Patent Application
20160237120
The current invention relates to the identification of B-cell epitopes (as linear peptides) from human polyoma virus proteins and their use in an immune diagnostic assay.
Progressive multifocal leukoencephalopathy (PML) is a rare but often fatal brain disease caused by reactivation of the polyomavirus JC. The monoclonal antibodies natalizumab, efalizumab, and rituximab—used for the treatment of multiple sclerosis, psoriasis, hematological malignancies, Crohn's disease, and rheumatic diseases—have been associated with PML. Worldwide 181 (as of November 2011) cases of natalizumab-associated PML have been reported. International studies and standardization of methods are urgently needed to devise strategies to mitigate the risk of PML in natalizumab-treated patients.
A new set of assay developments could lead to a better understanding of the virus reactivation, and that could lead to safe use of immune modulating agents (e.g. a Tysabri® (natalizumab)) and an optimized treatment algorithm.
The human neurotropic polyomavirus JCV is a non-enveloped DNA virus belonging to the group of polyomaviruses. JCV is the etiologic agent of progressive multifocal leukoencephalopathy (PML). Other members of this viral family are BK virus (mainly infecting the kidneys), and the non-human SV40 virus. JC and BK viruses have been named using the initials of the first patients discovered with the diseases.
Epidemiological studies showed that in certain populations, the seroprevalence of close to 90% by age 20. In those healthy immunocompetent individuals, JCV is establishing a lifelong sub-clinical infection.
The initial site of infection may be the tonsils, or possibly the gastrointestinal tract. The virus remains latent and/or can infect the tubular epithelial cells in the kidneys where it continues to reproduce, thereby shedding virus particles in the urine. JCV can cross the blood-brain barrier, and enters into the central nervous system where it infects oligodendrocytes and astrocytes.
Immunodeficiency or immuno-suppression allows JCV to reactivate. In the brain, this will cause the usually fatal PML by destroying oligodendrocytes.
Therefore, PML is a demyleating disease affecting the white matter, but is in process different from multiple sclerosis (MS), in which the myelin itself is destroyed. Whether the process behind PML is caused by the reactivation of JCV within the CNS or seeding of newly reactivated JCV via blood or lymphatics is unknown. PML progresses much more quickly than MS.
There are case reports of PML being induced by pharmacological agents (efalizumab, rituximab, infliximab, natalizumab . . . ) but the process how JCV interacts with these mAbs and cause PML is again not clearly understood.
PML is diagnosed by testing for JC virus DNA in cerebrosinal fluid, or in brain biopsy specimens. In addition, brain damage caused by PML has been detected on MRI images.
As of today, there is no known cure for PML, but the disease can be slowed or stopped, dependent on improvement of the patient's immune restoration (e.g. HAART in AIDS patients). A rare complication of immune reconstitution is known as “immune reconstitution inflammatory syndrome (IRIS), in which increased immune system activity increases the damage caused by the infection. IRIS can be managed by pharmacological intervention, but it is extremely fatal if it occurs in PML.
In order to study the correlates of JCV and PML, a large collection of clinical samples is needed, inclusive with the individual's clinical background.
JCV replicates in several different types of tissues (tonsils, gastro-intestinal tract, kidney, brain). In order to obtain a representative set of genetic variants and the corresponding serological markers, it is aimed to start with the collection of a large sample set from urine, blood, CSF, bone marrow, and paraffin embedded brain biopsy material, and potentially tonsil biopsy. Blood cells can be separated into different compartments (FACS). PML is a rare disease present only in immune suppressed individuals, and access to these precious materials is foreseen to be limited. Most of the study objectives for assay design can be completed on samples from infected healthy individuals.
Sequencing of the JCV genome indicates at least seven major genotypes and numerous subtypes. The type distribution was found to be as follows: Type 1: in Europeans; Types 2 and 7: in Asians; Types 3 and 6: in Africans; Type 4: in the United States, the whole genome of Type 4 strains was found to be most closely related to Type 1; and Type 5: a single natural occurring recombinant strain of Type 6 in VP1 gene with Type 2B in the early region. These genotypes and subtypes have been defined in three ways: namely by i) a 610 bp region spanning the 3′ ends of the VP1 and T-antigen genes, ii) a 215 bp region of the 5′ end of the VP1 gene and iii) based on the sequence of the entire coding region of the genome (5130 bp in strain MAD-1, Accession number: PLYCG MAD-1) including untranslated regions except the archetypal regulatory region to the late side of ori.
Besides the genotypic variations, the regulatory domain and the VP1 region contains mutations that are found more frequently in PML patients. From the frequency of observation, it is thought that these mutations are positively selected, and are not just present by chance. Analysis of the VP1 sequences isolated from PML patients were compared to control samples from healthy individuals showing that the mutated residues are located within the sialic add binding site, a JC virus receptor for cell infection. It is therefore likely that a more virulent PML-causing phenotype of JC virus is acquired via adaptive evolution that changes viral specificity for its cellular receptor(s).
On the other hand, on the basis of the survival time (less or more than 6 months) from the onset of the disease, patients were grouped in slow and fast PML progressors (SP and FP PML). It was suggested that VP1 outer loops can contain polymorphic residues restricted to four positions (aa 74, 75, 117 and 128) in patients with slow PML progression, VP1 loop mutations are associated with a favorable prognosis for PML.
The genomic organization and variability of JCV in the transcriptional control region (TCR), a 400 base pare non-coding regulatory region, were described by Jensen (2001). In addition, distinctive point mutations or deletions in the regulatory region also provide useful information to supplement coding region typing.
Rearranged JCV regulatory regions (RR), including tandem repeat patterns found in the central nervous system (CNS) of PML patients, have been associated with neurovirulence.
In HIV-infected patients with virologically confirmed PML, highly active antiretroviral therapy (HAART) leads to a partial immune-mediated control of JCV replication in CSF. However, the virus may tend to escape through the selection of rearrangements in the RR, some associated with enhanced viral replication efficiency, other resulting in multiplication of binding sites for cellular transcription factors (Macrophage Chemoattractant Protein MCP-1, cellular transcription factor NF-1). In a case of PML in an HIV-1 infected individual that did not respond to HAART therapy, there was a simultaneous presence of JCV strains with four different TCR structures in urine, peripheral blood cells, serum, and CSF samples, for which the authors suggested that the archetype TCR is restricted to urine, while the degree of the rearrangement varies and increases from the peripheral blood to CSF.
It is currently not clear if PML is more frequently found within certain genotypes, or if certain genotypes are excluded from PML. Also the genetic polymorphisms in VP1 and the RR need further analysis in the context of the different genotypes, tissue distribution, and presence/absence of PML.
While infection is very common in most human populations, this is usually subclinical since the virus is readily controlled by the immune system. After the initial infection is resolved, JCV nonetheless persists in the body and enters a state of latency which is poorly understood. However, under circumstances in which the immune system becomes impaired, e.g., AIDS, the virus reactivates and replicates in the central nervous system (CNS) to cause PML. The mechanisms involved in this reactivation are not known but it is possible that changes in the levels of cytokines and immunomodulators, such as TNF-α, MIP-1α and TGF-β, that are associated with immunosuppression, elicit changes in intracellular signal transduction pathways that, in turn, modulate the activities of transcription factors (e.g. Sp1 and Egr-1) that bound to the GG(A/C)-rich sequences in the TCR. These transcription factors are involved in regulating the expression of JCV genes.
JCV DNA is frequently, but intermittently detected in peripheral blood, supporting the hypothesis of viral reservoirs. In addition, mRNAs were seldom associated with DNA, suggesting that JCV reactivation does not take place in peripheral blood. JCV might remain latent in the peripheral reservoir, and immune suppression might enable reactivation, thereby facilitating the detection of JCV DNA in blood. However, circulating virus might have no link to the emergence of PML.
Antibody titers to JCV were measured in the past with hemagglutination inhibition (HI) assays. Nowadays, hemagglutination- and HI-assays are only used to study modifications in Vp1 and the effect of these mutations on receptor recognition. HI assays are replaced by antibody detection technologies. The detected antibodies to JCV are against Vp1 epitopes, the protein that makes up 75% of the total virion protein.
Recently, in addition to the previously characterized viruses BK and JO, three new human polyomaviruses have been identified: KIV (respiratory tract infection), WUV (respiratory tract infection), and MCV (merkel cell carcinoma). It was determined that initial exposure to KIV, WUV, and MCV occurs in childhood, similar to that for the known human polyomaviruses BKV and JCV, and that their prevalence is high. In order to study exposure to these viruses in humans, recombinant polyomavirus VP1 capsid proteins were expressed in E. coli in an ELISA assay.
Sera of 1501 adult individuals were tested for the presence of 7 polyomaviruses (including SV40=primate virus, in humans through the SV40-contaminated polio vaccine: and LPV=lymphotropic polyoma virus in African green monkeys) and the authors indicated that there may be an age-related waning of BKV VP1 specific antibodies, but not for the other 6 polyomaviruses tested. Also, a difference in sero-prevalence with respect to gender for any of the 7 polyomaviruses tested was not found (Kean et al., 2009). Of the 195 samples exhibiting initial SV40 seroreactivity, only 7 (3%) were cross reactive with JCV Vp1 protein. No other cross reactivity with JCV Vp1 was observed.
Since there is a causal relationship of reactivation of JCV in CSF and the development of PML, knowing the JCV serological status of individuals with decreased immunological status is crucial. Theoretically, uninfected individuals (seronegative) should not be at risk for developing PML, while seropositive individuals are. There are case reports of PML being caused by pharmacological agents, although there is some speculation this could be due in part to the existing impaired immune response or ‘drug combination therapies’ rather than individual drugs. These include efalizumab, rituximab, belatacept, infliximab, natalizumab, chemotherapy, corticosteroids, and various transplant drugs such as tacrolimus.
Epidemiological studies suggest that the JCV infection occurs primarily in childhood, but the infection in adults is not excluded. Seronegative individuals undergoing immunosuppression and/or therapy should in generally not at risk, but they might be in the seroconversion window where antibodies are not yet properly available. Hence this population would require further attention and analysis by molecular diagnostic means. The sensitivity and specificity of a JC virus serology assay is of substantial interest because such an assay is now being considered as a means to assess the risk of PML in patients treated with natalizumab.
Current available immune-assays are based on VP1 only, expressed in a baculovirus expression system, in an E. coli expression system or in a yeast expression system. No other viral proteins are available in such an assay meaning that only so-called conformational epitopes, but not linear epitopes present in the three dimensional structure of the virus, are part of the immune assay. As a consequence thereof human samples potentially containing antibodies directed against the missing part as such, will not be detected.
As a final Tysabri treatment algorithm would require the knowledge of the infection status, there is a high unmet medical need to:
The current invention therefore relates to human polyoma virus peptide sequences possessing an immune activity towards human antibodies in human samples.
More specifically the current invention makes it unexpectedly possible to use the human polyoma viral small T antigen for immune response diagnostic purposes.
The 63 specific sequences identified in Table 9 are considered human polyoma viral immune-dominant epitopes as indicated for the several polyoma viruses and can be used for immune diagnostic purposes accordingly.
In addition the human polyoma virus peptide sequences can be used for B-cell epitope studies i.e. the identification of linear peptides present in the three dimensional structure of the virus involved. In addition the human polyoma virus peptide sequences can be used for B-cell stimulation and/or B-cell functionality studies.
The human polyoma virus peptide sequences of the invention can also be part of a device or kit further containing means for measuring antibodies in a human test sample, like serum, plasma or whole blood.
In addition, the human polyoma virus peptide sequences mentioned in Table 9 can be used, directly or indirectly, for the manufacture of a medicament to treat progressive multifocal leukoencephalopathy (PML).
A peptide array representing human polyoma virus proteins has been prepared. The following proteins are covered by the peptide array: agnoprotein, small T antigen, large T antigen, VP1, VP2, VP3 and VP4 of the viruses BK, JC, KI, WU, MC and SV40. In addition, the VP1 protein of the viruses HPyV6, HPyV7, HPyV9, IPPyV and TSV are also included in this study. In total 4284 15-mer peptides overlapping by 11 residues are displayed in triplicates on one single array chip.
In order to prepare the peptide microarrays, polyoma virus protein sequences were retrieved from the NCBI (National Center for Biotechnology) database. The best covering sequence for each of the proteins of each virus was calculated. Then, each sequence was divided in all possible 15-mer peptides and coverage of related sequences by the peptides was calculated. The protein sequence providing the best covering peptides was determined. Mosaic sequences, which further increase the coverage of related sequences, were generated as well. The mosaic algorithm assembles artificial best covering sequences for a given sequence pool. The number of sequences that were retrieved from the NCBI database is given in Table 1 and Table 2.
For the design of the 15-mer peptides, the following proteins were included:
A total of 49 plasma samples from healthy volunteers (HV) have been tested on the peptide microarrays.
Peptides from the microarray that were reactive against antibodies present in the HV plasma samples were aligned against consensus sequences retrieved from the NCBI database. Table 3 provides the accession numbers for the sequences used in the analysis. For analysis purposes, the different proteins for the different organisms were labeled with a unique code (ID). Table 4 gives an overview of these unique identifiers.
A total of 49 clinical samples were tested on the peptide microarrays in triplicate (each peptide array contains 3 identical subarrays of 4284 peptides). Data from the subarrays were pooled, and only the median value (in case of 3 valid subarray data points), or the average of 2 data points (in case one of the subarray data points was excluded for quality reasons) were retained for further analysis. This will result in 209,916 data points (4284×49).
As a negative control, hybridization buffer without addition of human plasma was run alongside. Analysis of these 4284 control data points showed the following boxplot parameters:
For further analysis, the value of the 75th quartile is used as a cut-off, because it is reasonable to assume that from that moment onwards meaningful biological data might be available with the HV samples.
The following arbitrary classes of signal intensity were generated and represented in Table 5:
The most important results are found in the FU group of >30,000, with a total of 1,148 data points. However, the presentation of this result does not educate on the number of peptides that are responsible for this hybridization signal. Therefore, a further analysis of these data points was needed (given in Table 6).
A total of 635 peptides are responsible for the 1148 data points with an FU value >30,000. The 635 peptides are distributed over different classes of organisms and genes, with strong response to small T antigen peptides being the most prevalent for KIV, WUV, MCV, and JCV, followed by large T antigen and VP1, and a strong signal is the least prevalently found in VP2, VP3, and Agnoprotein. The sequence of these 635 peptides is given in Table 19. For interpretation of the origin of the peptides see Table 20
IDs given in table 19 which are not defined in table 20 do not represent further specified polyoma virus peptide sequences.
Subsequently, an analysis towards the immuno-dominancy of these peptides was conducted. Therefore, for each of the 4284 peptides the number of hits was searched for with a FU of >10,000 in each of the 49 HV samples.
The analysis retrieved the following result: 2424 peptides had at least “one out of the 49” HV samples a FU-value >10,000. As a consequence, 1860 peptides were having FU values below the arbitrary cut-off of 10,000 for all the samples tested (Note: this does not mean that for certain disease states these peptides might not show reaction with available antibodies). In addition, subgroups of prevalence were defined in blocks of 5 HV (Table 7). For the purpose of this exercise, we considered reaction on a peptide as immunodominant from >21 reactions (out of 49 HV) onwards.
A total of 63 peptides were identified for which the label of immunodominant epitope would be applicable (according to the above assumptions) (Table 8). The sequence of these 63 immuno dominant peptides is given in Table 9.
Detection of Peptides with Average FU Values >10000 Across the 49 HV
The dataset of 209,916 data points was analyzed for average values per peptide. This means that for each peptide, the average of FU values was calculated across the 49 HV reaction patterns. A total of 106 peptides were retrieved with values >10,000. The distribution of these peptides per organism is given in Table 10. In Table 11 to 18 the peptide sequences per organism are given.
Peptide arrays (15-mer peptides) were prepared covering all proteins of human polyoma viruses including BK virus, JC virus, KI virus, WU virus, MC virus, SV40, HPyV6, HPyV7, HPyV9, IPPyV and TSV.
Serum samples from 49 healthy volunteers were tested for the presence of antibodies against these peptides. As a result a set of potential B-cell epitopes were identified as described above.
| Number | Date | Country | Kind |
|---|---|---|---|
| 11193083.0 | Dec 2011 | EP | regional |
| 12798747.7 | Dec 2012 | EP | regional |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14364776 | Jun 2014 | US |
| Child | 15044340 | US |
