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CHAPTER 8
General Discussion
185
Chapter 8
EBV infection is ubiquitous in mankind. The great majority of adult
humans are lifelong virus carriers from childhood age onwards (72). EBV has
various biological behaviours. EBV initially infects submucosal resting naive B
lymphocytes and drives the activation and proliferation of these cell (“growth
program”). On the other hand, the virus can reside quietly in circulating resting
memory B cells (latency program), and occasionally, the infected memory B cell
can be activated to Ig-secreting plasma cells and then becomes a virus producer
cell (lytic program). These behaviours correlate with the location of the infected
cell. Lytic virus production is active in the nasopharyngeal lymphoid system,
including the tonsils, whereas the virus is dormant in the peripheral circulation
(44, 123, 138). When B cells with dormant EBV pass through lymph nodes, the
virus can be partially activated to promote host cell growth and survival (default
program). In addition, EBV can infect epithelial cells in latent and productive cycle
(44, 108). EBV in latently infected B cells can turn-off all latent gene expression (4),
and exit the tonsil into the circulation as resting memory cells, where the virus can
persist for life. Since the growth-promoting latent genes are not expressed (11,
113, 140), the cells are maintained in the periphery by normal memory B cell
homeostasis mechanisms. Moreover these cells are non-pathogenic and the virus
persists in a benign state (123). Furthermore, due to the lack of gene expression,
(11, 113, 140), the virus inside circulating memory B-cells is also hidden from the
immune response and cannot be eliminated.
EBV persistence is an
example of a dynamic equilibrium between the immune response and the various
states of infection. The numbers of virus-infected cells are increased by new
infection and by expansion of cells expressing the growth and latent (default)
programmes. This is counterbalanced by viral-neutralizing antibodies and
infected-cell death induced by cytotoxic T-cells (CTLs) directed against cells
expressing latent or lytic proteins (55, 138).
The different biological activities (latency, default, growth program) are
also seen in various EBV-associated malignancies. These can be of lymphoid or
epithelial origin, reflecting the dual cell tropism of EBV. EBV is officially recognized
as a human class I carcinogenic agent, with a proven role in Burkitt's lymphoma,
Hodgkin disease, extranodal T/NK cell lymphoma, immunoblastic B-cell
lymphomas in immunocompromised individuals, gastric carcinoma and
nasopharyngeal carcinoma (NPC). Nasopharyngeal carcinoma (NPC), one of the
most prevalent malignancies in certain regions, is an EBV-linked epithelial tumor
of the nasopharynx. It is a rare tumor in most parts of the world, but common in
certain geographic areas such as China, South East Asia and Northern Africa. The
etiology of NPC is multi-factorial, including genetic susceptibility, exposure to
carcinogens, and prior infection EBV. The virus is considered to give growth
advantage and apoptosis resistance to premalignant or (epi-)genetically modified
cells in the nasopharinx (107). Molecular analysis identified that undifferentiated
NPC biopsies are 100% positive for EBV by immunohistochemical staining for
EBNA1, LMP1, LMP2, EBER and BamHI A RNAs within all tumour cells. EBV
genome is clonal in NPC tumor cells, suggesting early onset involvement in the
carcinogenic process. The association is confirmed serologically by the elevated
antibody titer to capsid antigen (VCA) and a group of antigen (EA) synthesized
early in the viral replication process (114).
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General Discussion
Host immune responses are of central importance both in limiting
primary infection and in controlling lifelong virus carrier state. Under normal
circumstances in healthy individuals, EBV infections are not life-threatening and
are generally effectively controlled by the immune system through the action of
antigen-specific T lymphocytes (55, 120). EBV infection in human triggers both
humoral and cellular immunity (75). Immune control in viral infection is
predominantly mediated by CD8+ CTLs (120), essentially supported by helper and
regulatory T-cells (55). T cell responses to several peptide epitopes of immediate
early, early and late EBV antigens have been detected (53-55, 112, 129), but due to
difficulty of isolating cells in lytic cycle, these effectors have never been
demonstrated to specifically eliminate productively infected EBV+ target cells. To
date, there is no report on a protective role of antibody specific to lytic antigens EA
and VCA in eliminating EBV+ cells, except neutralizing antibodies to gp350/220
and gp85 which may also mediate killing of lytically infected cells via complement
or killer cells and protecting the virus-carrying host from subsequent infection
with a new exogenously transmitted virus strain (139, 146).
Control of virus replication and proliferation of transformed EBV-infected
B cells by virus specific-CTL is crucial for the host-virus relationship
(immunosurveillance). Virus production in the lytic cycle would benefit from
effective evasion of CTL responses (2, 47, 109, 117). As part of their normal
lifestyle, herpesviruses use general immune evasion approaches, such as blocking
the induction of programmed cell death and shutting down host protein synthesis
(121). In addition, herpesviruses specifically perturb recognition by virus specific
T cells via several mechanisms to thwart the MHC class I and class II processing and
presentation pathway. Down regulation of cell surface MHC class I is a common
strategy to escape from class I-restricted immunity (22, 117, 118). With regards to
EBV, the entry into lytic cycle is paralleled by reduced expressed of surface HLA
class I and II up to 5-fold. The BNLF2a protein inhibit TAP function, thus
preventing peptide-MHC I association (22). The viral G-protein-coupled receptor
(GPCR) encoded by BILF1 was shown to associate with MHC I in plasma
membrane, leading to its internalization and degradation (153). An early lytic gene,
BZLF1, inhibits upregulation of surface MHC class I expression (66). LMP1, -one of
latent type proteins, also expressed in the lytic cycle-, is known to induce
expression of MHC class II molecules, and numorous surface proteins in B cells
relevant for cell-cell contact (124, 144, 152). However, the viral gp42 directly
binds to MHC II, preventing proper maturation and blocking TCR recognition. The
BGLF5, viral DNAse serves as host shut-off protein, sufficient for reducing of all
these cellular MHC proteins (22, 119).
The detection of antibodies directed against viral structural proteins and
the EBV nuclear antigens is important for the diagnosis of EBV infection (75).
However, the simple detection of anti-EBV antibodies is not sufficient to
discriminate between healthy EBV carriers and patients with EBV associated
diseases. Analysis of the dynamics and diversity of anti-EBV responses are more
informative for diagnosis, since this better reflects the status of the virus-host
interplay (142).
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Previously, measuring humoral immune responses to EBV was done by
using indirect immunofluorescence assay (IFA) tests for antibody titers to four
serologically defined antigenic complexes of the virus life cycle, such as VCA, MA
including the viral envelope gp350/220 and gp85, EA and EBV nuclear antigen
(EBNA). Primary and persistent phases of infection are associated with different
combination of antibody reactivities. Primary infection is characterized by
detectable IgM and IgG antibodies to VCA and EA, a detectable IgM but relatively
weak IgG response to membrane antigen (MA), and the absence IgG reactivity to
EBNA1. The long term virus carrier stage is characterized by stable IgG reactivities
to VCA, MA, and EBNA, and weak or undetectable IgG anti-EA (48, 50, 51).
NPC is an aggressive tumor, with a high predisposition to metastasis,
following 85% of patients rapidly die of the disease and virtually all succumb
within 3 years (137). In Indonesia, close to 80% of the patients are diagnosed at late
stage (III & IV) [Tobing, unpublished] with poor prognosis which requires
combined chemo-radiotherapy, whereas early stage NPC may reach complete
remission by radiotherapy only (83). Therefore, better tumor markers are needed,
since the improvement of treatment and patients survival depends, at least in part,
on rapid diagnosis and early detection of relapse (34). Early studies showed that
NPC patients frequently have elevated serum antibodies against two lytic cycle
antigens, namely viral capsid antigen (VCA) and early antigen (EA) (49, 56).
Elevated IgG antibodies to EBV antigen have been used in NPC diagnosis (65), but
this response is also found in other EBV associated diseases such as EBV-related
lymphoma as well as normal healthy carriers (26, 65). EBV-IgA is an outstanding
marker for NPC rather than -IgG (49, 56), reflecting the tumor's origin in the
nasopharyngeal mucosal surface. Therefore, IgA-based ELISA is more suitable for
NPC serodiagnosis. Recent studies have further elucidated the role of EBV in the
pathogenesis of NPC and have demonstrated the utility of EBV markers in
diagnosis, prognosis, and post-therapeutic monitoring (136).
In this chapter, EBV based diagnostic aspects of NPC will be discussed
first. Subsequently we address immunological aspects of EBV tumor associated
antigens in NPC, and discuss options for inducing therapeutic antibodies in NPC
patients targeting tumor associated antigen expressed on the tumor cells.
1. EBV-based diagnostic aspects of NPC
From the first identification in 1960s (73, 74), there has been significant
interest in using antibody responses to various EBV proteins in the diagnosis and
prognosis of NPC. Quantitative analyses of EBV antibodies and EBV DNA have
been shown to be clinically useful (17, 103). In NPC diagnosis, indirect IFA for
measuring EBV specific antibody has proven very useful, but is also labor-intensive
and subjective (151). In recent years, based on progressing insights in the
molecular basis of anti-EBV antibody responses, more economical and objective
tests have been introduced using recombinant EBV proteins and enzyme-linked
immunosorbent assay (ELISA) technology, multiplexed microparticle basedimmunoassay (MMBI) and related methods (37, 151). Most recently, rapid filter
test have been introduced (EBV-TRU and Immunoquick) (61). IFA does not
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General Discussion
provide insight into the molecular basis of anti-EBV responses, because EBVinfected cells each contain multiple different EBV proteins that can serve as the
target for antibody (96, 97, 143).
In order to find detailed information about predominant markers for NPC,
we analyzed the molecular basis of IgG ang IgA antibody responses in patients with
NPC from different ethnic origin such as Javanese (Indonesia), Chinese
(Hongkong), and white (Europe), compared with non-NPC control subjects and
healthy EBV carriers as described in chapter 2. Regardless of the ethnic
background of patients, immunoblot analysis on extract of HH514.c16 cells
expressing lytic EBV antigens revealed the underliying complexity of anti-EBV
responses in NPC patients. Anti-EBV IgG diversity shows a positive correlation
with stage of malignancy, while IgA reactivity is more variable among patients. Our
results indicate that IgG and IgA-producing B cells are triggered differently, and
react to different EBV proteins and epitopes. This is probably related to the
location of EBV reactivation, paralleling NPC development. In normal EBV carriers,
EBV-IgG reactivity is characterized by dominant responses to EBNA1 (BKRF1; 72
kD) and VCA-p18 (BFRF3; 18 kD) with occasional weak responses to VCA-p40
(BdRF1; 40+45 kD) and Zebra (BZLF1; 36-38 kD). In higher stages of malignancy,
additional IgG responses to EBV proteins, including EA polypeptides p138
(BALF2), TK (BXLF1), DNAse (BGLF5), EAd-p47/54 (BMRF1) and Zebra (BZLF1)
were detected. The antibody diversity pattern in NPC differs from healthy EBV
carriers and other EBV associated diseases allowing this diversity profiles to be
used reliably as marker in NPC diagnosis.
Regarding the clinical use of different EBV lytic markers, IgA-EA is the
best marker for diagnosis of NPC, by showing the highest specificity compared to
VCA (141). Chapter 3 of this thesis confirms that EAd proteins, particularly EAdp47 and -p138 together with EBNA1 are best discriminator for NPC compared to
other proteins such as VCA-p18 and -p23. The results confirm previous findings
that the combination of IgG and IgA to EA and EBNA1 can be used to discriminate
NPC from healthy carriers and other EBV-related diseases (18, 60).
Most NPC patients have significantly higher antibody titers to EBV lytic
proteins, such as viral capsid antigen (VCA), ZEBRA, DNase, DNA polymerase, and
thymidine kinase (TK) (3, 16, 33, 43, 64, 85, 86). Some of these were considered to
be good indicators for prognosis of NPC because their levels fell after clinical
treatment and rose again upon recurrence (16, 27, 52, 87). IgG ZEBRA has been
proposed to be a prognostic marker for NPC patients after radiotherapy. Patients
with elevated IgG ZEBRA are predicted to develop distant metastasis or cranial
extension (25, 148). IgG ZEBRA is also a more sensitive marker for NPC diagnosis
in children (24). The combination of IgG ZEBRA with IgA EAd-p47+p138
improved the detection of NPC to 95% in overall of NPC population (23). However,
these ZEBRA findings could not be confirmed in our population. Neutralizing IgA
antibodies against DNAse is highly specific marker for NPC (12, 13), and it was
shown to be a valuable marker for screening. Individuals with elevated antibody
anti DNase have high risk to develop NPC in the future (12, 14). None of these
individual markers proved very useful in our hands, possibly related to the
differences in expression system and purity. Most recently, in parallel with this
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study, a further development was made by the construction of synthetic peptide
based ELISA test combining VCA- and EBNA1-IgA detection, with promising
diagnostic performance (30, 31).
Considering the diagnostic relevance of EA-specific antibody responses in
NPC, we developed a new way to isolate native EA protein from nuclei of induced
HH514.c16 cells by using a low salt extraction (Chapter 4). Native EA protein
extracted in a low salt yield an EA protein complex consisting of EAd-p138, TK,
DNase, EAd-p47/54 and ZEBRA. This native EA extract served as an excellent
antigen for NPC diagnosis compared to individual recombinant and synthetic EA
peptides, by showing sensitivity and specificity of 90.4% and 95.5% for IgG and
85.7% and 94% for IgA, respectively. These findings reflect most likely the
importance of native epitopes or post-translational modifications for interaction
with human antibodies, especially in NPC sera. Furthermore, it indicates that a
single EA marker might not be adequate to comprise the diverse anti-EBV antibody
responses for NPC diagnosis (33, 37). Therefore multiple EA proteins may be
required (15, 24, 33, 88). The use of multiple tests in combination is preferable,
since they provide better sensitivity and specificity (10, 18, 23, 31, 32, 60).
In order to promote improved treatment outcome and patients' survival,
screening of at risk population for having early stage NPC is important. The IgA-EA
ELISA system developed in chapter 4 is a promising method for NPC screening in
Indonesia. Besides NPC, IgA-EA is found significantly in IM patients (41.1%), but
low in other EBV related malignancies. For that reason, initial exclusion of IM is
proposed to be done prior the screening program to minimize the recruitment of
IM cases using this method. However, IM is hardly found in Indonesia. Therefore
IgA antibody detection using native EA antigen provides a highly sensitive and
specific for screening early stage NPC in population with low incidence of IM, such
as Indonesia. A disadvantage is that production of the native EA extract still
depends on culture cell, which is expensive and complicated. For that reason, the
IgA-EA ELISA is proposed only as confirmation test in a screening program, using
a cheap peptide EBNA1+VCA-p18 IgA ELISA as first step
Fachiroh et al [2006], developed an ELISA test was detecting IgA against two
synthetic peptides derived from immunodominant epitopes of EBNA1 and VCAp18 (IgA [EBNA1+VCA-p18]), which showed good sensitivity and specificity of
85.4% and 90.1%. This test may be suited as first step in an affordable (cheap)
mass-screening program. Therefore, we proposed to apply the peptide based IgA
ELISA as first line screening test and IgA-(native) EA extract as confirmation test.
In Chapter 5 we show that by using this two-step ELISA approach the sensitivity &
specificity increases from 85.4% to 96.7% and 90.1% to 98%, respectively.
Previous studies (149, 150) have clearly demonstrated the value of EBV-IgA
serology in NPC screening. This was recently further highlighted by the
demonstration that increased IgA-VCA levels precede NPC presentation by an
average of 2 years (63). These studies still used IFA-based methods, which should
be replaced by more standardized and objective methods such as ELISA. The twostep ELISA approach developed in this thesis is currently under evaluation in
prospective studies in several “high risk goups” in the Yogyakarta region [Hutajulu,
in prep].
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General Discussion
Based on good expertise in the (post-transplant) EBV+ lymphoma field
(130, 131), other biological markers have also been suggested in NPC
diagnosis/screening, such as EBV-DNA detection in serum or plasma (59, 102, 111,
126, 127). Recent developments in molecular technology and instrumentations will
provide new (affordable) tools for detecting tumor-derived nucleic acids in the blood.
EBV DNA serum or plasma has been hypothesized to be due to the leaking of DNA
from dead cells (102). This was proven by demonstrating that circulating EBV DNA
in NPC patients (but also in HD patients) largely consist of small fragmented DNA of
about 150 bp in length derived from apoptotic cells (9). Circulating EBV DNA in
plasma and serum may aid in the diagnosis of NPC (91, 102), prognostication (78,
82, 89), monitoring the progress of NPC patients (59, 90). Furthermore, it might
serve as a screening tool for individuals at high risk for developing NPC (147).
Serum EBV DNA represents released tumor DNA, not originating from other
latently infected cells such as epithelium or B lymphocytes, because active NPC
specific BARF1 RNA expression could not be detected in the circulation
(Chapter 6). Circulating EBV DNA is potentially useful for confirmation of serodiagnostic risk stratification in mass surveys. However, large variations in
peripheral blood EBV DNA positivity in NPC patients is reported by using different
DNA target size in PCR (Chapter 6). The percentage of NPC patients positive for
EBV DNA in peripheral blood varies from 30-98%, most having very low EBV DNA
loads (10, 57, 59, 102, 127). In this thesis, initially a 213-bp region of EBNA1 was
targeted by PCR for EBV DNA quantification in blood of NPC patients, 72.5%
positivity and only 29.5% had EBV DNA above CoV level. In addition, a 99-bp PCR
was developed showing 85.9% positivity, with 60.4% having EBV DNA loads above
CoV. In contrast to the initial study of Lo et al, we conclude that circulating EBV DNA
load quantification is not sensitive enough for NPC diagnosis, confirming
numerous other studies from regions where NPC is endemic (79, 102, 111, 127).
Therefore, we find that EBV DNA load measurement may have limited value for the
primary diagnosis of NPC in population screening in high incidence area
(chapter 6).
However, the disappearance of EBV DNA after radiotherapy in NPC
patients and its reappearance during recurrence may reflect the tumor burden in
selected patients. A transient increasing EBV DNA concentration occurs in patients
after receiving radiotherapy, within 3 days of the start of radiotherapy and reaches
the peak at 2 weeks after the start of radiotherapy. Following this initial surge in
circulating EBV DNA concentration, the decrease in EBV DNA concentration
occurred. The median half-life of EBV DNA decay was determined to be 3.8 days
(92). These data are confirmed in the recent study from Jakarta, Indonesia [Adham
and Middeldorp, pers. comm]. Therefore, monitoring EBV DNA concentration in
blood over a time during treatment may enable direct monitoring the in vivo effect
of treatment (7, 8, 57).
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2. Antibody against EBV-encoded tumor-associated antigens in NPC
Studies on EBV-specific antibody responses in different EBV-related
disorders have concentrated mainly on analyzing antibody reactivity to
components of the EBNA, EA, and VCA complexes, reflecting local (mucosal) lytic
virus replication which in some (direct or indirect) way is associated with the
oncogenic process. However in most EBV-related tumors that arise in
immunocompetent individuals, such as BL, HD, T-NHL, and NPC, only latent gene
products are expressed in the tumor cells.
NPC tumor cells consistently express EBNA1, LMP1 and LMP2, and
BARF1 with CD4+ and CD8+ T cell memory production to these antigens at levels
equivalent to those seen in healthy carriers (84). These “non-self” protein
expressed in the tumor are now becoming prime targets for immunologic
intervention (70).
HLA class I-restricted CTLs play an important role in controlling EBV
infection, and also the pathogenesis of EBV-associated tumors including NPC.
Adoptive transfer of EBV-specific CTL has been used for treatment of EBV-positive
tumors, such as PTLDs (69, 106, 122). The effector cells in these treatments
predominantly consist of CTL's targeting the dominant EBNA3 family of latent
proteins. NPC tumor cells only express LMP1, LMP2 and EBNA1. Although LMP1
and LMP2 are subdominant antigens, they provide targets for immunotherapeutic
approaches (134). However, immunotherapy targeting EBV in NPC might not
work as well as in PTLDs (136). Several investigations using EBV-specific CTLs
indicate that immune intervention in NPC is feasible. The first trial in NPC patients
with advanced diseases showed that infusion with autologous EBV CTLs safe and
led to increase the number of CTL and reduced plasma EBV DNA levels (19). A trial
by Comoli (20, 21) using LMP2 specific CTLs also observing the LMP2-specific
immune response in NPC patients. Another trial by Straathoff (133) indicated that
treatment using autologous CTLs showed remarkable anti-tumour effect.
However, there are some obstacles in the development of CTL based strategy. T cell
responses to viral proteins are restricted through HLA alleles, which hamper a
generic approach in the patient population. Furthermore, only weak CTL
responses to tumour-associated EBV antigens can be detected in the blood of a
minority of NPC patients, but remain undetectable at the tumour site (81).
Moreover, there is the possibility of CTL resistance and immune evasion by the
tumour cells, and there is no guarantee whether the effector T cells will home to the
tumour site. A clear understanding of important molecular mechanisms in this
process is limited (135).
LMP1 is expressed in most NPC cases (68). Therefore, LMP1 has become a
major target for vaccination against EBV-induced malignancies. Study by
Duraiswamy (29) propose an LMP1 epitope-based vaccination strategy to
enhance EBV-specific CD8+ CTLs as a preferred approach for the treatment of EBVassociated relapse HD and NPC. However, LMP1 protects against apoptosis by
increasing bcl-2 activity (46), thus increasing resistance to CTL therapy and LMP1
may have multiple additional roles in tumor immune evasion (98). Therefore,
LMP1 expressing tumors may resist CTL attack. Therefore, more knowledge is
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General Discussion
needed to provide a rationale for using anti-LMP1 T-cell responses as a therapy for
NPC.
NPC is characterized by a large cellular infiltrate comprised mainly of CD4+
+
and CD8 T lymphocytes that is intimately associated with the tumour cells. These
contains few, if any EBV-specific reactivities (77), but may be include significant
number of T regulatory cells (76), indicating immune evasion to occur. The effort
to target NPC should involve means to overcome the potentially suppressive effects
of the local tumour environment and deliver virus-specific T cells to the tumor site.
In addition, a study from our population show that close to 50% (10/21) patients
with more than 25% of granzyme B positive TILs did not reach complete
remission. The poor prognosis in cases with many granzyme B-positive TILs can
be explained by assuming that under the pressure of a strong CTL-mediated
immune response, selection for apoptosis-resistant tumour cells occurs, resulting
in resistance to both CTL and radio-therapy-induced apoptosis (105). More
detailed study of these cases indicated that apoptosis resistance may indeed be a
fundamental mechanisms underlying CTL resistance (104). Despite this, many
studies on cell-mediated immunity are ongoing using either vaccination or CTL
infusion treatment, although the efficacy is still in debate.
However, humoral immune responses to EBV tumour associated protein,
-when existing-, may provide a protective mechanisms mediating tumor lysis
effectuated via complement or FC-R bearing natural killer cells. This has not been
studied in detail before. Therefore, chapter 7 of this thesis explores the potential
presence and relevance in NPC patients of antibodies targeting tumor-associated
antigen expressed in or on the NPC tumor cells. In previous chapters it was shown
that NPC patients have high IgG and IgA antibody responses to a range of EA and
VCA antibodies, plus EBNA1. In contrast, analysis of natural humoral immune
responses (IgG and IgA) to individual EBV encoded tumor associated proteins (i.e.
EBNA1, LMP1, LMP2A and BARF1) revealed the presence of very low or mostly
undetectable natural antibody responses to these proteins. In particular, the
potentially extracellular “loop” domains of LMP1 and LMP2 seem rather nonimmunogenic, as well as secreted BARF1 protein. However, immunization of
rabbit using synthetic peptides of selected epitopes on “putative extracellular
domains” of those proteins result in the generation of specific antibodies, capable
binding to the surface of viable latent EBV infected cells. Furthermore, we show
that antibodies against these putative LMP1 and LMP2 extracellular domains can
mediate complement-driven cytolysis of LMP1 and LMP2 expressing cell lines.
This opens possibilities for inducing therapeutic antibodies in NPC patients
targeting tumor associated antigen expressed on the NPC tumor cells, either
vaccination or infusion of specific antibodies. This hypothesis recently proven in
a mouse model (28).
Antiviral drugs have not had a great impact on the treatment of EBV
related diseases (38). Therefore, a therapeutic EBV vaccine is worth considering
(99). A therapeutic vaccine would be much less expensive and more practical than
adoptive transfer of EBV-reactive T-cells (6). A peptide-based vaccine has recently
been designed to induce specific cellular responses (71). To overcome the problem
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of HLA specificity of viral CTL responses, the extensive allelic variation of human
HLA genes, and variation in epitopes between EBV strains, a “polytope” vaccine
containing multiple epitopes to which over 94% of the population should respond
has been formulated (29, 94). Again, this vaccine is not designed to induce “sterile
immunity” but in a hope that it will be effective in the prevention of disease states.
The circular synthetic peptides developed in chapter 7 may provide a new means
for inducing anti tumor immune responses, which needs to be explored in future
studies. A recent study using so-called “long peptide” of HPV E6 and E7 have
provided a strong supportive evidence for the ability of inducing effective immune
responses against weakly immunogenic intracellular proteins (67). This is the
first proof that therapeutic vaccination with peptides is feasible for treatment of
cancer.
Another option for EBV vaccination is the induction of antibody
responses. So far, the investigations aimed to explore the use of gp350 vaccination
for preventing primary infection in young children (42) and infectious
mononucleosis (101, 128), and recently to children with end stage renal failure
awaiting transplantation (116). The data supports the clinical feasibility of using
EBV vaccine to prevent acute primary infection and infectious mononucleosis in
teenagers and young adults. This vaccine induces specific antibody responses and
reduced the proportion of symptomatic primary EBV infection in teenagers and
young adults (101, 128). Additionally, the vaccine is able to induce the production of
a neutralizing antibody response, which inhibit the EBV transformation of B
lymphocyte (62, 100, 101).
Neutralizing antibody provides a humoral defense against cell entry of
EBV. Antibodies may penetrate mucosa and cell compartements, not hindered by
(local) immunosuppressive influences. This is an attractive feature for a
preventive vaccine. A preventive vaccine in PTLD has also been explored. EBVnaïve candidates for haematopoietic stem cell or solid organ transplantation who
are at the high risk developing PTLD were identified and immunized before
transplantation. These individuals could be monitored after transplantation, not
only for the development of PTLD but also to evaluate the effect of vaccination on
the quantity of EBV viral load (116). Unfortunately, the vaccine formulations used
so far do not provide long lasting immunity nor from exogenous EBV infection.
Endemic BL and NPC are also worthy targets for preventive trials, because of the
seriousness of these malignancies (115). Therefore, EBV-specific vaccination for
inducing antibody response against EBV tumor associated protein should be
considered. The recent initiation of prophylactic HPV vaccination, using structural
HPV proteins to prevent cervical cancer expressing only non-structural E6 and E7
is a promising example of the potential antiviral vaccination to prevent cancer (1,
39).
Alternatively, new treatment modalities using monoclonal antibody
targeting antigens on the surface of tumors cells are used for several types of
malignancies, including lymphomas and solid tumours, such as anti-CD20
(rituximab), -CD22 (epratuzumab), -CD80 (galiximab) (35) and EGFR (Erbitux,
Nimotuzemab) (5, 41). The action of rituximab appears to be mediated via three
potential humoral and cell-mediated effector mechanisms, including complement194
General Discussion
dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC) and
direct apoptosis signaling. CDC appears to be the predominant mechanism by
which rituximab exerts its therapeutic effect (40, 45). When CD22 binds to a
natural ligand or antibody will be rapidly internalized and provide pro-apoptotic
signal within B NHL cells (125). A preclinical study demonstrated that anti-CD80
antibodies inhibit lymphoma cell proliferation by inducing ADCC (110). Inhibition
of EGFR signaling with the monoclonal antibody cetuximab (Erbitux), or the
tyrosine kinase inhibitor gefitinib (Iressa), has been shown to retard cell growth
and induce apoptosis in NPC cells. The knowledge on kinase-mediated cell
signaling, which is commonly deregulated in epithelial cancers, and the ability to
pharmacologically inhibit such kinases, has led to the development of protein
kinase inhibitors in oncology. The rationale of targeting the EGFR- mediated
signaling in NPC is based on both preclinical and clinical groundwork. EGFR gene
amplifications can be found in 40% of NPC tissues, and EGFR overexpression is
associated with poor prognosis following chemoradiation in patients with
advanced NPC (93). The recent introduction of Nimotuzemab, which has less side
effect compare to Erbitux and is less expensive may be an important step forward
in treating NPC. However the price of this treatment is still prohibitory for many
developing countries.
The development of (monoclonal) antibody-based therapy for cancer and
our result with immunization of rabbits using LMP1, LMP2 synthetic peptides to
induce extracellular domain-specific antibodies as discussed in this thesis
(Chapter 7), creates the option of using peptide-based vaccination for inducing
antibody responses against external domains of LMP1, LMP2 proteins in NPC
patients. During natural infection or even in patients with LMP1 and LMP2
expressing tumors barely any anti-LMP1, 2 antibody responses is formed, in
particular not against the evolutionary well conserved extracellular domains
(Chapter 7). This provides an open window for vaccination. Induced antibodies
against the putative LMP1 and LMP2 extracellular domains, when having sufficient
avidity, can mediate complement-driven cytolysis of LMP1 and LMP2-expressing
cell lines. This may allow inducing therapeutic antibodies targeting virus-encoded
antigen expressed on the NPC tumor cells, either using active or passive
immunization. For passive immunization, anti-LMP1 and -LMP2 loop reactive
human antibodies may be selected from phage libraries using the peptides
described here in this thesis. Phage library technology has shown strong potential
in recent years (58). Further technological developments led to advances in the
generation of chimeric antibodies (65-90% human), partially humanized
antibodies (95% human), and most recently fully humanized antibodies (58, 80,
145). An obstacle in treating cancer cells with targeted therapy is the ability of
cancer cells to become resistant to a targeted therapy by activating alternative
pathway to evade apoptosis. It suggests the use of several antibodies or
combination of antibodies with chemotherapy. The study of using combination
targeted therapy in NPC has been done by combining cetuximab and platinum in
NPC cell lines, cetuximab and carboplatin in patients with metastatic NPC (93).
Antibody based therapy targeting EBV tumor associated antigen is considered to
be the advantageous option and can be used as a component of an
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immunotherapeutic for NPC. However the considerable cost of (human) antibody
therapy, precludes its widespread use in developing countries (95). Therefore,
more work aiming for less costly “small molecule” therapeutic agent is still needed.
On the other hand, recent studies have indicated the option using “common” drugs
for epigenetic modification of latent viral promoters to trigger EBV into lytic cycle
production, thus becoming sensitive to antiviral drugs and more potent immune
responses. This so-called “lytic” induction therapy was proven successful in
mouse models and awaits further evaluation in humans (36, 132).
In conclusion, the aberrant humoral immune responses of NPC patients
against EBV, mainly to components of the EBNA, EA, and VCA complexes, is
important for diagnosis and prognosis and may find application in screening for
early-stage NPC. However, IgG antibodies to EBNA1 and VCA frequently present in
healthy carriers. Therefore, IgG and particularly IgA antibodies to EA are proposed
to be specific marker for NPC. The latter appear to be directed to conformational
rather than linear epitopes (this thesis). Together with detection of IgA specific for
VCA and EBNA1 peptide, IgG/IgA-EA can be used as NPC discriminator from
healthy carrier and other EBV-related diseases. The EBV-encoded tumor
associated antigens are marginally immunogenic for humoral immune responses
in NPC patients. However, this may be stimulated specifically using exogenous
LMP peptide constructs to generate such responses. These “anti-loop” antibodies
can mediate killing activity through antibody dependent cytotoxicity and open
options for peptide-based tumor vaccination in patients carrying EBV latency-II
type tumors such as NPC.
General Discussion
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