Introduction

The interplay between malignancy, infection and immunity is best illustrated by the neoplasms related to KSHV (Boshoff and Weiss, 2002): Kaposi sarcoma (KS) is approximately 100 times more common during immunosuppression and can be resolved when iatrogenic immunosuppression is stopped (Euvrard et al., 2003) and during highly active antiretroviral treatment (HAART) of HIV-1 infected individuals (Boshoff and Weiss, 2002). Primary effusion lymphoma (PEL) and plasmablastic multicentric Castleman’s disease (MCD) also occur predominantly during immunosuppression. Like other gammaherpesviruses, KSHV persists as a latent episome in B-lymphocytes (Ambroziak et al., 1995; Cesarman et al., 1995; Renne et al., 1996), without provoking host responses that would eliminate infected cells. KSHV acquired a fascinating repertoire of decoys to trick the host immune response enabling establishment of lifelong infection in humans with very few clinical manifestations. When the balance between viral infection and host immunity is disturbed, some of the molecular pathways employed by KSHV to evade host immune responses are directly involved in driving oncogenesis (Moore and Chang, 2003). KSHV is an excellent model to study the coevolution of pathogen attack and mechanisms of host counter attack.

KS is most aggressive in the immunosuppressed and resolves with partial restoration of the immune system (Gill et al., 2002). Since the introduction of HAART, there has also been a dramatic fall in the incidence of KS (Jacobson et al., 1999). Although non-immune mechanisms may contribute to this drop in KS cases and the resolution of established lesions, it is reasonable to propose that immune reconstitution is a major factor in the control of this neoplasm (Box 52.1).

Box 52.1

Kaposi’s sarcoma, immunity and anti-retroviral treatment.

We are only starting to understand how KSHV avoids these host responses, which viral epitopes are targets for adaptive immune responses, and how anti-KSHV immunity is altered during immunosuppression (Table 52.1).

Table 52.1

Overview of host responses to KSHV infection and future questions.

Adaptive immunity

T-lymphocytes

T-lymphocytes are in the forefront of the battle of the host’s immune system with invading pathogens. Activation of the innate arm of the immune system results in the direct killing of invading pathogens and the initiation and direction of efficient adaptive immune responses, which, if successful, lead to the establishment of immunological memory. Through the MHC machinery CD8+ and CD4+ T-lymphocytes specifically recognize viral peptide antigens, proliferate, and either directly lyze the infected cells (cytotoxic CD8+ T-lymphocytes, CTL) or further orchestrate the adaptive immune response (helper CD4+ T-lymphocytes, Th cells). However, during infection a variety of mechanisms are used by herpesviruses to escape T-cell responses (Ploegh, 1998; Yewdell and Hill, 2002).

Cytotoxic T-lymphocytes

CTL are primed by dendritic cells (DC) and by other professional antigen presenting cells, which present viral antigens through the MHC-I machinery. Following clonal expansion, the primed CTL act against virus by killing infected cells via perforin- and/or Fas-dependent pathways, before new virus particles are made, whereas they also release cytokines and chemokines with antiviral activity.

CTL epitopes

Considering the size of the KSHV genome, only a relatively small number of KSHV specific MHC class Ⅰ-restricted CTL epitopes have been identified thus far (Fig. 54.1(a)) (Osman et al., 1999; Wang et al., 2000, 2001, 2002; Brander et al., 2001; Micheletti et al., 2002; Wilkinson et al., 2002; Stebbing et al., 2003a; Ribechini et al., 2006). Although the use of these epitopes led to the detection of KSHV-specific CTL responses, these responses are in general weak compared to those seen against other herpesviral antigens. Whether this is due to viral immune escape from CTL, or whether help from autologous antigen presenting cells is necessary for efficient CTL responses (as shown for DC (Wang et al., 2002)) or whether the KSHV genome has just not yet been exhaustively screened for CTL epitopes remains to be elucidated. One difficulty is that in the West, KSHV is predominantly present in HIV-1 infected individuals who exhibit suppressed CTL activity. The majority of CTL epitopes have therefore been identified in HIV-1 infected individuals during HAART, where the immune system is partly restored. Interestingly, T cell responses to LANA and ORF 65 are also detectable, even in KSHV-seronegative HIV-1 infected individuals, implying that KSHV-specific cellular immunity can occur in the absence of antibody responses (Woodberry et al., 2005). However, the peptide epitopes present in these two viral proteins were not identified and it is not clear whether the observed responses were due to CTL or CD4⁺ cells.

Fig. 52.3

KSHV specific CTL epitopes and CTL recovery during HAART. (a) KSHV specific CTL epitopes: CTL peptide epitopes have been identified in five KSHV encoded proteins. The K1 epitopes all cluster in the most variable region (VR1) and are associated with specific (more...)

In KSHV, as in other herpesviruses, most of the CTL epitopes have been identified in conserved sites. It was demonstrated that functional CTL epitopes cluster in a positively selected region of the most variable KSHV gene. K1 is a positional homologue of LMP-1 of EBV and contains the two most variable regions (VR1 and VR2) across the entire KSHV genome which are used to classify KSHV into four clades (A, B, C, and D) (Zong et al., 1999). Every viral isolate studied thus far is unique to an infected individual. However, unlike the situation in retroviruses, K1 mutations have not been detected within an infected individual over time. Several, HLA class Ⅰ restricted epitopes within the VR1 were identified with the use of autologous overlapping peptides corresponding exactly to a patient’s own viral sequence (Fig. 52.1(a)). These CTL epitopes are conserved within a specific strain (e.g. A or B), but not between strains. Based on these observations it appears that part of the genetic variability occurring in K1 is driven by a positive selection for CTL recognition, rather than due to CTL escape (Stebbing et al., 2003a). Furthermore, the observed variability does not appear to be due to escape from humoral immunity (a mechanism employed by other viruses such as influenza and HIV-1 where variability in viral surface proteins is driven by escape from humoral immunity resulting a -antigenic shift’). It seems likely that this selection prevents the complete evasion of all host immune control mechanisms, which would lead to overwhelming viral infection with subsequent death of the host and, therefore, of the virus. It seems that K1, which is an early-lytic product, serves as a “suicide” protein allowing CTL recognition of cells reactivating from latency. Of note, CTL epitopes were also identified in vMIR1 and vMIR2 (Ribechini et al., 2006), two lytic KSHV genes that downregulate expression of MHC-I and other co-stimulatory proteins (Chapters 31 and 62), whereas vFLIP is able to upregulate expression of MHC-I and ICAM-1 (Lagos et al., unpublihsed data). In addition to K1 recognition by CTL, the above findings reveal two more mechanisms employed by KSHV to regulate immune escape, limit viral dissemination and establish equilibrium in the virus–host co-speciation.

Effects of HAART on CTL

HAART promotes long-term immune reconstitution in patients with and without KS. This reconstitution is also KSHV-specific (Wilkinson et al., 2002; Bourboulia et al., 2004). It has been demonstrated that there is a significant decline of KSHV DNA load in PBMC and plasma during HAART and this correlates with a significant increase of anti-KSHV-specific CD8+ T-cell responses (Bourboulia et al., 2004) (Fig. 52.1(b)). It appears that prolonged HAART (more than 12 months) is necessary for these anti-KSHV effects to be established and maintained in most HIV-1 infected individuals. In individuals with KS, resolution of KS is also associated with significant increases of KSHV specific CD8+ T-cell responses during the first 6–9 months on HAART. Future work has to determine whether other KSHV specific epitopes are able to elicit more potent CD8+ T cell responses and contribute to the restoration of KSHV specific T-cells and whether such responses are stronger and occur earlier than those observed for K8.1 and K12. Moreover, detailed phenotyping of KSHV-specific CD8+ T-cells would give further insight into the anti-KSHV responses in comparison to other viruses that establish persistent infections (Appay et al., 2002).

Helper T-lymphocytes

The final stages of herpesvirus virion assembly occur in endosomal cellular compartments with extensive targeting of viral proteins to endosomes. During this process viral proteins can be efficiently sampled by the MHC-II, leading to the presentation of viral antigens to CD4+ T-lymphocytes.

The association of KS with low CD4+ T-cell count (mainly below 200/mm³; although on average these values are higher than those associated with other AIDS-associated cancers) (Crowe et al., 1991; Cannon et al., 2003; Engels et al., 2003; Mbulaiteye et al., 2003) and the rapid KS resolution during HAART suggest a potential role for anti-KSHV CD4+ T-cell responses in the control of KSHV infection. However, although CD4+ T-cell proliferation as a response to KSHV has been reported (Strickler et al., 1999), a correlation between CD4+ T-cell number and KS resolution during HAART has not been observed (Gill et al., 2002). Future studies should determine the KSHV specific CD4+ T-cell epitopes and the patterns of these responses during primary and persistent infection comparing also with other herpesviruses (Amyes et al., 2003).

B-lymphocytes

Antibody responses from B lymphocytes play a major role in anti-viral immunity (Burton, 2002). Antibodies can bind to viral proteins and block viral entry to the host cells (neutralizing antibodies), inhibit release of viral particles from infected cells, or trigger effector functions through their Fc domain causing the lysis of free virions or infected cells by NK cells (antibody-dependent cellular cytotoxicity, ADCC) or by the complement (complement dependent cytotoxicity, CDC).

The importance of humoral immune responses against human herpesviruses has been shown (Chapters 34, 43, 51 and 72). Furthermore, antibody responses against MHV68, in the absence of CD8+ and CD4+ T-cells, can efficiently control MHV 68 replication (Kim et al., 2002).

Antibody responses are recognized and detectable against KSHV latent and lytic proteins. However, the role of these antibodies in controlling KSHV replication or infection remains to be elucidated.

Antibody epitopes and serological assays for anti-KSHV antibody detection

The ORF73 of KSHV encodes for the major latent nuclear antigen of KSHV. More than 70% of infected individuals have detectable anti-LANA antibodies (Gao et al., 1996; Kedes et al., 1996). These humoral immune responses are directed against epitopes in the C-terminus. Detection of anti-LANA antibodies correlates in different populations with the KS burden (Chapter 54), and the detection of anti-LANA antibodies by indirect immunofluorescence is a useful assay for serological surveys (Fig. 52.2(a)). Several studies have suggested that seroconversion and/or a high antibody titer against LANA correlates with risk of KS development in HIV-1 infected individuals (Gao et al., 1996; Sitas et al., 1999; Sitas and Newton, 2001; Ziegler et al., 2003). However, the association between KSHV load in sera or in PBMC and anti-LANA antibody titer is still unclear.

Fig. 52.4

Humoral responses to KSHV and reconstitution during HAART (a) Immunofluorescent assay for anti-LANA antibodies: The KSHV LANA elicits antibody responses and is used for serological studies in an indirect IFA. Sera from KSHV infected individuals recognize (more...)

Strong anti-lytic antibody responses are directed against K8.1, which encodes for an envelope glycoprotein that is the positional homologue of EBV gp220/350 (Chandran et al., 1998). Anti-K8.1 antibodies are detectable in approximately 80% of individuals with AIDS-KS and in more than 90% of individuals with non-AIDS KS (Fig. 52.2(b)). A K8.1 peptide epitope, with no known sequence similarity to any other pathogen has been used in the form of a multiple antigenic peptide (MAP) for the development of a highly sensitive and specific ELISA to detect KSHV infection (Lam et al., 2002). The sensitivity of this ELISA is improved by combining the K8.1 epitope with a known LANA epitope in a MAP (D. Lagos, unpublished data).

Another lytic antigen that generates humoral responses is the small viral capsid antigen encoded by ORF65 (Simpson et al., 1996; Lin et al., 1997). A combination of an ORF65 peptide epitope with a K8.1 epitope is employed in one of the most sensitive and specific, commercially available assays (Schatz et al., 2001) (Fig. 52.2(b)).

Specific antibody responses among individuals with KS have also been reported against ORF26 and K12, although significantly less common than LANA, K8.1, and ORF65 (Schatz et al., 2001).

A variety of serological assays for KSHV detection have been described. These include immunofluorescent assays using PEL cell lines and ELISA using virions, purified recombinant proteins, peptide epitopes, mixtures of

peptide epitopes, and multiple antigenic peptide epitopes as antigens. These assays have been successfully used in large epidemiological studies providing insight into KSHV biology, transmission, risk factors for KSHV infection and KS development (Ablashi et al., 1999; Sarid et al., 1999; Dedicoat and Newton, 2002; Dukers and Rezza, 2003; Martin, 2003) (Fig. 52.2(c)). The sensitivity of these assays ranges from less than 80% for individuals with AIDS-KS to more than 90% for HIV-1 negative individuals with KS (Rabkin et al., 1998; Enbom et al., 2000; Spira et al., 2000; Schatz et al., 2001; Dukers and Rezza, 2003; Pellett et al., 2003) (Fig. 52.2(b)). There are some AIDS-KS cases where anti-latent and anti-lytic antibodies are not detectable, but KSHV copies can be detected in plasma and/or PBMCs by quantitative PCR (Lallemand et al., 2000). Whether this is due to low sensitivity of the current serological assays or the defect in the humoral immunity of these individuals remains to be elucidated.

Effects of HAART on humoral responses

Although it is not clear if HAART has any significant effect on anti-LANA antibody titer (Wilkinson et al., 2002; Bourboulia et al., 2004), an increase in the number of individuals with detectable anti-K8.1 antibodies coincides with plasma virus clearance during HAART (Bourboulia et al., 2004) (Fig. 52.2(d)). Based on these results it could be proposed that the humoral arm of the immune system plays an important role in the control of the KSHV replication.

How antibodies contribute to KSHV immunity

It has been shown that sera from KSHV-seropositive individuals can block KSHV infection in vitro (Dialyna et al., 2004; Kimball et al., 2004). Moreover, individuals with AIDS-KS display reduced levels of neutralizing antibodies, despite the fact that higher total binding antibody levels are observed in this group (Kimball et al., 2004). This implies that the humoral immunity plays a crucial role in the prevention of KS development and that the most immunogenic KSHV antigens are not necessarily the targets of neutralizing antibodies. However, the contribution of KSHV neutralizing antibodies in controlling in vivo infection needs to be addressed further, as the neutralizing titers are low and the specific epitopes not known. K8.1 could be such a neutralizing antibody target as it is directly involved in the process of viral entry into host cells by binding to heparan sulfate (Birkmann et al., 2001; Chapter 23) and reconstitution of anti-K8.1 humoral responses during HAART coincides with virus clearance. Of note, gp 220/350, the EBV positional homologue of K8.1, elicits neutralizing antibody responses and has been investigated as a target for development of an EBV vaccine (Chapter 72). Another candidate for KSHV-specific neutralizing antibodies is glycoprotein B, which is also involved in viral entry (Chapter 23). Rabbit polyclonal antibodies raised against glycoprotein B neutralize infectivity in vitro (Akula et al., 2002). In addition to their neutralizing potential, antibodies can control KSHV infection through their effector functions. It has been reported that the genotype of the low affinity Fc receptor FcγRIIIA can contribute protection or conversely be a risk factor for KS development (Lehrnbecher et al., 2000). This receptor is expressed on the surface of NK cells and could be an important factor influencing ADCC activity. Further studies are necessary to elucidate the role of the humoral immunity in the host defense against KSHV.

Box 52.2Transmission of cancer, immunity and Kaposi’s sarcoma

Until the end of the eighteenth century, it was believed by many that cancer was a contagious disease. James Nooth, an English surgeon, and Jean Louise Alibert, the founder of the French School of Dermatology and personal physician of King Louis XVIII, were the first to independently challenge this hypothesis: both injected themselves with breast cancer cells, which resulted in short-lived local inflammatory responses, but no tumour establishment. They concluded that cancer was not contagious.

The discovery in 1908 by Peyton Rous that cell-free filtrates from tumors could transmit cancer between Plymouth Rock hens heralded the era of viral oncology and sparked a debate whether viruses were responsible for most cancers. The first evidence that a virus was indeed involved in the pathogenesis of a human cancer only emerged in 1964 with the discovery of Epstein–Barr virus. Two years later Peyton Rous was awarded the Nobel Prize in Physiology of Medicine for his discovery of “tumor inducing viruses.”

In the 1950s, selective immunosuppressant drugs were developed and organ transplantation became a life-saving reality. However, the first case of a kidney transplant recipient who developed iatrogenic Kaposi’s sarcoma was reported soon afterwards. It was also noticed that Kaposi’s sarcoma can disappear when immunosuppression is stopped, linking immunity closely with Kaposi’s sarcomagenesis. The transmission of other cancers during organ transplantation, e.g., melanoma, has also been documented.

Until recently, it was widely accepted that iatrogenic Kaposi’s sarcoma was due to the loss of immunesurveillance against KSHV: It was thought that KSHV could be transmitted from donor to host, or that immunosuppressive drugs led to the reactivation of KSHV in already infected recipients. The provocative finding in 2003 that certain cases of post-organ transplant Kaposi’s sarcoma are due to the transmission of KSHV-infected precancerous cells, revived the debate on the transmission of cancer. Ironically, this is a tumor induced by a virus, where both host cell and pathogen are transmitted simultaneously. These findings infer that KSHV not only infect endothelial cells during periods of immunodeficiency, but that KSHV-infected circulating endothelial cells are present in otherwise healthy individuals. Notably, the concept of cancer cell transmission is further supported by the identification in 2006 of the oldest known somatic mammalian clonal cell population as the causative agent of the canine transmissible venereal tumor.

The wider implications of this are that in populations such as certain sub-Saharan African countries with a large HIV-1 burden where many are immunosuppressed, the transmission of cancer cells between infected individuals, e.g., Kaposi’s sarcoma or anogenital cancer may represent a reality. The study of iatrogenic Kaposi’s sarcoma would provide further insight into the immunological control of cancer.

Further reading

• Barozzi P., Luppi M., Facchetti F., et al. Post-transplant Kaposi’s sarcoma originates from the seeding of donor-derived progenitors. Nat. Med. 2003;9:554–561. [PubMed: 12692543]
• Birkeland S. A., Storm H. H. Risk for tumor and other disease transmission by transplantation: a population-based study of unrecognized malignancies and other diseases in organ donors. Transplantation. 2002;74:1409–1413. [PubMed: 12451241]
• Fitzgerald P. J. From Demons and Evil Spirits to Cancer Genes. Washington: American Registry of Pathology Publications; 2000.

Conclusions and future perspectives

Excluding pox viruses, KSHV has pilfered an unprecedented array of cellular genes, mainly to impede the function of host antiviral immune responses. During immunosuppression, both the innate and adaptive anti-KSHV immune responses are hampered, allowing the uncontrolled proliferation of KSHV-infected cells that belong to the B-lymphocyte and EC lineages.

KSHV proteins that elicit humoral and cellular immune responses are being identified and the consequences of immunodeficiency (HIV-1 induced or iatrogenic) and immune-restoration are being elucidated. Such studies will allow the understanding of the anti-KSHV host defence mechanisms and could eventually lead to the generation of an effective vaccine. During the next couple of years, the study of KSHV immunobiology should also provide further insight into tumor and transplant immunology.

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