Thrombotic thrombocytopenic purpura (TTP) is clinically characterized by the occurrence of thrombocytopenia and microangiopathic hemolytic anemia [1, 2, 3]. TTP was first described as a pathological entity in 1924 by Moschcowitz  and was clearly identified as an autoimmune disorder by Harrington et al. in 1951 . Currently, after 70 years, we know that the majority of TTP patients suffer from acquired TTP caused by the presence of autoantibodies (AAbs) against ADAMTS13 [5, 6, 7], a protease that cleaves the von Willebrand factor (vWF) multimers into smaller forms thereby controlling vWF-mediated platelet thrombus formation . However, the molecular basis and the mechanism(s) that lead to the loss of immunotolerance toward ADAMTS13 and permit the production of anti-ADAMTS13 AAbs are still unknown .
Therefore, it assumes a scientific relevance the clinical observation by Kosugi et al. , that influenza infection triggers TTP by producing anti-ADAMTS13 IgGs. The observation is crucial in light of the fact that the association between influenza infection and TTP is clinically well known [11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29], so making feasible the hypothesis that the anti-influenza immune responses that follow influenza infection may cross-react with ADAMTS13 protein thus generating anti-ADAMTS13 AAbs. To prove or disprove the cross-reactivity hypothesis, the present study analyzed the peptide commonality between influenza virus and ADAMTS13 proteins since, actually, peptide sharing can lead to autoimmunity through cross-reactivity phenomena following pathogen infection. It was found a wide influenza virus vs ADAMTS13 peptide overlap that can represent a cross-reactive molecular platform underlying anti-ADAMTS13 AAb production.
Sequence analyses were conducted on ADAMTS13 (or A disintegrin and metalloproteinase with thrombospondin motifs 13 or vWF-cleaving protease) and cytoplasmic and perinuclear autoantigens of antineutrophil cytoplasmic antibodies, ie, c-ANCA myeloblastin and p-ANCA myeloperoxidase. The three human proteins are described in detail in the Uniprot database at https://www.uniprot.org [30, 31]. Protein primary sequences were decomposed into overlapping pentapeptides offset by one residue, ie, MGVPF, GVPFF, and VPFFS. Next, each pentapeptide was analyzed for occurrences in viral proteomes using Peptide Match program (https://research.bioinformatics.udel.edu/peptidematch) .
Proteomes from nine viruses were analyzed: influenza A virus, H1N1 (NCBI:txid211044), influenza A virus, H3N2 (NCBI:txid385580), influenza A virus, H5N1 (NCBI:txid93838), influenza A virus, H10N7 (NCBI:txid382838), influenza B virus (NCBI:txid518987), and influenza C virus (NCBI:txid11553). Tobacco mosaic virus (NCBI:txid12243), human parvovirus B19 (NCBI:txid648237), and dengue virus (NCBI:txid11059) were used as controls.
ADAMTS13 protein sequence was analyzed for peptide sharing with influenza virus proteomes. In addition, c-ANCA myeloblastin and p-ANCA myeloperoxidase, two autoantigens that characterize a group of small vessel vasculitis [34, 35], were analyzed for comparison.
Analyses were extended to human parvovirus B19 because B19 infection, although predominantly affects erythrocytes, shows clinically nonsignificant lymphopenia, neutropenia, and thrombocytopenia  and to dengue virus because thrombocytopenia and clotting abnormalities are at the heart of dengue pathology . Tobacco mosaic virus was utilized as a negative TTP-unrelated viral control.
Operationally, the pentapeptide was used as a probe in sequence analyses since, in immunobiology, a functional/structural unit is generally represented by five amino acid (aa) residues . In fact, biological interactions can be described by peptide–protein interactions involving a pentapeptide [39, 40, 41, and pertinent references therein]. Alike, the capability of generating immune responses (immunogenicity) as well as the immune recognition process (antigenicity) appears to be circumscribed to the space of five residues [42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52].
4 Peptide sharing between influenza viruses and ADAMTS13
The pentapeptide sharing between the viral proteomes and c-ANCA myeloblastin, p-ANCA myeloperoxidase, and ADAMTS13 is described in Table 1. Main points are as follows:
Description of the pentapeptide sharing between B19, dengue, and influenza proteomes and the ANCA and ADAMTS13 autoantigens
|Virus||c-ANCA myeloblastin||p-ANCA myeloperoxidase||ADAMTS13|
|Tobacco mosaic virus||–||–||–|
|Human parvovirus B19||–||FEQVM||ALVRP|
|Dengue virus 1||–||ARASF, SGSAS||AGEKA, TLRVL AGILA, GAGLA, GANAS, SLRTT,|
|Influenza A virus, H1N1||–||–||GDMLL, GHADL, GILHL, LESSL, PGHAD|
|Influenza A virus, H5N1||–||–||ALTED, GDMLL, GHADL GILHL, PGHAD|
|Influenza A virus, H3N8||–||–||ALTED, PGHAD, ELLVA, QGSLL GDMLL, GHADL, GILHL,|
|Influenza A virus, H10N7||–||–||ALTED, PGHAD ELLVA, GDMLL, GHADL, GILHL,|
|Influenza B virus||VTVVT, TVVTF||LERKL, RLATE||GGVLL, RQRQR, TGTID|
|Influenza C virus||–||–||–|
- –•On the whole, the viral vs human peptide overlap consists of 24 pentapeptide matches, 17 of which occur in ADAMTS13 and mainly involve influenza A and dengue viruses.
- –•The viral peptide sharing with the two ANCA autoantigens amounts to seven pentamers and is restricted to influenza B, B19, and dengue viruses.
- –•Influenza C virus and the negative control tobacco mosaic virus have no pentapeptide sequences in common with any of the three analyzed human proteins.
In commenting data from Table 1, two further observations merit notice. First, the extent of the viral vs human peptide overlap is mathematically unexpected since the probability that two proteins may share a pentapeptide is equal to 20˗5 (ie, probability 1 out of 3,200,000 or 0.0000003125). Second, the present study examines only reference influenza proteomes, ie, selected proteomes that cover well-studied model organisms [30, 31], and neglects the hundreds of existing influenza subtypes and variants. That is to say that the cross-reactivity scenario between influenza infection and ADAMTS13 might be more intense and varied than that summarized in Table 1. As an example, the influenza A virus, H5N1 (NCBI:txid176674) shares the additional pentapeptide SLEPC with ADAMTS13 when compared to the, here, analyzed influenza A virus, H5N1 (NCBI:txid93838).
5 Immunologic potential of the peptide sharing between influenza viruses and ADAMTS13
The peptide sharing as summarized in Table 1 has a high immunologic potential. In fact, using IEDB , a catalog of experimentally validated epitopes, and searching within IEDB epitopes, it was found that almost all of the shared pentapeptides described in Table 1 repeatedly occur in immunoreactive epitopic sequences. Table 2 lists the immunopositive epitopes containing sequences shared between B19, dengue, and influenza proteomes and the ANCA and ADAMTS13 autoantigens and highlights the disproportionately high number of epitopes containing ADAMTS13 pentapeptides.
Epitopes containing peptides shared between B19, dengue, and influenza proteomes and ANCA and ADAMTS13 autoantigens
|c-ANCA myeloblastin||p-ANCA myeloperoxidase||ADAMTS13|
|IEDB ID1||Epitope sequence2,3||IEDB ID1||Epitope sequence2,3||IEDB ID1||Epitope sequence2,3|
Numerous pentapeptides are shared between influenza A and B viruses and ADAMTS13, the autoantigen of TTP (Table 1). The peptide sharing is higher than expected by being very low the chance for two proteins to share a pentapeptide. Moreover, the peptide sharing is immunologically significant by being most of the shared peptides also part of experimentally validated epitopes (Table 2). Hence, the present study substantiates the hypothesis of cross-reactivity involvement in the generation of anti-ADAMTS13 AAbs following influenza infection, in this way flanking previous findings [53, 54, 55, 56, 57] that indicate autoimmune cross-reactions as a basic mechanism in the generation of autoimmune diseases following infections.
The data warrant further collaborative research efforts, especially in light of the fact that low levels of ADAMTS13 protease are a risk factor for the development of myocardial infarction [58,59], stroke [59, 60, 61], preeclampsia , disseminated intravascular coagulation , cerebrovascular disease , etc.
This research received no specific grants from any funding agency in public, commercial, or not-for-profit sectors.
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