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Hypothesis: Could Hepatitis B vaccine act as an immune adjuvant in glioblastoma? Clues to conduct further epidemiological analyses
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Hypothesis: Could Hepatitis B vaccine act as an immune adjuvant in glioblastoma? Clues to conduct further epidemiological analyses ⁎
Meric A. Altinoza, , Aysel Ozpinara, Alp Ozpinarb, Emily Hackerb, İlhan Elmacic a
Department of Medical Biochemistry, Acibadem University, Istanbul, Turkey Department of Neurosurgery, Pittsburgh University, Pittsburgh USA c Department of Neurosurgery, Acibadem University, İstanbul, Turkey b
A B S T R A C T
A failure of neurodevelopmental differentiation at the level of oligodendroglial-astrocytic biprogenitors (O2A) is shown to be involved in the pathogenesis of both multiple sclerosis (MS) and glioblastoma multiforme (GBM). In this review article, we suggest that certain antigens of Hepatitis B Virus (HBV) and HBV-Vaccine (HBV-V) could act as immune stimulants in GBM treatment based on several lines of evidence. HBV-Vs may cause rare but prominent neuroimmune side effects including demyelination and multiple sclerosis, which may be associated with HBV-proteins creating antigenic mimicry of oligodendroglial progenitors. The combined prevalance of HBV and Hepatitis C Virus-carrier state is less in patients with brain tumors compared to healthy subjects. Furthermore, within the population of patients with brain tumors, the prevalence is even about two times lesser in GBM in comparison to those with a diagnosis of meningioma. Although indirectly, this epidemiological data may indicate that the immune response triggered against hepadnavirus antigens would eliminate aberrantly differentiating O2A progenitor cells giving rise to GBMs. Moreover, Hepatitis B surface antigen-antibody variable domain is among the top 100 differentially expressed transcripts in fresh frozen and formalin-fixed paraffin-embeded specimens obtained from pediatric GBM tissues in comparison to the control brain tissues. However, the provided evidence is still premature and we think that HBV-V warrants investigation first by epidemiological studies and then by animal experiments to determine whether it reduces the risk of GBM and whether it could slow GBM growth via immune stimulation.
1. Introduction Glioblastoma multiforme (GBM) is the most grave malignancy among the brain cancers, with a 5-year survival of less than < 4% [1]. Unfortunately, GBM also accounts for about 80% of primary brain cancers and affects more than 17,000 patients annually in the USA alone [2,3]. Prominent invasiveness is one of the major features of GBMs which hinders total surgical resection. For more than a century, it is well established that certain infections or vaccinations could induce very rare but dramatic instances of “spontaneous cancer healing” [4,5]. In this manuscript, we propose that Hepatitis B Virus Vaccines (HBVVs) may be new adjuvants in immunotherapy of GBM. We will explain several features to support our proposal: 1- HBV-Vaccines, which have ample benefit, also cause rare side effects mostly manifested as demyelinating diseases such as Multiple Sclerosis (MS). We believe that this feature may be associated with the antigenic similarity between certain HBV proteins and O2A (oligodendroglia-astrocyte biprogenitor cell) surface proteins. 2- O2A cell pathology is a common feature for both MS and GBM. In MS, there is evidence suggesting immunity against oligodendroglial progenitor cells and a differentiation failure of oligodendrocyte progenitors are involved in the pathology. In parallel, certain evidence showed a blockage of differentiation at O2A stage in
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GBM oncogenesis. 3- HBV- and HCV-carrier state is less in GBM patients, which may suggest that the developed immunity against proteins of hepadnaviri (including HBV and HCV) may also trigger immune responses against undifferentiated/aberrant O2A biprogenitors, subsequently causing their elimination, and reducing the likelihood of GBM formation. 4- Hepatitis B surface antigen-antibody variable domain is among the top 100 differentially expressed transcripts in specimens obtained from pediatric GBM tissues in comparison to the control brain tissues. Below, we will summarize the relevant data, but we will first describe the features of HBV and the neuro-immune side effects of HBVV. 2. HBV antigens exert similarities to malignant and benign tissue antigens The serum specimens taken from carriers of HBV (17 out of 21) crossreact with the basal cell layer of squamous epithelia and intensely with thymic stellate epithelia [6]. In the thymic tissue, stellate epithelia reside in the cortex and medulla and their morphological and residential features exert similarities to the epithelia producing the immunostimulant molecule α1-thymosin [6]. The immune network theory suggests that there is a production of various anti-idiotype (anti-id)
Corresponding author at: Acibadem University, Atasehir, Istanbul, Turkey. E-mail address: [email protected] (M.A. Altinoz).
https://doi.org/10.1016/j.intimp.2019.106038 Received 18 August 2019; Received in revised form 7 October 2019; Accepted 7 November 2019 1567-5769/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Meric A. Altinoz, et al., International Immunopharmacology, https://doi.org/10.1016/j.intimp.2019.106038
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more than any public health intervention and antibiotics [13]. Nonetheless, serious side effects have been reported following vaccination, including with HBV-vaccines [14]. The adverse effects comprise of disorders with autoimmune features and neural manifestations. In a period of six years, 35 primary demyelinating events 8 weeks after HBV vaccinization were witnessed in Paris [13]. Completing a mean followup of 3 years, half of these cases developed Multiple Sclerosis (MS) [13]. The retrospective case-control study was performed at the Salpêtrière on patients experiencing their first episode of CNS demyelination (FECD) after HBV-vaccination [13]. There were 121 cases with a FECD and adjusted odds ratios (OR) between a FECD and HBV-vaccinization of 1.7 during the previous 60 days and 1.5 during the previous 61–180 days [13]. An investigation of 753 MS patients over a 2 years period identified 92 subjects who were vaccinated against HBV. Among these patients, 46 developed a FECD, 20 in less than 8 weeks following HBV vaccination [14]. From a DR2+ subject who suffered from MS after HBV vaccination, a T lymphocyte clone specific for HBV antigen was found that reacts with a peptide existing in the structure of proteolipid protein (PLP) (aminoacids 179 to 197) [13]. A nested case–control study defined a association between HBV vaccination and an increased incidence of MS in the 3 years following vaccination [15]. This investigation revealed that 11 of the 143 MS cases were vaccinated with HBV-V within a 3-year interval, indicating that longer periods should be assessed when determining vaccination safety in patients with MS [15]. Also, according to the VAERS database (US Vaccine Adverse Events Reporting System), adults vaccinated against HBV exerted significantly higher odds ratios (OR) for MS (OR = 5.2; p < 0.0003), and optic neuritis (OR = 14; p < 0.0002) in comparison to those vaccinated against tetanus [16]. Bogdanos et al. investigated aminoacid similarities between the sHBsAg, and the MS-autoantigens myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG) [17]. Peptides of 20 amino acid-length were synthesized spanning 4 sHBsAg/MOG and 1 sHBsAg/ MBP mimicking pair and evaluated with ELISA. 147 samples from 58 adults were obtained prior to HBV vaccination (58/58), and after HBV vaccination [18]. Reactivity to at least one of the MOG mimics existed in 4 (8%) pre-HBV vaccinated and in 30 (60%) post-HBV vaccinated subjects (p = 0.001). 30/50 (60%) of the vaccinated subjects had sHBsAg/MOG double reactivity compared to none before vaccination. No reactivity was found against the sHBsAg/MBP mimics. Some investigators indicated that those who developed autoimmunity after HBV-V had exerted certain patterns of human leukocyte antigens (HLA) [18]. Acute disseminated encephalomyelitis (ADEM) is a very rare neuroinflammatory demyelinating disorder of the central nervous system (CNS) [19]. ADEM can develop in any age but is mainly a disease of children and young adults. A mildly enhanced risk for ADEM was demonstrated by Touze and Mikaeloff specifically after Engerix B HBV-vaccine [19]. Reports also exist on isolated optic neuritis following various vaccinations including Hepatitis A and B [20]. Acute transverse myelitis is a rare acute inflammatory spinal disorder in childhood [20]. An analysis of all English literature published between 1970 and 2009, revealed 37 transverse myelitis cases following different vaccinizations including 13 after HBVvaccination [20]. HBV-Vs are also the second most common vaccines reported to the VAERS [21]. A retrospective analysis of the VAERS database in 2004 revealed 120 reports of myelitis after HBV vaccination. A structural mimicry of virus antigens to host antigens was assumed to cause these adverse events [21].
antibodies during an immunological response to a specific antigen. Certain subtypes of “internal-image” anti-id antibodies are anti-paratopic and exert the 3D-molecular structure of the original antigen. Hence, it is possible that anti-idiotype antibodies against HBV-surface antigens may bind and activate thymic stellate cells. HBV polymerase (HBVP) shares six amino acids His-Tyr-Gly-Ser-Leu-Pro-Gln with the encephalitogenic epitopes of MBP (rabbit myelin basic protein) [7]. As we will discuss below, vaccination against HBV may cause neuroimmune side effects including MS. In MS, it is well established that there exists auto-immunity against myelin proteins. There may also exist other molecular mimicries between HBV proteins and proteins of aberrantly-dedifferentiating oligodendroglial progenitorss. Such mimicries may eliminate these cells and subsequently lower the risk of GBM which will be detailed below. Monoclonal antibodies were produced against a cell lineage obtained from a carcinogen-induced large bowel tumor. One of these antibodies (MAb E4/pE4) slightly stained the healthy small and large bowel but not other the normal organs, as detected with immunohistochemical staining (IHC) [8]. However, in the colon carcinoma cells, pE4 reacted with the cell membranes. The first 20 amino-terminal amino acids of pE4 were the same as residues 5 to 25 from glycoprotein-30 (gp30) from HBsAg [8]. Antigenic epitopes linked with HLA class I molecules on a bladder cancer cell line were defined and 1 of the 3 peptides (VTDPGNLLY) exerted partial homology to HBV (corresponding to aa residues 122–128) [9]. Altogether, these finding may indicate that malignant cells express HBV epitopes and thus, antibodies against HBV epitopes could have triggered immunity against them. Indeed, there exist anecdotal reports of regression of neuroectodermal cancers such as neuroblastomas. Additionally, glioblastoma risk is lower in carriers of HBV and HCV. These facts are discussed below. 3. Regression of neuroblastoma coincident with HBsAg seroconversion In 1982, a dramatic regression of a far advanced neuroblastoma was documented which began following acute infection with HBV [10]. At that time, spontaneous remisson of neuroblastoma in infants was fairly known, whereas total remission in patients older than two years was very rarely reported. A 13-year-old boy had many bony, retroperitoneal, and liver metastases, with a diagnosis of an anaplastic type neuroblastoma. Chemotherapy including adriamycin, cyclophosphamide and dacarbazine and radiotherapy did not lead to any healing [10]. However, 14 months after diagnosis, the patient’s general condition became strikingly better, and 3 months later, his tests demonstrated serum conversion of HBsAg with increased liver enzymes and γglobulin levels [10]. Afterwards, abdominal and osteolytic metastates vanished in a progressive manner. Moreover, a PET scan revealed complete vanishing of hepatic metastases and urine levels of VMA decreased to normal ranges [10]. When this case was published, the patient was 16 years old and free of detectable disease three years after diagnosis [10]. At that time, the survival of neuroblastomas for children over seven years was 8%. Moreover, survival was even more rare in patients with disseminated skeletal metastases and such a circumstance involving a child over 6 years old had not been published at the time of the report. The authors proposed that the antigenic epitopes of HBV triggered anticancer immune responses and rejection of the tumor [10]. Peculiarly, HBV DNA was determined in a human neuroblastoma and when DNA samples from the neuroblastomas and nephroblastomas were tested, HBV DNA was found in 6 out of 9 of these [11]. In parallel, SK-N-SH human neuroblastoma cells were shown to express HBV receptors [12].
5. Oligodendroglial-Astrocytic biprogenitor (O2A) cells in demyelinating disease. A neurodevelopmental differentiation failure?
4. Rare neuroimmune/demyelinizing side effects of HBV-V
Archelos et al. analyzed CNS-specific antigens in MS and whether antigens exist against oligodendrocyte-precursor cells [22]. To achieve this goal, they constructed a λ-phage protein expression library from a cell line with oligodendrocyte-precursor features [22]. The library was
The health benefits of current vaccination programs are beyond question, since vaccinations have reduced infectious disease mortalities 2
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through astrocyte-origined CXCL10 [31]. Postmortem investigations of brain lesions from MS patients demonstrated that in 30% of the lesions remyelination did not occur as a result of malfunctions in oligodendroglial progenitor cell recruitment, In 70%, inhibition of oligodendroglial progenitor cell diferentiation was to blame [32].
then analyzed to determine the presence of crossreactivity with cerebrospinal fluid (CSF) from 54 subjects with MS. Importantly, they isolated a B-cell epitope that reacts in 44% of cases [22]. Another significant finding included Wren and Noble’s demonstration that oligodendrocytes and O2A oligodendrocyte/astrocyte biprogenitor cells obtained from the optic nerves of rats bind and induce complement activation in the absence of antibodies in vitro, causing their demise [23]. Moreover, this feature was not observed for perinatal O2A biprogenitors, astrocytes, meningeal cells, or Schwann cells [23]. Chang et al analyzed oligodendrocyte progenitor cells in normal adult cerebral tissues and MS lesions by employing anti-NG2 antibodies [24]. Oligodendrocyte progenitors express NG2, chondroitin sulfate proteoglycan, in their integral membrane [24]. These cells also express PDGFαR (platelet-derived growth factor-α receptor). NG2-positive cells of stellate shape abundantly exist in the white and gray matter of brain specimens obtained from normal human brain tissue and exert features distinct from glia, oligodendroglia, and microglia. In many of the MS lesions, there was a dramatic reduction of NG2 cells, which supports their specific death [24]. Some cells positive for NG2 expressed the proapoptotic p75 NTR, yet this expression did not coincide with TUNELpositivity labelling apoptotic cells. Hence, the authors have presumed that p75 NTR expression did not directly relate to oligodendrocyteprogenitor cell apoptosis [24]. Nonetheless, it is also possible that these cells may lose the expression of this receptor when the execution of apoptosis is at a later stage. Armstrong et al. demonstrated the presence of “pre-oligodendrocytes” in adult human brain with features similar to that of O2A biprogenitors [25]. They also underlined that early oligodendrocyteprogenitors may also be accepted as O2A cells and express gangliosides such as A2B5 and GD3 [25]. Scolding et al. supported these findings and encountered A2B5+ O2A biprogenitor cells with processes and A2B5+ type-II astrocytes in adult human white matter in situ by analyzing fresh tissue print preparations [26]. Kuhlmann et al. demonstrated that in healthy human CNS tissues the Nkx2.2 and Olig2 transcription factors are robustly expressed in oligodendroglial progenitors while mature oligodendrocytes express lower levels of these transcription factors [27]. In early MS lesions, Olig2+ progenitor cells exist at all lesion stages and in periplaque white matter (PPWM) in higher amounts than the normal white matter [27]. Strikingly, in chronic lesions of MS, progenitor cells still existed but in significantly lower density compared to MS lesions at the beginning stage [27]. The authors attributed these observations to the differentiation failure of oligodendrocyte progenitors [27]. In parallel, reduced differentiation capability of NG2-positive oligodendroglial progenitor cells was demonstrated in another demyelinizing model, Theiler’s murine encephalitis, which may associate with aberrant activation of STAT3 pathway [28,29]. Importantly, the STAT3 pathway is one of the most potent protumorigenic cascades because it induces stem cell-like features, proliferation, survival and metastatic capabilities of cancer cells. In addition, the STAT3 pathway aids in fueling tumor cells by activating metabolic pathways important in carcinogenesis [30]. In situ observations of active MS lesions revealed that oligodendroglial progenitor cells are more susceptible to injury than mature oligodendrocytes even within the same lesion area [31]. In vitro and animal studies demonstrated increased susceptibility of oligodendroglial progenitor cells to inflammatory molecules, including TNFα, in comparison to more mature oligodendrocytes [31]. More recent studies have demonstrated that molecules released by proinflammatory immune cells could both block the differentiation and induce cell death in oligodendroglial progenitor cells. Supernatants obtained from cell cultures of M1-polarized myeloid cells and inflammatory T cells (Th1/Th17) exerted direct cytotoxicity on human A2B5+ progenitors, causing decreased O4+ and GalC+ oligodendrocytes [31]. Again, supernatants obtained from cell cultures of astrocytes exposed to inflammatory supernatants also hindered differentiation of oligodendrocyte progenitor cells which was mediated
6. Oligodendroglial progenitor and O2A cells in GBM O2A Oligodendrocyte-Astrocyte Biprogenitor Cells were first characterized with the studies of Noble et al. [33]. They successfully isolated O2A cells from the optic nerve of rats which could differentiate into both oligodendroglia and type-2 astrocytes. The O2A cells demonstrated that the simultaneous presence of growth factors (i.e. FGF and PDGF) cause their robust proliferation in absence of differentiation [33]. The same group demonstrated the existence of human glioblastoma cells which possess features very similar to rat O2A cells [27]. Hu-O-2A/Gb1 (Human O-2A lineage Glioblastoma-1) cells responded to the same mitogens and differentiation inducers as rat O2A progenitors, and additionally gave rise to cells with the properties of precursor cells, glia and oligodendroglia [34]. Moreover, 1H NMR analysis of amino acid content revealed a prominent overlap of free amino acid types and levels between the rodent primary O2A cells and human GBM cells [34]. Barnett et al. demonstrated that c-myc and H-ras transformed O2A biprogenitor cells formed GBMs after stereotactic injection into the rat brain [35]. These tumors were highly cellular, composed of poorly differentiated cells with fusiform, round or pleomorphic morphology, and included necrotic areas with variable GFAP expression [35]. The authors also suggested that some previously characterized glial tumor cell lines, Ln18 and SB18 clone-2 had features similar to O2A cells [35]. Supporting the O2A-origin of at least a fraction of high grade glial tumors, olig1 and olig2 transcription factors are also expressed in astrocytic tumors independent of grade [36]. Liu et al. modeled gliomagenesis in mice by inducing simultaneous Nf1/p53 mutations in neural stem cells (NSCs) [37]. Of significance, prominent aberrant growth before gliomagenesis occured only in oligodendrocyte precursor cells, but not in NSCs or in any other NSC-derived cell lineages [37]. Following tumorigenesis, phenotypic and transcriptomic characterization of malignant cells demonstrated clear features of oligodendroglial progenitor cells. Moreover, inducing the same Nf1/p53 mutations into oligodendroglial progenitor cells steadily led to glioma formation [37]. The authors underlined the fact that oligodendroglial progenitor cells constitute the largest proliferative pool in the rat and human brain [37]. Moreover, oligodendroglial progenitor cells at early developmental stages express common cell markers, including Sox2 and nestin [37]. Peculiarly, human oligodendroglial progenitor cells from subcortical white matter inherently behave like NSCs; revealing the likelihood that primate oligodendroglial progenitor cells are more plastic than their counterparts in rodents [37]. The injection of viral vectors expressing PDGF-BB into the corpus callosum, the forced induction of PDGD-BB expression under CNPase (an oligodendrocyte specific enzyme) promoter, and the stimulated expression of v-erbB under human S100β promoter caused gliomagenesis with oligodendroglial progenitor cell features [37]. Certainly, these data do not provide definitive proof of the hypothesis suggesting that oligodendroglial progenitor cells constitute the origin of gliomagenesis. However, it is clearly established that oligodendroglial progenitor cells are the cell-of-origin for gliomas if they harbour relevant gene mutations [37]. In 2014, Lindberg et al. demonstrated that oligodendroglial progenitor cells can give rise to astrocytomas or oligodendrogliomas depending on their transduced oncogenes [38]. Lastly, by 2018, it was firmly established that the gene expression profile of so called “proneural” glioblastomas exerted extensive resemblance with that of oligodendrocytes. Additionally, it was proposed that IDH1 mutant GBMs may originate from oligodendrocyte progenitor cells, rather than from astrocytic cells undergoing dedifferentiation [39]. According to many observations, it is possible to assume that at least a certain percentage of high grade glial tumors and 3
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Pilot Animal Studies With HBV-V: Second-generation HBV-Vs include several recombinant DNA products either based on the coding sequence for the S gene or on the coding sequence for the pre-S2 and S genes [13,44]. We will compare different HBV-Vs to determine whether the presence of pre-S2 segment could change any immunostimulant efficacies. We will employ intracranially-implanted C6 glioblastoma models as proof-of-principle experiments. We have experience in establishing and studying this model [41,42]. More importantly, dependent on the differentiation-inducing micromillieu, C6 glioblastoma cells may express both early and late differentiation markers of O2A biprogenitors, oligodendroglial cells, astrocytes and even neurons including A2B5, GalC, CNPase, GFAP and βIII-tubulin, respectively [43–45]. Thus, we will not only determine the effects of HBV-V on glioma growth (as will be assessed with survival data and volumetric analysis); but we will also stain for these differentiation markers with immunohistochemistry to reveal which pool of GBM cells are particularly vulnerable to be eradicated with this immunoadjuvant application. If we obtain significant differences, we will step-by-step detail our studies including FACS analysis of peripheral and tumor-infiltrating immunocytes and extend our studies with transgenic mice developing glial tumors.
oligodendrogliomas have the same origin, but de-differentiate into different lineages with differing mutations. As suggested in this hypothetical review article, we presume that immune reactions against hepadnavirus antigens can cause both harm and benefit. The mechanisms involve stimulating immunotoxicity against O2A cells leading to demyelination and immune-eradication of aberrantly-differentiating O2A cells, which may give rise to the development of GBM. If the second scenario is also relevant, one should expect lower prevalance of GBM in patients immune to hepadnaviri. Indeed, this is the case. 7. Reduced prevalance of GBM in Hepatitis B and Hepatitis C carriers Cabanne et al. employed a retrospective analysis to determine the prevalence of HBV and HCV in subjects suffering from meningioma or GBM [1]. Patient’s data were gathered by a retrospective chart review at two different institutions in Southern California. The first institution was the City of Hope National Medical Center (COH), a comprehensive cancer center located in Duarte (CA, USA), and the second was the Arrowhead Regional Medical Center (ARMC) in Colton (CA, USA). Data available from 1998 to 2009 at the COH and 2001–2010 at the ARMC were searched electronically for all patients with brain tumors and their serological testing results within the medical record were compared with the normal population. The combined prevalence of HBV and HCV in the USA from 1999 to 2008 was 5.7% [1]. The prevalence of HBV and HCV was 2.4% and 1.38% in patients with meningiomas and GBMs, respectively; which indicates a protective role of viral hepatitis infections against brain tumors. Furthermore, the OR of having HBV and HCV in glioblastoma versus meningiomas was 0.56, indicating that viral hepatitis has a profound preventive effect on glioblastomas having much more malignant character and profound mortality rates. Compared with historical controls, the total HBV and HCV prevalance in meningioma and GBM patients was found to be less [1].
10. Limitations of the current hypothesis and further evidence from gene expression studies In the available epidemiological literature, the immune parameters which assessed the development of immunity against HBV are not detailed and we only have seroprevalance data in regard to brain tumors. Hence, we do not have current solid evidence for our hypothesis. Additionally, we do not know the prevalence of GBM tumors that expressed similar antigenic epitopes as HBV. On the other hand, there exist some gene expression studies which provide some further evidence for our hypothesis. Haque et al. extracted RNA from 16 pediatric GBM and 3 control brains and on this RNA source, they performed RTPCR on housekeeping and tumor-associated genes and microarray analyses [47]. Despite RNA degradation, microarray analysis was possible on 16 of 19 samples and reproduced the pattern of findings obtained on fresh frozen pediatric GBM tissues. Strikingly, Hepatitis B surface antigen-antibody variable domain was found among the top 100 differentially expressed transcripts in fresh frozen and formalin-fixed paraffin-embeded specimens obtained from pediatric GBM tissues in comparison to the control brain tissues [47]. Here, it would not be illogical to presume that immune cells infiltrating GBM tissues somehow triggered immune responses against HBV surface antigens likely due to a similarity between glioma surface antigen epitops and HBV surface antigenic epitops. The nucleotide sequence of this transcript is known [48] (UniGene ID: Hs.449598; https://www.ncbi.nlm.nih.gov/ nuccore/M88310?report=GenBank); and shared antigens between GBM and HBV may be purified from the deduced peptide sequences.
8. Harnessing the side effects of HBV-V as side benefits against GBMs Above, we provided several lines of evidence that O2A progenitors may be the target in demyelinizing conditions and O2A cells may give rise to at least a fraction of GBMs. Here, we hypothesize that HBV-V may kill O2A progenitors with aberrant differentiation in a small fraction of vaccinized patients harbouring ill-differentiated O2A cells. This hypothesis is consistent with both phenomena of: i) HBV-V associated demyelinizing conditions being rare events; ii) HBV prevalance is lesser in brain tumor patients and especially so in GBM patients. While HBV-V antigens may induce immunological death of O2A cells in patients without manifest brain tumors (and those harbouring ill-differentiated progenitor cells), it may also kill and eradicate malignant O2A cells in later stages of tumor initiation. Very strikingly, a study published in 2015 found that, with a broad analysis of rAb (recombinant antibody) binding to human U87MG GBM cells, more than 50% of MS CSF (cerebrospinal fluid)-derived rAbs bound strongly to GBM cells [40]. This finding also supports our hypothesis that both diseases may associate with an aberrant cell differentiation pattern.
11. Conclusions In recent years, immunotherapy of cancer received a renewed interest and impetus with the discovery of immune-checkpoint inhibitors and their profound clinical success in melanoma [46]. HBV-vaccines may be good candidates to increase the efficacy of novel immune treatment strategies against cancer.
9. Future plans to analyze potentials of HBV-V as antineoplastic immunostimulants In the near future, we will employ two different studies to determine the potential efficacies of HBV-Vaccines against cancer: i) epidemiological analyses, ii) animal experiments in rats inoculated with GBM cells. Epidemiological Analyses: We will perform case-control studies to determine the prevalance of HBV in our patient cohort (more than 1000 patients with GBM) and compare this with the HBV prevalance in Istanbul, Turkey. If any significant differences are found, a multi-centric analysis will be planned to conduct larger epidemiological studies.
Source of funding None. Declaration of Competing Interest None to declare. 4
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Hypothesis: Could Hepatitis B vaccine act as an immune adjuvant in glioblastoma? Clues to conduct further epidemiological analyses ⁎
Meric A. Altinoza, , Aysel Ozpinara, Alp Ozpinarb, Emily Hackerb, İlhan Elmacic a
Department of Medical Biochemistry, Acibadem University, Istanbul, Turkey Department of Neurosurgery, Pittsburgh University, Pittsburgh USA c Department of Neurosurgery, Acibadem University, İstanbul, Turkey b
A B S T R A C T
A failure of neurodevelopmental differentiation at the level of oligodendroglial-astrocytic biprogenitors (O2A) is shown to be involved in the pathogenesis of both multiple sclerosis (MS) and glioblastoma multiforme (GBM). In this review article, we suggest that certain antigens of Hepatitis B Virus (HBV) and HBV-Vaccine (HBV-V) could act as immune stimulants in GBM treatment based on several lines of evidence. HBV-Vs may cause rare but prominent neuroimmune side effects including demyelination and multiple sclerosis, which may be associated with HBV-proteins creating antigenic mimicry of oligodendroglial progenitors. The combined prevalance of HBV and Hepatitis C Virus-carrier state is less in patients with brain tumors compared to healthy subjects. Furthermore, within the population of patients with brain tumors, the prevalence is even about two times lesser in GBM in comparison to those with a diagnosis of meningioma. Although indirectly, this epidemiological data may indicate that the immune response triggered against hepadnavirus antigens would eliminate aberrantly differentiating O2A progenitor cells giving rise to GBMs. Moreover, Hepatitis B surface antigen-antibody variable domain is among the top 100 differentially expressed transcripts in fresh frozen and formalin-fixed paraffin-embeded specimens obtained from pediatric GBM tissues in comparison to the control brain tissues. However, the provided evidence is still premature and we think that HBV-V warrants investigation first by epidemiological studies and then by animal experiments to determine whether it reduces the risk of GBM and whether it could slow GBM growth via immune stimulation.
1. Introduction Glioblastoma multiforme (GBM) is the most grave malignancy among the brain cancers, with a 5-year survival of less than < 4% [1]. Unfortunately, GBM also accounts for about 80% of primary brain cancers and affects more than 17,000 patients annually in the USA alone [2,3]. Prominent invasiveness is one of the major features of GBMs which hinders total surgical resection. For more than a century, it is well established that certain infections or vaccinations could induce very rare but dramatic instances of “spontaneous cancer healing” [4,5]. In this manuscript, we propose that Hepatitis B Virus Vaccines (HBVVs) may be new adjuvants in immunotherapy of GBM. We will explain several features to support our proposal: 1- HBV-Vaccines, which have ample benefit, also cause rare side effects mostly manifested as demyelinating diseases such as Multiple Sclerosis (MS). We believe that this feature may be associated with the antigenic similarity between certain HBV proteins and O2A (oligodendroglia-astrocyte biprogenitor cell) surface proteins. 2- O2A cell pathology is a common feature for both MS and GBM. In MS, there is evidence suggesting immunity against oligodendroglial progenitor cells and a differentiation failure of oligodendrocyte progenitors are involved in the pathology. In parallel, certain evidence showed a blockage of differentiation at O2A stage in
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GBM oncogenesis. 3- HBV- and HCV-carrier state is less in GBM patients, which may suggest that the developed immunity against proteins of hepadnaviri (including HBV and HCV) may also trigger immune responses against undifferentiated/aberrant O2A biprogenitors, subsequently causing their elimination, and reducing the likelihood of GBM formation. 4- Hepatitis B surface antigen-antibody variable domain is among the top 100 differentially expressed transcripts in specimens obtained from pediatric GBM tissues in comparison to the control brain tissues. Below, we will summarize the relevant data, but we will first describe the features of HBV and the neuro-immune side effects of HBVV. 2. HBV antigens exert similarities to malignant and benign tissue antigens The serum specimens taken from carriers of HBV (17 out of 21) crossreact with the basal cell layer of squamous epithelia and intensely with thymic stellate epithelia [6]. In the thymic tissue, stellate epithelia reside in the cortex and medulla and their morphological and residential features exert similarities to the epithelia producing the immunostimulant molecule α1-thymosin [6]. The immune network theory suggests that there is a production of various anti-idiotype (anti-id)
Corresponding author at: Acibadem University, Atasehir, Istanbul, Turkey. E-mail address: [email protected] (M.A. Altinoz).
https://doi.org/10.1016/j.intimp.2019.106038 Received 18 August 2019; Received in revised form 7 October 2019; Accepted 7 November 2019 1567-5769/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Meric A. Altinoz, et al., International Immunopharmacology, https://doi.org/10.1016/j.intimp.2019.106038
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more than any public health intervention and antibiotics [13]. Nonetheless, serious side effects have been reported following vaccination, including with HBV-vaccines [14]. The adverse effects comprise of disorders with autoimmune features and neural manifestations. In a period of six years, 35 primary demyelinating events 8 weeks after HBV vaccinization were witnessed in Paris [13]. Completing a mean followup of 3 years, half of these cases developed Multiple Sclerosis (MS) [13]. The retrospective case-control study was performed at the Salpêtrière on patients experiencing their first episode of CNS demyelination (FECD) after HBV-vaccination [13]. There were 121 cases with a FECD and adjusted odds ratios (OR) between a FECD and HBV-vaccinization of 1.7 during the previous 60 days and 1.5 during the previous 61–180 days [13]. An investigation of 753 MS patients over a 2 years period identified 92 subjects who were vaccinated against HBV. Among these patients, 46 developed a FECD, 20 in less than 8 weeks following HBV vaccination [14]. From a DR2+ subject who suffered from MS after HBV vaccination, a T lymphocyte clone specific for HBV antigen was found that reacts with a peptide existing in the structure of proteolipid protein (PLP) (aminoacids 179 to 197) [13]. A nested case–control study defined a association between HBV vaccination and an increased incidence of MS in the 3 years following vaccination [15]. This investigation revealed that 11 of the 143 MS cases were vaccinated with HBV-V within a 3-year interval, indicating that longer periods should be assessed when determining vaccination safety in patients with MS [15]. Also, according to the VAERS database (US Vaccine Adverse Events Reporting System), adults vaccinated against HBV exerted significantly higher odds ratios (OR) for MS (OR = 5.2; p < 0.0003), and optic neuritis (OR = 14; p < 0.0002) in comparison to those vaccinated against tetanus [16]. Bogdanos et al. investigated aminoacid similarities between the sHBsAg, and the MS-autoantigens myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG) [17]. Peptides of 20 amino acid-length were synthesized spanning 4 sHBsAg/MOG and 1 sHBsAg/ MBP mimicking pair and evaluated with ELISA. 147 samples from 58 adults were obtained prior to HBV vaccination (58/58), and after HBV vaccination [18]. Reactivity to at least one of the MOG mimics existed in 4 (8%) pre-HBV vaccinated and in 30 (60%) post-HBV vaccinated subjects (p = 0.001). 30/50 (60%) of the vaccinated subjects had sHBsAg/MOG double reactivity compared to none before vaccination. No reactivity was found against the sHBsAg/MBP mimics. Some investigators indicated that those who developed autoimmunity after HBV-V had exerted certain patterns of human leukocyte antigens (HLA) [18]. Acute disseminated encephalomyelitis (ADEM) is a very rare neuroinflammatory demyelinating disorder of the central nervous system (CNS) [19]. ADEM can develop in any age but is mainly a disease of children and young adults. A mildly enhanced risk for ADEM was demonstrated by Touze and Mikaeloff specifically after Engerix B HBV-vaccine [19]. Reports also exist on isolated optic neuritis following various vaccinations including Hepatitis A and B [20]. Acute transverse myelitis is a rare acute inflammatory spinal disorder in childhood [20]. An analysis of all English literature published between 1970 and 2009, revealed 37 transverse myelitis cases following different vaccinizations including 13 after HBVvaccination [20]. HBV-Vs are also the second most common vaccines reported to the VAERS [21]. A retrospective analysis of the VAERS database in 2004 revealed 120 reports of myelitis after HBV vaccination. A structural mimicry of virus antigens to host antigens was assumed to cause these adverse events [21].
antibodies during an immunological response to a specific antigen. Certain subtypes of “internal-image” anti-id antibodies are anti-paratopic and exert the 3D-molecular structure of the original antigen. Hence, it is possible that anti-idiotype antibodies against HBV-surface antigens may bind and activate thymic stellate cells. HBV polymerase (HBVP) shares six amino acids His-Tyr-Gly-Ser-Leu-Pro-Gln with the encephalitogenic epitopes of MBP (rabbit myelin basic protein) [7]. As we will discuss below, vaccination against HBV may cause neuroimmune side effects including MS. In MS, it is well established that there exists auto-immunity against myelin proteins. There may also exist other molecular mimicries between HBV proteins and proteins of aberrantly-dedifferentiating oligodendroglial progenitorss. Such mimicries may eliminate these cells and subsequently lower the risk of GBM which will be detailed below. Monoclonal antibodies were produced against a cell lineage obtained from a carcinogen-induced large bowel tumor. One of these antibodies (MAb E4/pE4) slightly stained the healthy small and large bowel but not other the normal organs, as detected with immunohistochemical staining (IHC) [8]. However, in the colon carcinoma cells, pE4 reacted with the cell membranes. The first 20 amino-terminal amino acids of pE4 were the same as residues 5 to 25 from glycoprotein-30 (gp30) from HBsAg [8]. Antigenic epitopes linked with HLA class I molecules on a bladder cancer cell line were defined and 1 of the 3 peptides (VTDPGNLLY) exerted partial homology to HBV (corresponding to aa residues 122–128) [9]. Altogether, these finding may indicate that malignant cells express HBV epitopes and thus, antibodies against HBV epitopes could have triggered immunity against them. Indeed, there exist anecdotal reports of regression of neuroectodermal cancers such as neuroblastomas. Additionally, glioblastoma risk is lower in carriers of HBV and HCV. These facts are discussed below. 3. Regression of neuroblastoma coincident with HBsAg seroconversion In 1982, a dramatic regression of a far advanced neuroblastoma was documented which began following acute infection with HBV [10]. At that time, spontaneous remisson of neuroblastoma in infants was fairly known, whereas total remission in patients older than two years was very rarely reported. A 13-year-old boy had many bony, retroperitoneal, and liver metastases, with a diagnosis of an anaplastic type neuroblastoma. Chemotherapy including adriamycin, cyclophosphamide and dacarbazine and radiotherapy did not lead to any healing [10]. However, 14 months after diagnosis, the patient’s general condition became strikingly better, and 3 months later, his tests demonstrated serum conversion of HBsAg with increased liver enzymes and γglobulin levels [10]. Afterwards, abdominal and osteolytic metastates vanished in a progressive manner. Moreover, a PET scan revealed complete vanishing of hepatic metastases and urine levels of VMA decreased to normal ranges [10]. When this case was published, the patient was 16 years old and free of detectable disease three years after diagnosis [10]. At that time, the survival of neuroblastomas for children over seven years was 8%. Moreover, survival was even more rare in patients with disseminated skeletal metastases and such a circumstance involving a child over 6 years old had not been published at the time of the report. The authors proposed that the antigenic epitopes of HBV triggered anticancer immune responses and rejection of the tumor [10]. Peculiarly, HBV DNA was determined in a human neuroblastoma and when DNA samples from the neuroblastomas and nephroblastomas were tested, HBV DNA was found in 6 out of 9 of these [11]. In parallel, SK-N-SH human neuroblastoma cells were shown to express HBV receptors [12].
5. Oligodendroglial-Astrocytic biprogenitor (O2A) cells in demyelinating disease. A neurodevelopmental differentiation failure?
4. Rare neuroimmune/demyelinizing side effects of HBV-V
Archelos et al. analyzed CNS-specific antigens in MS and whether antigens exist against oligodendrocyte-precursor cells [22]. To achieve this goal, they constructed a λ-phage protein expression library from a cell line with oligodendrocyte-precursor features [22]. The library was
The health benefits of current vaccination programs are beyond question, since vaccinations have reduced infectious disease mortalities 2
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through astrocyte-origined CXCL10 [31]. Postmortem investigations of brain lesions from MS patients demonstrated that in 30% of the lesions remyelination did not occur as a result of malfunctions in oligodendroglial progenitor cell recruitment, In 70%, inhibition of oligodendroglial progenitor cell diferentiation was to blame [32].
then analyzed to determine the presence of crossreactivity with cerebrospinal fluid (CSF) from 54 subjects with MS. Importantly, they isolated a B-cell epitope that reacts in 44% of cases [22]. Another significant finding included Wren and Noble’s demonstration that oligodendrocytes and O2A oligodendrocyte/astrocyte biprogenitor cells obtained from the optic nerves of rats bind and induce complement activation in the absence of antibodies in vitro, causing their demise [23]. Moreover, this feature was not observed for perinatal O2A biprogenitors, astrocytes, meningeal cells, or Schwann cells [23]. Chang et al analyzed oligodendrocyte progenitor cells in normal adult cerebral tissues and MS lesions by employing anti-NG2 antibodies [24]. Oligodendrocyte progenitors express NG2, chondroitin sulfate proteoglycan, in their integral membrane [24]. These cells also express PDGFαR (platelet-derived growth factor-α receptor). NG2-positive cells of stellate shape abundantly exist in the white and gray matter of brain specimens obtained from normal human brain tissue and exert features distinct from glia, oligodendroglia, and microglia. In many of the MS lesions, there was a dramatic reduction of NG2 cells, which supports their specific death [24]. Some cells positive for NG2 expressed the proapoptotic p75 NTR, yet this expression did not coincide with TUNELpositivity labelling apoptotic cells. Hence, the authors have presumed that p75 NTR expression did not directly relate to oligodendrocyteprogenitor cell apoptosis [24]. Nonetheless, it is also possible that these cells may lose the expression of this receptor when the execution of apoptosis is at a later stage. Armstrong et al. demonstrated the presence of “pre-oligodendrocytes” in adult human brain with features similar to that of O2A biprogenitors [25]. They also underlined that early oligodendrocyteprogenitors may also be accepted as O2A cells and express gangliosides such as A2B5 and GD3 [25]. Scolding et al. supported these findings and encountered A2B5+ O2A biprogenitor cells with processes and A2B5+ type-II astrocytes in adult human white matter in situ by analyzing fresh tissue print preparations [26]. Kuhlmann et al. demonstrated that in healthy human CNS tissues the Nkx2.2 and Olig2 transcription factors are robustly expressed in oligodendroglial progenitors while mature oligodendrocytes express lower levels of these transcription factors [27]. In early MS lesions, Olig2+ progenitor cells exist at all lesion stages and in periplaque white matter (PPWM) in higher amounts than the normal white matter [27]. Strikingly, in chronic lesions of MS, progenitor cells still existed but in significantly lower density compared to MS lesions at the beginning stage [27]. The authors attributed these observations to the differentiation failure of oligodendrocyte progenitors [27]. In parallel, reduced differentiation capability of NG2-positive oligodendroglial progenitor cells was demonstrated in another demyelinizing model, Theiler’s murine encephalitis, which may associate with aberrant activation of STAT3 pathway [28,29]. Importantly, the STAT3 pathway is one of the most potent protumorigenic cascades because it induces stem cell-like features, proliferation, survival and metastatic capabilities of cancer cells. In addition, the STAT3 pathway aids in fueling tumor cells by activating metabolic pathways important in carcinogenesis [30]. In situ observations of active MS lesions revealed that oligodendroglial progenitor cells are more susceptible to injury than mature oligodendrocytes even within the same lesion area [31]. In vitro and animal studies demonstrated increased susceptibility of oligodendroglial progenitor cells to inflammatory molecules, including TNFα, in comparison to more mature oligodendrocytes [31]. More recent studies have demonstrated that molecules released by proinflammatory immune cells could both block the differentiation and induce cell death in oligodendroglial progenitor cells. Supernatants obtained from cell cultures of M1-polarized myeloid cells and inflammatory T cells (Th1/Th17) exerted direct cytotoxicity on human A2B5+ progenitors, causing decreased O4+ and GalC+ oligodendrocytes [31]. Again, supernatants obtained from cell cultures of astrocytes exposed to inflammatory supernatants also hindered differentiation of oligodendrocyte progenitor cells which was mediated
6. Oligodendroglial progenitor and O2A cells in GBM O2A Oligodendrocyte-Astrocyte Biprogenitor Cells were first characterized with the studies of Noble et al. [33]. They successfully isolated O2A cells from the optic nerve of rats which could differentiate into both oligodendroglia and type-2 astrocytes. The O2A cells demonstrated that the simultaneous presence of growth factors (i.e. FGF and PDGF) cause their robust proliferation in absence of differentiation [33]. The same group demonstrated the existence of human glioblastoma cells which possess features very similar to rat O2A cells [27]. Hu-O-2A/Gb1 (Human O-2A lineage Glioblastoma-1) cells responded to the same mitogens and differentiation inducers as rat O2A progenitors, and additionally gave rise to cells with the properties of precursor cells, glia and oligodendroglia [34]. Moreover, 1H NMR analysis of amino acid content revealed a prominent overlap of free amino acid types and levels between the rodent primary O2A cells and human GBM cells [34]. Barnett et al. demonstrated that c-myc and H-ras transformed O2A biprogenitor cells formed GBMs after stereotactic injection into the rat brain [35]. These tumors were highly cellular, composed of poorly differentiated cells with fusiform, round or pleomorphic morphology, and included necrotic areas with variable GFAP expression [35]. The authors also suggested that some previously characterized glial tumor cell lines, Ln18 and SB18 clone-2 had features similar to O2A cells [35]. Supporting the O2A-origin of at least a fraction of high grade glial tumors, olig1 and olig2 transcription factors are also expressed in astrocytic tumors independent of grade [36]. Liu et al. modeled gliomagenesis in mice by inducing simultaneous Nf1/p53 mutations in neural stem cells (NSCs) [37]. Of significance, prominent aberrant growth before gliomagenesis occured only in oligodendrocyte precursor cells, but not in NSCs or in any other NSC-derived cell lineages [37]. Following tumorigenesis, phenotypic and transcriptomic characterization of malignant cells demonstrated clear features of oligodendroglial progenitor cells. Moreover, inducing the same Nf1/p53 mutations into oligodendroglial progenitor cells steadily led to glioma formation [37]. The authors underlined the fact that oligodendroglial progenitor cells constitute the largest proliferative pool in the rat and human brain [37]. Moreover, oligodendroglial progenitor cells at early developmental stages express common cell markers, including Sox2 and nestin [37]. Peculiarly, human oligodendroglial progenitor cells from subcortical white matter inherently behave like NSCs; revealing the likelihood that primate oligodendroglial progenitor cells are more plastic than their counterparts in rodents [37]. The injection of viral vectors expressing PDGF-BB into the corpus callosum, the forced induction of PDGD-BB expression under CNPase (an oligodendrocyte specific enzyme) promoter, and the stimulated expression of v-erbB under human S100β promoter caused gliomagenesis with oligodendroglial progenitor cell features [37]. Certainly, these data do not provide definitive proof of the hypothesis suggesting that oligodendroglial progenitor cells constitute the origin of gliomagenesis. However, it is clearly established that oligodendroglial progenitor cells are the cell-of-origin for gliomas if they harbour relevant gene mutations [37]. In 2014, Lindberg et al. demonstrated that oligodendroglial progenitor cells can give rise to astrocytomas or oligodendrogliomas depending on their transduced oncogenes [38]. Lastly, by 2018, it was firmly established that the gene expression profile of so called “proneural” glioblastomas exerted extensive resemblance with that of oligodendrocytes. Additionally, it was proposed that IDH1 mutant GBMs may originate from oligodendrocyte progenitor cells, rather than from astrocytic cells undergoing dedifferentiation [39]. According to many observations, it is possible to assume that at least a certain percentage of high grade glial tumors and 3
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Pilot Animal Studies With HBV-V: Second-generation HBV-Vs include several recombinant DNA products either based on the coding sequence for the S gene or on the coding sequence for the pre-S2 and S genes [13,44]. We will compare different HBV-Vs to determine whether the presence of pre-S2 segment could change any immunostimulant efficacies. We will employ intracranially-implanted C6 glioblastoma models as proof-of-principle experiments. We have experience in establishing and studying this model [41,42]. More importantly, dependent on the differentiation-inducing micromillieu, C6 glioblastoma cells may express both early and late differentiation markers of O2A biprogenitors, oligodendroglial cells, astrocytes and even neurons including A2B5, GalC, CNPase, GFAP and βIII-tubulin, respectively [43–45]. Thus, we will not only determine the effects of HBV-V on glioma growth (as will be assessed with survival data and volumetric analysis); but we will also stain for these differentiation markers with immunohistochemistry to reveal which pool of GBM cells are particularly vulnerable to be eradicated with this immunoadjuvant application. If we obtain significant differences, we will step-by-step detail our studies including FACS analysis of peripheral and tumor-infiltrating immunocytes and extend our studies with transgenic mice developing glial tumors.
oligodendrogliomas have the same origin, but de-differentiate into different lineages with differing mutations. As suggested in this hypothetical review article, we presume that immune reactions against hepadnavirus antigens can cause both harm and benefit. The mechanisms involve stimulating immunotoxicity against O2A cells leading to demyelination and immune-eradication of aberrantly-differentiating O2A cells, which may give rise to the development of GBM. If the second scenario is also relevant, one should expect lower prevalance of GBM in patients immune to hepadnaviri. Indeed, this is the case. 7. Reduced prevalance of GBM in Hepatitis B and Hepatitis C carriers Cabanne et al. employed a retrospective analysis to determine the prevalence of HBV and HCV in subjects suffering from meningioma or GBM [1]. Patient’s data were gathered by a retrospective chart review at two different institutions in Southern California. The first institution was the City of Hope National Medical Center (COH), a comprehensive cancer center located in Duarte (CA, USA), and the second was the Arrowhead Regional Medical Center (ARMC) in Colton (CA, USA). Data available from 1998 to 2009 at the COH and 2001–2010 at the ARMC were searched electronically for all patients with brain tumors and their serological testing results within the medical record were compared with the normal population. The combined prevalence of HBV and HCV in the USA from 1999 to 2008 was 5.7% [1]. The prevalence of HBV and HCV was 2.4% and 1.38% in patients with meningiomas and GBMs, respectively; which indicates a protective role of viral hepatitis infections against brain tumors. Furthermore, the OR of having HBV and HCV in glioblastoma versus meningiomas was 0.56, indicating that viral hepatitis has a profound preventive effect on glioblastomas having much more malignant character and profound mortality rates. Compared with historical controls, the total HBV and HCV prevalance in meningioma and GBM patients was found to be less [1].
10. Limitations of the current hypothesis and further evidence from gene expression studies In the available epidemiological literature, the immune parameters which assessed the development of immunity against HBV are not detailed and we only have seroprevalance data in regard to brain tumors. Hence, we do not have current solid evidence for our hypothesis. Additionally, we do not know the prevalence of GBM tumors that expressed similar antigenic epitopes as HBV. On the other hand, there exist some gene expression studies which provide some further evidence for our hypothesis. Haque et al. extracted RNA from 16 pediatric GBM and 3 control brains and on this RNA source, they performed RTPCR on housekeeping and tumor-associated genes and microarray analyses [47]. Despite RNA degradation, microarray analysis was possible on 16 of 19 samples and reproduced the pattern of findings obtained on fresh frozen pediatric GBM tissues. Strikingly, Hepatitis B surface antigen-antibody variable domain was found among the top 100 differentially expressed transcripts in fresh frozen and formalin-fixed paraffin-embeded specimens obtained from pediatric GBM tissues in comparison to the control brain tissues [47]. Here, it would not be illogical to presume that immune cells infiltrating GBM tissues somehow triggered immune responses against HBV surface antigens likely due to a similarity between glioma surface antigen epitops and HBV surface antigenic epitops. The nucleotide sequence of this transcript is known [48] (UniGene ID: Hs.449598; https://www.ncbi.nlm.nih.gov/ nuccore/M88310?report=GenBank); and shared antigens between GBM and HBV may be purified from the deduced peptide sequences.
8. Harnessing the side effects of HBV-V as side benefits against GBMs Above, we provided several lines of evidence that O2A progenitors may be the target in demyelinizing conditions and O2A cells may give rise to at least a fraction of GBMs. Here, we hypothesize that HBV-V may kill O2A progenitors with aberrant differentiation in a small fraction of vaccinized patients harbouring ill-differentiated O2A cells. This hypothesis is consistent with both phenomena of: i) HBV-V associated demyelinizing conditions being rare events; ii) HBV prevalance is lesser in brain tumor patients and especially so in GBM patients. While HBV-V antigens may induce immunological death of O2A cells in patients without manifest brain tumors (and those harbouring ill-differentiated progenitor cells), it may also kill and eradicate malignant O2A cells in later stages of tumor initiation. Very strikingly, a study published in 2015 found that, with a broad analysis of rAb (recombinant antibody) binding to human U87MG GBM cells, more than 50% of MS CSF (cerebrospinal fluid)-derived rAbs bound strongly to GBM cells [40]. This finding also supports our hypothesis that both diseases may associate with an aberrant cell differentiation pattern.
11. Conclusions In recent years, immunotherapy of cancer received a renewed interest and impetus with the discovery of immune-checkpoint inhibitors and their profound clinical success in melanoma [46]. HBV-vaccines may be good candidates to increase the efficacy of novel immune treatment strategies against cancer.
9. Future plans to analyze potentials of HBV-V as antineoplastic immunostimulants In the near future, we will employ two different studies to determine the potential efficacies of HBV-Vaccines against cancer: i) epidemiological analyses, ii) animal experiments in rats inoculated with GBM cells. Epidemiological Analyses: We will perform case-control studies to determine the prevalance of HBV in our patient cohort (more than 1000 patients with GBM) and compare this with the HBV prevalance in Istanbul, Turkey. If any significant differences are found, a multi-centric analysis will be planned to conduct larger epidemiological studies.
Source of funding None. Declaration of Competing Interest None to declare. 4
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