal epithelial cells in vivo. Nevertheless, it has been shown recently, using polarized tongue and orophayngeal epithelial cells in vitro, that EBV-infected donor cells in saliva are very efficient at infecting recipient epithelial cells at their apical surface by cell–cell contact. However, these same epithelial cells are refractory to infection with free virus at their apical surface. It is also shown that neighboring epithelial cells are infected by cell–cell transmission and free virus is produced at both the apical and basolateral epithelial surfaces (Tugizov et al., 2003). Presumably, it is the latter cell-free virus that subsequently infects B-cells circulating within the oropharyngeal epithelium and oropharyngeal lymphoid tissues.

EBV infects and transforms B-cells in vitro and six EBV nuclear antigens, EBNAs 1 to 6 and two viral latent membrane-proteins, LMP1 and 2, expressed in the transformed B-cells are responsible for their changed growth and phenotype. These latency antigens potentially offer a range of targets for vaccine-induced cell-mediated immune responses. Healthy seropositive individuals carry CD8+ T-cells that are specific for epitopes in the latent antigens, in particular EBNA3, 4 and 6. However, EBV gene expression in infected B-cells in vivo is quite different and the growth-transformed phenotype has only been detected in the B-cell follicles of tonsils while in peripheral blood, EBV is detected only in a very small number of resting memory B-cells when EBV gene expression is restricted to LMP2 or does not occur at all (Thorley-Lawson, 2001).

A normal healthy immune response appears to be essential in maintaining the asymptomatic carrier-state as demonstrated by the occurrence of post-transplant lymphoproliferative disease (PTLD) in immunosuppressed organ transplant recipients and Non-Hodgkin’s B-cell lymphomas in AIDS patients whose immune systems are seriously impaired. This strongly indicates that some aspects of EBV infection are under immune control and that vaccine-induced immune responses may be able to regulate primary infection and/or modify subsequent persistent infection. Most primary infections occur in the first few years in life and any prophylactic vaccine to control EBV diseases must allow for this. It would be a daunting task to deliver an EBV vaccine to large populations in Africa and China where primary infection occurs soon after birth.

Each EBV-associated disease probably arises for a complex set of different reasons and each may require a different vaccination strategy. Present approaches can be divided into those that seek to prevent or modify infection and those that might be used therapeutically to direct existing or de novo immune responses against the EBV-associated tumors. The therapeutic approach may prove to be particularly difficult, as, despite extensive efforts over a number of years, no tumors to date have consistently been controlled in humans by vaccine-induced immune responses. Modification of EBV infection may yet prove to be the correct approach and an interesting parallel may be drawn with a recent trial of a human papillomavirus (HPV) virus-like particle vaccine. In a trial involving 2392 young women, no cases of HPV 16 infection or cervical intraepithelial neoplasia were detected in vaccinated women in contrast to the control group (Koutsky et al., 2002). Immunotherapeutic approaches to the treatment of cervical carcinoma have so far been unsuccessful. The logistics and costs associated with mass HPV vaccination of populations at risk will probably exceed those likely to be incurred with a prophylactic EBV vaccine and would at least set an important precedent if adopted as a public health measure.

Infection by more than one strain of EBV in both healthy and immunocompromised individuals appears to be unexceptional (Walling et al., 2003). This raises difficult questions about the induction of immunity by EBV infection itself and about what vaccine-induced immune responses could realistically be expected to achieve. Infection by more than one strain simultaneously from one donor would not rule out the presence of subsequent virus-induced protective immune responses. However, if a second strain were able to infect an individual following an earlier infection with another strain, then it would suggest that the second EBV strain is able to evade the broad spectrum of humoral and cellular immune responses induced by the first EBV infection. A precedent for this situation has been found earlier with cytomegalovirus (Bale et al., 1996). Another consequence of the existence of several strains of EBV is that strain differences will have to be incorporated into any prophylactic or therapeutic vaccine formulations that contain elements that differ between strains.

Since EBV infection is almost completely non-permissive in vitro, it is still not technically feasible to produce EBV itself on a large enough scale to support even a small vaccine trial where killed or attenuated forms could be used. Moreover, since EBV has been formally classified as a Grade I carcinogen (Ablashi et al., 1997), and a number of its genes can independently transform certain cell types, the use of killed, attenuated or recombinant EBV vaccines can probably be ruled out for the time being. The possible composition of an EBV vaccine must be restricted to viral components that are non-transforming, however, this need not exclude non-transforming derivatives of viral transforming gene products such as synthetic peptides.

EBV causes infectious mononucleosis (IM), post-transplant lymphoproliferative disease (PTLD) and is associated with undifferentiated nasopharyngeal carcinoma (NPC), certain types of Hodgkin’s lymphoma (HL), certain T-cell lymphomas, a subset of gastric carcinomas, and endemic Burkitt’s lymphoma (BL). It is conceivable that a prophylactic vaccine or a postinfection vaccine that could modify but not prevent EBV infection could reduce the incidence of all diseases associated with EBV. The fact that increased antibody levels against virus capsid antigen (VCA) are prognostic for both BL and NPC suggests that immune intervention prior to the onset of disease could be beneficial. The success of such approaches will depend on whether the apparent reactivation of EBV is associated with the cause of NPC or is simply a consequence of tumor development.

Figure 72.1 illustrates the possible targets for vaccine-induced immune responses. These include free virus itself that might be susceptible to inactivation by vaccine-induced circulating or mucosal neutralizing antibodies. Naturally occurring virus-neutralizing antibodies are generated mainly against the major envelope glycoprotein, gp350 and much early work in EBV vaccine development focused on producing recombinant forms of this molecule (Morgan, 1992). Gp350 is also a target for cell-mediated responses (Khanna et al., 1999a,b,c; Wilson et al., 1999; Adhikary et al., 2006). Other lytic cycle products such as gp85, gp42, the gB homologue and the products of the BZLF1, BMRF1, BDLF3 and BILF2 open reading frames have not been investigated in the context of vaccine development. CD8+ T-cell responses against BZLF1 predominate in healthy seropositives and vaccine-induced immune responses against BZLF1 in EBV seronegatives might have a role to play. It is not known how many EBV-infected B-cells enter the lytic cycle or the location of the cells that do. Very few B-cells infected and transformed with EBV in vitro enter the lytic cycle, being less than 5%. Since free virus is shed in the oropharynx, it is assumed that epithelial cells and/or B-cells in this region are responsible and might therefore be regulated by immune responses. It must be assumed for the time being that vaccine-induced immune responses against lytic cycle products would act on these cells and/or the virus they produce. The increased oral shedding of EBV in immunosuppressed patients is consistent with this view (Preiksaitis et al., 1992). Current strategies to control EBV disease by vaccination are described below, while adoptive T-cell immunotherapy for these diseases is described elsewhere in this volume.

Fig. 72.1. Potential targets for EBV vaccine-induced immune responses.

Fig. 72.1

Potential targets for EBV vaccine-induced immune responses. In addition to the virus itself there are at least six different potential cellular targets for vaccine-induced immune responses. (a) Resting memory B-cells in which only LMP2 transcripts have (more...)

Vaccines to prevent infectious mononucleosis

In the wealthier Western societies primary EBV infection is often delayed until adolescence whereupon it gives rise to IM in 30% and 50% of individuals. It is still an open question as to why the remaining 50% to 70% of individuals become infected without symptoms or disease. However, it has been found that asymptomatic individuals display broad expansions of their TcR repertoire, while acute IM donors show oligoclonal expansion of TcR families (Silins et al., 2001). Perhaps of even greater interest is the small minority who apparently never become infected. A higher production of IFNα and IL-6 and a greater number of monocytes were detected in cultures of peripheral blood lymphocytes from EBV-seronegative adults (Jabs et al., 1996). The genetic and immunological differences between EBV seropositives and seronegatives remains obscure but would be informative as far as vaccine design is concerned. Since these immunological parameters are unknown, a vaccine cannot be designed on an entirely rational basis. IM almost always resolves itself over a relatively short time and is only very rarely fatal. The question arises as to whether large-scale vaccination of otherwise healthy children is justified when set against the actual risks of illness and its socio-economic consequences.

Which stages in the process and maintenance of EBV infection are susceptible to control by vaccination? What target antigens need to be recognized by vaccine-induced immune responses to prevent IM? The differentiation pathways of EBV-infected cells, the types of B-cell that are infected and the EBV proteins expressed in vivo have been tentatively identified and include a memory B-cell that may only express LMP2, a proliferating and activated B-cell expressing the full panel of EBV latent genes in the growth program or Latency Ⅲ, and B-cells in Latency Ⅱ which may subsequently generate the memory reservoir B-cells (Thorley-Lawson, 2001). Cells in Latency Ⅲ have only been found in the germinal center of lymphoid follicles and these are regions in which cytotoxic T-cells (CTL) are poorly represented probably because EBV-specific CD8 T-cells lack homing receptors for lymphoid infection sites (Chen et al., 2001). CD4+ T-cells are detectable at low frequency within B-cell follicles and may, therefore, interact directly with EBV-infected B-cells at this site. It is possible that CD4+ T-cells primed by gp350 or latency antigen vaccination would become reactivated on viral challenge (Adhikary et al., 2006). Such cells could influence the course of IM by inducing apoptosis of EBV-infected B-cells within infected lymph nodes and by down regulating the large monoclonal or oligoclonal populations of CD8+ T-cells that account for much of the lymphocytosis that is symptomatic of IM.

Prophylactic EBV vaccine development has focused on the gp350 and EBNA3 antigens. Systemic virus neutralizing antibodies can be easily induced by vaccination with gp350 and an appropriate adjuvant and, while epithelial cell infection may be unaffected, it is conceivable that free virus liberated on the basolateral surface of infected oropharyngeal epithelium could be neutralized and minimize transmission to circulating naïve B-cells. While infection itself will not be prevented, the effective virus dose at the B-cell level would be greatly diminished. The induction of mucosal immune responses in the orapharynx may prove to be more effective and the oral/nasal administration of gp350 in conjunction with mucosal adjuvants such as Iscoms (Wilson et al., 1999), cholera toxin B-subunit or the E.coli heat labile enterotoxin B-subunit, EtxB (Williams et al., 1999), should be investigated. However, there is some evidence that mucosal IgA specific for gp350 actually enhances infection of certain epithelial cells in vitro (Gan et al., 1997).

The strategy would be to prevent or modify primary EBV infection by vaccination of children before primary infection. The disease itself is caused by excessive CD8+ T-cell responses to EBV infection, and in particular, to EBV lytic antigens. Any vaccine that could allow a more rigorous control of the primary infection phase may therefore prevent the disease or reduce its severity. Other explanations for the pathogenesis of IM, unconnected to the dose of virus at primary infection, are possible and include differences in NK and CD4+ T-cell responses (Wilson & Morgan, 2002), expansion of a CD28 subset of CD4+ T-cells (Uda et al., 2002) and autoimmune responses (McClain et al., 2003). Until these issues are better understood, it will be difficult to take account of them.

Gp350

The major EBV envelope glycoprotein, gp350, binds to the CR2 complement receptor on B-cells and is consistent with it being a target for neutralizing antibodies. Following attachment the virus infects the target cell through envelope fusion events involving other EBV envelope glycoproteins gp85, gp42 and gp25 (Borza & Hutt-Fletcher, 2002). More recent work has shown that gp350 is not an absolute requirement for EBV infection to take place. A recombinant EBV in which the gp350 gene had been deleted was able infect a range of B-cell lines and epithelial cells, albeit at a lower efficiency (Janz et al., 2000). The early observation that serum EBV neutralizing antibodies largely recognized the major viral envelope glycoprotein, gp350 set the scene for subsequent work over a number of years involving a New World primate, the cottontop tamarin (Saguinus oedipus Oedipus). Oligoclonal B-cell lymphomas closely resembling those seen in post-transplant lymphoproliferative disease (PTLD) can be routinely induced in these animals by injection of EBV. The characterization and purification of tractable quantities of the gp350 viral envelope glycoprotein from both natural sources and by recombinant DNA methods was carried out. Recombinant gp350 in combination with adjuvants or when expressed in vaccinia or adenovirus vectors, induced protective immunity in cottontop tamarins susceptible to EBV-induced B-cell lymphoma. Protective immunity was not dependent on the induction of gp350-specific neutralizing antibodies but was achieved through cell-mediated immune responses (Morgan, 1992). Gp350 vaccine formulations have since been shown to induce CTL responses as well as neutralizing antibody (Khanna et al., 1999c, Wilson et al., 1996). The mechanism of protection in this animal model is unknown. Did the protective responses induced in the tamarin act on the virus or on tumor cells? Since the tumor cells have a Latency Ⅲ phenotype and do not express gp350, the induced immune responses were probably able to reduce the effective virus dose on challenge since the induction of tumors by EBV is dose dependent. Derivatives of the gp350 vaccines described above (Jackman et al., 1999) have been evaluated in human trials (Denis, 2005). The results of these trials strongly indicate that gp350 vaccination of seronegative young adults prevents IM but does not prevent EBV infection. Surprisingly, little attention has been placed on developing DNA vaccines coding for gp350. Immunization of mice with such a vector gave rise to antibodies to gp350, antibody-dependent cellular cytotoxicity (ADCC) and gp350-specific CTLs (Jung et al., 2001).

The cottontop tamarin has a number of shortcomings as a model of EBV infection and disease. This species is not infected by the oral route and does not sustain a persistent infection. A further complication is that the tamarin has an unusually restricted histocompatibility complex polymorphism and only expresses the alleles G, F. and E, associated primarily with NK cell function (Cadavid et al., 1999). Moreover, contrary to earlier beliefs, tamarins and marmosets have been found to carry their own resident γ-herpesviruses (de Thoisy et al., 2003).

A recombinant derivative of the Chinese vaccinia Tien Tan strain expressing gp350 was used to vaccinate a small group of both seronegative and seropositive children in Southern China. Antibody levels to gp350 were raised in seropositive subjects and were induced in those who were seronegative at the beginning of the trial. Six out of nine vaccinated children who were seronegative for EBV at the time of vaccination remained seronegative for at least three years after vaccination (Gu et al., 1995). The particular vaccinia recombinant used in this trial would not be currently acceptable for large-scale use on safety grounds. The Modified Vaccinia Ankara (MVA) strain would provide an acceptable alternative vector (Stittelaar et al., 2001).

Some development work has been carried out on the generation of a recombinant varicella zoster virus (VZV) vaccine vector for the delivery of EBV genes and VZV recombinants were produced which are able to express EBV gp350 (Lowe et al., 1987). It may be timely to explore this option further given the success of the Oka VZV vaccine strain and its incorporation into national vaccine programs in the USA, Japan and elsewhere.

A murine γ-herpesvirus (MHV 68) is increasingly being used to model EBV biology and this virus induces a mononucleosis-like syndrome in mice (Blackman et al., 2000). Vaccination studies have been carried out using this model and the MHV 68 major envelope glycoprotein gp150, an analogue of EBV gp350 (Stewart et al., 1996) was incorporated into a recombinant vaccinia virus vector and used to vaccinate mice prior to intranasal challenge with MHV 68. Virus-neutralizing antibodies were induced and the mononucleosis-like syndrome normally caused by MHV 68 was almost completely eliminated. MHV 68 latency was established in the vaccinated mice nevertheless (Stewart et al., 1999). These results are a cause for some optimism in that appropriately administered EBV gp350 could prevent IM even if the establishment of latent, persistent EBV infection was unaffected.

EBNA3

Another approach using synthetic peptides based on the EBNA3 latent antigen, to induce cell-mediated immune responses, was developed in parallel and has also been the subject of small-scale human trials (Khanna et al., 1999a,b,c). This approach utilizes latent antigen epitopes restricted through common MHC class Ⅰ alleles to induce CD8+ T-cell responses in the vaccinee. Strong support for this approach came from the demonstration that autologous CD8+ T-cells against EBNA3, propagated ex vivo and introduced into immunosuppressed patients at high risk of PTLD, were able to prevent PTLD and in some cases cause PTLD to regress (Gottschalk et al., 2002). In other words, CTLs specific for some EBV latent antigens can control the propagation of EBV-infected B-cell tumors in vivo. A Phase I human trial has been carried out to establish whether an EBNA 3 synthetic peptide, FLRGRAYGL, incorporated into a water-in-oil emulsion adjuvant, can be safely used to induce epitope-specific CTL responses (Moss et al., 1998). Ultimately, a collection of epitopes will be used to span the majority of HLA types and encompass strain variation in target populations. Similar strategies may be adopted with peptide epitopes from lytic cycle antigens such as gp350 and BZLF1. Until it is known which T-cell specificities are important in protecting against IM, there may be a case for constructing an epitope vaccine using epitopes from both lytic and latent antigens. Vaccination with a latency antigen inserted into a DNA plasmid expression vector has been tested in the MHV 68 murine γ-herpesvirus model using the M2 latency-associated gene. M2 DNA vaccination had no effect on virus replication in the lung but did reduce the latently infected cell burden in the early, but not the later, stages of infection (Usherwood et al., 2001).

One serious hurdle for peptide-based vaccines is the relatively large number of epitopes that would need to be incorporated into a single vaccine so that it could be delivered to a wide range of individuals with different HLA types. A vaccine formulation with so many components may face difficulties with regulatory approval. This problem can be overcome using polyepitope constructs (Thomson et al., 1996). The polyepitope corresponds to the linking of minimalized CTL epitopes within a single coding sequence as a “string of beads” and has been shown to be highly efficient in inducing protective CTL responses when delivered as part of a live viral vector or a recombinant DNA plasmid (Thomson et al., 1998, 1996). Gp350 subunit vaccines avoid these problems since all possible epitopes are contained within the whole protein and sequence variation between different EBV isolates appears to be minimal. To what extent do CTLs reactivated from memory T-cells by autologous LCLs and assayed against peptide-coated targets, or targets infected with vaccinia recombinants, reflect in vivo CTL activities? The therapeutic effects on PTLD, of EBV-specific CTLs grown ex vivo, support the view that CTLs of one or other latent antigen are responsible for these effects (Sherritt et al., 2003). Which cell types and which antigen specificities are responsible for the beneficial effects of infusion of ex vivo grown T-cells, is not yet clear.

Vaccines to prevent post-transplant lymphoproliferation disease

Approximately 10% of seronegative children receiving solid organ transplants develop PTLD during the first year after transplant. The five-year survival of patients who develop PTLD is poor being only 35% for renal transplant recipients and 26% for heart transplant recipients. The risk of developing significant morbidity or PTLD following primary EBV infection in non-immune transplant recipients is about 20 times greater than in seropositive transplant recipients. Pediatric transplant patients are much more likely to be seronegative than adult patients since EBV infection increases with increasing age. Primary EBV infection occurs in about 70% of all seronegative recipients during the first 6 months following transplantation. This is the period during which the most intensive immunosuppression occurs. Immunization of seronegative patients before transplantation provides a realistic opportunity to test whether the presence of antibody to EBV or residual EBV-specific T-cell responses still active during immunosuppression will protect against infection, spread or pathogenic effects of EBV following transplantation.

Therapeutic vaccines to prevent Hodgkin’s disease and nasopharygeal carcinoma

Of the interventions designed to treat human malignancies, immunotherapy with CTL is increasingly being recognized as potentially the most efficient strategy with minimal side effects (Savoldo et al., 2000; Bollard et al., 2004; Straathof et al., 2005). The key factors in the development of a CTL-based therapeutic strategy are the characterization of therapeutic immune correlates, and the delineation of the specific portions of the tumor-associated antigens that elicit these responses. CD8+ CTLs are considered important for protection against various virus-associated malignancies and have emerged as the major element in the immune control of malignant cells (Khanna et al., 1999b). Indeed, a number of studies have recently been published, which have shown that adoptive immunotherapy can be successfully used to reverse the outgrowth of polyclonal B-cell lymphomas in transplant patients (Khanna et al., 1999a,b,c; Rooney et al., 2002).

Approximately half of HD cases involve EBV-positive tumor cells while all cases of undifferentiated NPC are EBV positive. Prophylactic vaccination of seronegative children or seropositive adults could lead to the reduction or elimination in the incidence of HD and NPC. However, the logistics, potential costs, and timescale of such a program may be disproportionate when seen in the context of other health priorities such as hepatitis, cervical carcinoma, malaria, HIV and others.

Despite being quite different cell types and at different locations, HD and NPC tumor cells express the EBV latent antigens, EBNA1, LMP1 and LMP2 and these are potential targets for the immune system. It is, therefore, surprising that the immune system is not able to kill these cells and indicates that viral immune evasion mechanisms are operating. One aspect of this phenomenon is already partly understood in so far as EBNA1 includes a glycine-alanine repeat (GAr) domain that not only may block its proteasomal degradation but also inhibits EBNA1 mRNA translation in cis (Yin et al., 2003). Whether EBNA1 is degraded and processed in an MHC class Ⅰ pathway in epithelial cells seems to depend on the type of epithelial cell. When EBNA1 is artificially expressed in epithelial cells of squamous origin, it appears to both inhibit growth and be degraded in a way that renders the cells targets for HLA-matched EBNA1-specific CTLs. Neither of these phenomena were observed when EBNA1 was expressed in epithelial cells of glandular origin (Jones et al., 2003). Despite the presence of significant numbers of EBNA1-specific T-cells generated by cross-priming (Blake et al., 2000), NPC and HD cells are not effectively targeted. More recent studies have shown that epitopes from EBNA1 can be endogenously processed and presented on the cell surface. These epitopes are primarily derived from newly synthesized protein as defective ribosomal products (Tellam et al., 2004). LMP1 and LMP2 are potential targets and CTLs specific for epitopes in these proteins are found in the circulation, albeit at relatively low levels and CTL responses to LMP epitopes are said to be subdominant in relation to CTL responses to epitopes in EBNA3 and in BZLF1. T-cell responses and antibody responses against LMP1 have been difficult to detect in humans (Khanna et al., 1998a; Meij et al., 2002). Both LMP1 and LMP2 may interfere with their own MHC class Ⅰ presentation and may be poor targets and/or weak inducers of CTLs (Dukers et al., 2000; Ong et al., 2003). LMP1 and LMP2 have a plethora of effects on the biochemistry of EBV-infected cells some of which could impact on antigenic processing and presentation as well as on cell growth and differentiation (Dawson et al., 2003; Portis and Longnecker, 2003). Another important observation in regard to CTL recognition of LMP1 and LMP2 is that the majority of the T-cell epitopes from these proteins are processed through a TAP-independent pathway (Khanna et al., 1996; Lee et al., 1996). TAP-deficient LCLs expressing LMP proteins are more efficiently recognized by LMP-specific CTLs than TAP-positive LCLs. These observations suggest that in the absence of TAP, ER generated LMP epitopes are presented more efficiently. On the other hand, presentation of these epitopes may be significantly reduced if TAP is expressed, as the peptides originating from the cytoplasmic compartment compete with ER generated LMP epitopes. It is therefore tempting to speculate that LMP-positive NPC and HD cells may have evolved to maintain TAP expression which limits the presentation of CTL epitopes from LMP1 and LMP2 and thus allows these tumors to escape CTL recognition in vivo.

Broadly speaking, two types of approach to therapeutic vaccination to treat NPC, HD and BL can be adopted. First, specific enhancement of effector cell responses to EBV proteins expressed in these cancers and secondly, enhancement of the presentation of the antigens in question by the tumor cells themselves. CTLs specific for LMP2 are detected in the circulation of NPC patients but are not found in the tumor lymphocyte infiltrate. NPC and HD cells also appear to have a normal MHC class Ⅰ presentation pathway, at least in terms of MHC class Ⅰ and TAP expression (Khanna et al., 1998b; Lee et al., 1998). Since both these cancers express identical viral proteins, it is anticipated that common immunotherapeutic protocols may be developed. It is important to remember that even if antigen presentation is unimpeded and specific CTLs are present at the tumor site in sufficient numbers, numerous other mechanisms, not necessarily related to EBV products, may prevent tumors cells being removed by the immune system (Gandhi et al., 2006). There are many elements of the CTL recognition and target cell destruction process that have not yet been evaluated for NPC and HD tumor cells. These could include the expression of coreceptors and adhesion molecules, production of cytokines that provide an inappropriate milieu for CTLs, production of T-cell inhibitory receptors such as Fas, altered proteasome function and other tumor cell-specific factors.

Recent studies on HL patients have indicated that regulatory T-cells and LAG-3 play a pivotal role in suppressing EBV-specific T cell immunity (Gandhi et al., 2006.)

LMP polyepitope vaccines

Although adoptive transfer of EBV-specific T cells has recently been tested for the treatment of relapsed HD, only a limited long-term therapeutic benefit was observed (Roskrow et al., 1998). One of the major limiting factors in the development of an efficient therapeutic strategy is the viral antigens expressed in these malignancies are not only poorly immunogenic but also in some cases have the potential to initiate an independent neoplastic process in normal cells. Thus a strategy which can overcome both these potential limitations is likely to provide a safe and long-term therapeutic benefit to cancer patients.

One such strategy involves the delivery of immunogenic determinants from LMP1 and LMP2 as a polyepitope vaccine. Indeed, initial studies from one of our laboratories have shown that multiple HLA class-I-restricted LMP1 CTL epitopes, when used as a polyepitope vaccine in a poxvirus vector, efficiently induced a strong CTL response and this response could reverse the outgrowth of LMP1-expressing tumors in HLA-A2 Kb mice (Duraiswamy et al., 2003b, 2004). A polyepitope-based vaccine for HD and NPC has a number of advantages over the traditionally proposed vaccines, which are based on full-length LMP antigens. Previous studies from our laboratory have indicated that polyepitope proteins are extremely unstable within the cytoplasm and may be rapidly degraded as a result of their limited secondary and tertiary structure. In contrast, full-length LMP antigens are unlikely to be degraded rapidly and may initiate various intracellular signalling events leading to the development of secondary cancers at the site of injection. Another important advantage includes the ability of a polyepitope vaccine to induce long-term protective CTL responses against a large number of CTL epitopes using a relatively small construct without any obvious need for a cognate help. Finally, the polyepitope-based vaccine is also likely to overcome any potential problem with the prevalence of LMP1 genetic variants in different ethnic groups of the world (Duraiswamy et al., 2003a).

Although the poxvirus polyepitope vaccine vector provides long-lived expression of encoded epitopes, there are concerns in terms of its safety profile with adverse side effects including postvaccine encephalitis when used in humans. Moreover, the poxvirus-based LMP1-polyepitope vaccine contained only HLA A2-restricted epitopes and the HLA A2 allele is carried by only about 55% of the individuals in most populations. If a CTL-based therapy for NPC and HD is to be applicable to a significant number of patients, the target population must be presented through HLA alleles present at high frequency in the patient population. In this context, in addition to LMP1, LMP2-specific responses restricted through A11, A24 and B40 are of particular interest because these alleles are very common in the Southern Chinese population (A11, 56%; A24, 27%; B40, 28%), particularly where NPC is endemic. To overcome these potential limitations, a novel approach has been devised of activating LMP-specific CTL responses with a replication-incompetent adenovirus encoding multiple epitopes from LMP1 and LMP2 (Duraiswamy et al., 2004). This replication-incompetent adenovirus vaccine contains both LMP1 and LMP2 epitopes restricted through HLA alleles common in different ethnic groups including NPC endemic regions (HLA A2, A11, A23, A24, B27, B40 and B57). It has been estimated that these optimally selected MHC class Ⅰ-restricted epitopes would include more than 90% of the Asian, African and Caucasian populations. Attractive features of adenovirus-based vaccines are their well-characterized genetic arrangement and function, as well as their extensive and safe usage in North American army recruits without inducing adverse side effects (Imler, 1995). Adenovirus-based vectors are being increasingly recognized for high efficiency and low toxicity and have been used in multiple human gene therapy clinical trials and preclinical vaccine applications. These vectors are also increasingly being used for cancer immunotherapy (Kusumoto et al., 2001). Two of the most promising recent reports were studies in non-human primate models of the Ebola virus and HIV (Shiver et al., 2002; Sullivan et al., 2000). In each study, an immunization regimen that included priming with plasmid DNA followed by boosting with adenovirus vector particles showed the induction of effective CTL responses when compared with the plasmid DNA alone (Sullivan et al., 2000). This new polyepitope vaccine is based on an E1/E3-deleted recombinant adenovirus comprising a chimeric Ad5/F35 vector that has been engineered to substitute the shorter-shafted fiber protein from Ad35 strain. This expression system provides an advantage over previous Ad5 vectors with respect to efficiency of expression of recombinant protein in hematopoietic stem cells and dendritic cells (Yotnda et al., 2001). Our studies with the Ad5F35 LMP polyepitope vaccine have shown that each of the epitopes in this vaccine is not only efficiently processed endogenously by the human cells but also recalls memory CTL responses specific for LMP antigens in healthy virus carriers and HD patients. Furthermore, the adenoviral polyepitope vaccine is capable of inducing a primary T-cell response, which was shown to be therapeutic in a tumour challenge system (Smith et al., 2006).

Altered antigen processing of LMPs using bacterial toxins

Since NPC is an epithelial tumor at a mucosal surface, the question of whether mucosal vaccine adjuvants might have a role to play in their treatment has been considered. One such adjuvant that has reached an advanced stage of development is the cholera toxin-like E. coli heat labile enterotoxin B-subunit (EtxB) (Williams et al., 1999). Bacterial protein toxins are molecules that combine unique cell binding with efficient cytosolic delivery properties. Toxoid derivatives of the adenylate cyclase toxin of Bordetella pertussis, pertussis toxin, anthrax toxin, and Shiga toxin B subunit have been investigated as potential vehicles for delivery of exogenous peptides or proteins into the MHC class Ⅰ presentation pathway (de Haan and Hirst, 2002). Recent work has established that EtxB possesses important features that makes it uniquely placed to be used as a delivery vehicle for MHC class Ⅰ-restricted T-cell responses (De Haan et al., 2002).

LMP-1 and LMP-2 are colocalized within plasma membrane GM1-rich lipid rafts in LCLs (Higuchi et al., 2001). LMP1 can be ubiquitinated and degraded through a proteasome pathway (Aviel et al., 2000) while the LMP2 intracellular N-terminal polypeptide binds Nedd4-like ubiquitin ligases and is also subject to proteasomal processing (Ikeda et al., 2002). MHC class Ⅰ-restricted antigen presentation of LMP2 is unusual as some, but not all, epitopes are presented independently of TAP (Khanna et al., 1996; Lee et al., 1996). It is unclear at present why LMPs are not always effectively presented to CTL by LCLs in the absence of peptide epitope pre-sensitization or LMP2 expression by recombinant vaccinia. However, it has been shown that the treatment of EBV-positive LCLs with EtxB results in colocalization, capping and internalisation of LMP1 and 2. EtxB itself binds the ganglioside, GM1, at the plasma membrane, enters the cell by endocytosis, and then the trans-Golgi network or endoplasmic reticulum by retrograde trafficking. LCLs treated with EtxB show a greatly increased susceptibility to killing by LMP1 and LMP2-specific CTLs (Ong et al., 2003). The mechanism by which EtxB causes this enhancement of antigen presentation by LCLs requires further investigation but these results indicate that EtxB interferes with the normal distribution and pathways of LMP turnover (Ong et al., 2003). The possibility therefore exists that EtxB could serve both as a mucosal adjuvant in the conventional sense by enhancing mucosal immune responses in the nasopharynx and also by enhancing the presentation of LMP1 and LMP2 in NPC tumor cells in vivo. Further work is needed to establish whether EtxB can enhance the CTL killing of EBV-infected epithelial cells expressing LMP1 and/or LMP2 as it does for LCLs. To this end, it has recently been shown in one of the authors’ laboratories that EtxB enhances the killing by LMP2-specific CTLs of H103 oral epithelial carcinoma cells expressing LMP2 (O. Salim, A. D. Wilson and A. J. Morgan, unpublished data).

One explanation for the failure of the immune system to kill HD and NPC tumor cells is that expression levels of the main potential immunological target, LMP2, are too low. Indeed, the unequivocal detection of LMP2 protein as opposed to RNA transcript in NPC cells has yet to be achieved. An EBV-transformed LCL, stably transfected with a plasmid expressing LMP2A under the control of an ecdysone analogue Ponasterone A (No et al., 1996), has been created in one of our laboratories. Following exposure to Ponasterone A, the level of LMP2A was increased ten fold and was localized in the plasma membrane. Increased LMP2A expression resulted in the up-regulation of LMP1 expression, and had a blocking effect on the EBV spontaneous lytic cycle by down-regulating the expression of both the BZLF1 and BRLF1 genes. In normal LCL, LMP2 is not efficiently processed or presented to CTLs by MHC class Ⅰ (Dukers et al., 2000; Ong et al., 2003). The tenfold increase in LMP2A expression induced by Ponasterone A did not result in any increase in lysis by an MHC class Ⅰ restricted LMP2A specific CTL line. Susceptibility to CTL lysis was enhanced by treatment with EtxB but the enhancement was only marginally higher in the Ponasterone A-induced targets compared to the controls with normal levels of LMP2 (G. Patsos, A. D. Wilson, and A. J. Morgan, unpublished data). These data suggest that LMP2 processing and presentation is impaired in some way and that access to different, more efficient presentation pathways, will render LMP2-expressing cells more susceptible to specific CTLs.

Therapeutic vaccines for Burkitt’s lymphoma

Whether prophylactic vaccination with EBV gp350 or latent antigen polyepitope formulations would prevent or reduce the incidence of endemic BL remains an open question. Raised levels of antibody against VCA are prognostic for BL and it has been suggested that control of EBV replication by vaccination before primary infection or before ant-VCA levels rise may have a protective effect. The prospect of devising a therapeutic vaccine for BL is rather problematical because of the absence of MHC class Ⅰ-mediated antigen targets. Endemic BL cells were thought to express EBNA1 only but it has recently been found that three out of fifteen EBV-positive endemic BLs tested also expressed a truncated EBNA LP, EBNA3a, 3b and 3c (Kelly et al., 2002). Although CTLs specific for the EBNA3 family may be abundant in these patients, it has been argued that the absence of LMP1 expression in the tumor cells does not allow the up regulation of antigen processing machinery. LMP1 may upregulate antigen-presenting functions in B-cells through an NF-κB pathway (Pai et al., 2002).

The restriction of EBV gene expression to EBNA1 and a consistent loss of antigen processing function through the MHC class Ⅰ pathway in BL cells, severely restricts the potential use of antigen-specific immunotherapeutic strategies. However, recent studies have provided some promising alternative therapeutic strategies for these malignancies. One such strategy involves targeting the tumor cells through virus-specific CD4+ CTLs. Previous studies from one of our laboratories have shown that BL cell lines displaying antigen processing defects through the MHC class Ⅰ pathway are efficiently recognized by EBV-specific CD4+ CTLs. Furthermore, these tumor cells also express normal levels of all the essential components involved in the processing of T-cell epitopes through the MHC class Ⅱ pathway. The importance of these studies has been further strengthened by the observation that CD4+ EBNA1-specific CTLs from healthy virus carriers can efficiently recognize virus-infected normal B-cells as well as BL cells expressing EBNA1 only (Munz et al., 2000; Paludan et al., 2002). These observations demonstrate that it may be possible to target EBNA1 through the MHC class Ⅱ pathway (Paludan et al., 2005). It raises the possibility that a vaccine based on EBNA1 that induces a strong CD4 T cell response may provide therapeutic benefit to BL patients.

One attractive way to deliver EBNA1 through the MHC class Ⅱ pathway is to enable this antigen to gain access to endosomal or lysosomal compartments. There are two major pathways by which antigens are targeted to these compartments. The traditional pathway involves the phagocytosis or endocytosis of exogenous antigens, followed by degradation by acid proteases in the endosomal or lysosomal compartments. On the other hand, MHC class Ⅱ-restricted presentation of endogenously synthesized proteins mainly involves membrane antigens that are thought to enter the endosomal or lysosomal pathway by internalization from the cell surface. The lysosome-associated membrane protein (LAMP-1) and the invariant chain are transmembrane proteins, which are predominantly localized in the lysosomes and endosomes, respectively. The cytoplasmic domains of these proteins contain specific targeting or address signals that mediate their translocation to the specific compartments. Previous studies from one of our laboratories have shown that these targeting signal sequences can be utilized to direct multiple MHC class Ⅱ-restricted CTL epitopes into the endosomal and lysosomal compartments (Thomson et al., 1998). This approach not only preferentially translocates the polyepitope protein to these compartments but also enhances endogenous presentation of CTL epitopes. Furthermore, this strategy was successfully used to activate a virus-specific memory CTL response from peripheral blood lymphocytes. Endosome/lysosome-targeted EBNA1 is currently being tested as a therapeutic vaccine in a non-immunogenic murine B cell tumor model in which the tumor cells express EBNA1. If this vaccine strategy is successful in reversing the outgrowth of these tumor cells, it is possible that an EBNA1 vaccine may not only be applicable to BL but also against other EBV-associated malignancies such as HD and NPC.

Another interesting strategy involves treatment of the BL cells with soluble CD40 ligand (CD40L) (Khanna et al., 1997). This treatment is highly effective in reversing the down-regulated expression of antigen processing genes involved in MHC class Ⅰ-restricted presentation. Moreover, CD40L-treated BL cells regain susceptibility to EBV-specific CTL-mediated lysis. These data suggest that direct infusion of soluble CD40L at tumor sites or cytokine-mediated induction of CD40 ligand on bystander lymphocytes should be considered as an alternative approach to restoring immunogenicity of malignant cells. Taken together, these preclinical studies provide an important platform for the development of a therapeutic strategy for BL based on a combination of EBNA1 vaccination and CD40L injection.

Recently, a novel approach to override the GAr-mediated proteosomal block on EBNA1 by specifically targeting this antigen for rapid degradation by a process of cotranslational ubiquitination combined with N-end rule targeting, has been explored (Tellam et al., 2001). These studies showed that enhanced intracellular degradation of EBNA1 was coincident with an induction of a very strong EBNA1-specific CTL response, and restored the endogenous processing of MHC class Ⅰ-restricted CTL epitopes within EBNA1 for immune recognition by EBV-specific CTLs. It has also been shown recently that EBNA1 can be degraded in some epithelial cells of squamous origin and these cells are subsequently susceptible to MHC class Ⅰ-restricted, EBNA1-specific CTLs (Jones et al., 2003). These observations raise the possibility of developing therapeutic strategies to modulate the stability of EBNA1 in normal and malignant cells. These schemes may involve treatment of virus-infected cells with synthetic or biological mediators capable of enhancing stable ubiquitination and rapid intracellular degradation of EBNA1 in vivo. Because the substrate specificity of the ubiquitin-proteasome pathway is conferred by the E3 ubiquitin-protein ligases, then one approach may involve manipulation of the ubiquitin-dependent proteolytic machinery by targeting specific E3 ubiquitin-protein ligases to direct the degradation of otherwise stable cellular proteins, such as EBNA1. Potentially, this engineered proteolysis system could be utilized as a therapeutic method to counteract the proteasomal block conferred on EBNA1 through the cis-acting inhibitory GAr domain.

The MHV 68 model of γ-herpesvirus infection has indicted a role for CD4+ T-cells in both the control of infection (Hogan et al., 2001) and in the control of tumor cells expressing MHV 68 antigens. When MHV 68-infected S11 cells were injected subcutaneously into nude mice, adoptively transferred lymphocytes caused the regression of S11 tumors and CD4+ T-cells were most effective in preventing tumor formation. CD4+ T-cells were also present in the regressing tumors (Robertson et al., 2001).

Conclusions

The limitations imposed on EBV vaccine design include (1) the behavior of EBV in vivo is poorly understood such that the anatomical location of infected cells, the site at which they become infected, and the site at which new virus is produced, are not known with confidence; (2) the correlates of immune protection against, or modification of, in vivo EBV infection are unknown; (3) killed or attenuated variants of EBV cannot be used as vaccines because of the oncogenic potential of the virus and a number of its components; (4) the virus infection persists for life in the face of humoral and cell mediated immune responses; (5) although numerically very significant, only a very small proportion of the infected population will develop disease. Despite the fact that recent human trials have indicated that gp350 vaccination can prevent IM, but not EBV infection, in seronegative young adults (Denis, 2005), the immunological basis for this is remains unclear. Until further human trials have taken place it is unlikely that any correlates of protective immunity against either infection or disease will become known with only limited information coming from animal models of related viruses. It is impossible to say at this stage what effects gp350 vaccine-induced mucosal or systemic immune responses might have on primary EBV infection or existing EBV infection. Once latency has been established, will immune responses in the vaccinee be more effective in preventing EBV disease than in a normal unvaccinated seropositive? Large scale and unselective prophylactic mass vaccination are presently impractical given current public health priorities and may even be unwise. Unless a clear benefit of such vaccination in a sizeable population can be demonstrated, such as in young seronegative adults in Western countries, resources will inevitably be focussed on therapeutic strategies for those who have already developed disease. Our increased knowledge of the antigen presentation and processing pathways of the main EBV-associated tumours, NPC and HD, has given rise to rational and novel immunotherapeutic strategies based on the enhancement of either LMP-specific CTLs or enhancement of LMP antigen presentation in the tumours themselves. Until recently, there were no immunotherapeutic strategies for treating endemic BL but the induction of appropriate CD4+ T-cells by vaccination may offer a way forward. The conducting of properly controlled human trials to evaluate the options set out above is clearly the first priority and little further progress can be expected until they take place.

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