Immunohistochemical detection of enteroviruses in pancreatic tis- sues of patients with type 1 diabetes using a polyclonal antibody against 2A protease of Coxsackievirus

The need for antiserum for immunohistochemical (IHC) detection of enterovirus (EV) in formaldehyde fixed and paraffin-embedded (FFPE) specimens is increasing. The standard monoclonal antibody against EV-envelope protein (VP1), clone 5D8/1, was proven to cross-react with other proteins. Another candidate marker of EV proteins is 2A protease (2A), which is coded by the EV gene and translated by host cells during EV replication. We raised polyclonal antiserum by immunizing rabbits with an 18-mer peptide of Coxsackievirus B1 (CVB1)-2A protease (2A) and examined the specificity and sensitivity for EV on FFPE tissue samples. ELISA study showed a high titer of antibody for CVB12A. IHC demonstrated that antiserum against 2A reacted with CVB1-infected Vero-cells. Confocal microscopy demonstrated that 2A labelled by the antibody located in the same cell with VP1 stained with 5D8/1. IHC demonstrated dense positive reactions pancreatic islets of EV-associated fulminant type 1 diabetes (FT1DM), and located in the same cell stained positive with 5D8/1. Specificity of IHC staining FT1DM pancreas was confirmed by absorption with an excessive concentration of immunized peptide. In conclusion, our study provides a new polyclonal antiserum against CVB1 2A which may be useful for detection of EV-infected human tissues stored as archive of FFPE tissue samples.

It is also implicated as an inflammatory factor for neighboring host proteins that trigger autoimmune cell destruction in CV-induced chronic myocarditis [4,5] and type 1 diabetes [1]. However, there are no commercially available polyclonal or monoclonal antibodies against 2A pro of EV for IHC analysis [6]. In addition, the gold standard antiserum against EV-capsid protein 1 (VP1) exhibits problematic cross reactions with other protein epitopes [7,8]. Thus, the establishment of complicated optimized conditions that preclude cross-reactions with non-target protein epitopes is required in IHC analysis [7][8][9]. It is difficult to obtain positive results using the in situ hybridization (ISH) technique because EV RNA, especially in pancreatic tissues, is degraded by many digestive enzymes during the prolonged warm postmortem conditions that precede fixation of the samples [10]. In addition, the density of IHC by antibody (5D8/1) is relatively weak, which is a common characteristic of monoclonal antibodies.
We raised polyclonal antiserum against EV-2A pro and validated its specificity and reactivity against a CVB1-infected cell line and human pancreatic tissues with or without type 1 diabetes using IHC. We found that the polyclonal antibody proved to be specific to EV-2A pro and useful as a diagnostic tool for IHC of EV-infected formaldehyde-fixed paraffin embedded (FFPE) tissues, including type 1 diabetic pancreas.

2.1.Preparation of antigen and immunization of antigen
2.1.1. Preparation and immunization of CVB1-2A pro antigen 18-mer peptide of CVB1 2A pro (Fig. 1) was synthetized and conjugated with keyhole-limpet-hemocyanin (KLH). Two rabbits (weight 2.5 kg, age: 7 months, housed for12 hours light/dark cycles and free access to food and water) were immunized 5 times (1month intervals) with CVB1 2A pro conjugated to KLH (500 μg), mixed with Complete-Freund's adjuvant (Sigma-Aldrich Japan, Japan). Prior to each injection, blood samples were obtained from the marginal vein of the rabbit ear, centrifuged at 2000 rpm for 10 min at 4°C and the sera was used to determine antibody titer by ELISA.
Serum was incubated with KHL at 20 μg/ml overnight at 4°C and spin down at 3000 rpm for 20 min at 4°C for evaluation of validity of antibody.

Cell lines and viruses
Vero-cells which were grown in Dulbecco's modified Eagle medium (Gibco) at 37°C and 5% CO2 were used for viral infection.
Vero-cells were infected with CVB1 (strain number: 16-20289) at multiplicity of infection (MOI) of 0.1 and 3.0 for 24 hours and fixed with 5% formaldehyde-PBS for 24 hours and subjected to IHC examination.   was performed with 0.5% gelatin/PBS-T (200 µL/well) for 1 h. After 3 washes with PBS, antiserum diluted at a range from x500 -x7,812,500 (concentration range) of 100 µL/well was applied and incubated for 1 hour as the primary reaction. HRP-labeled antirabbit IgG (1:5000) was added 100 µL/well and incubated for 1 h at room temperature. After 3 washings, 0.5 mg/mL ophenylenediamine dihydrochloride (Sigma-Aldrich Japan) was added at 100 µL/well and incubated for 20 min. The reaction was stopped by the addition of 50 µL/well 2 N H2SO4 and absorbance was measured at 490 nm.

3. IHC of formalin-fixed cultured cell tissues and FFPE tissues
The raised rabbit polyclonal antiserum (ET2112) was diluted at x100, x200, x400, x1600 and x3200. Vero-cells grown on 24-well glass plates were infected with CVB1 for 24 hours. The cell tissues were fixed with 5% formaldehyde-PBS. Cells were stained with Japan) and fixed by 5% formaldehyde-PBS were stained as a positive control. As disease control, autopsied pancreas from three patients who died 3-5 days after onset of fulminant type 1 diabetes (FT1DM) case due to diabetic ketoacidosis [10] was stained by IHC [10 -12]. The presence of CVB-RNA in the pancreatic islets and acinar cells of these FT1DM cases was ascertained by in situ hybridization (ISH) [13].

3. 3. Sensitivity examination on FFPE tissues and formaldehyde-fixed cultured cells using IHC
IHC analysis on staining of 5% FFPE tissues or formaldehyde-fixed tissues and localization of CVB1 2A pro and EV VP1 was done as previously reported [10 -12]. Optimized condition for VP1 IHC staining was described [11]. The stained samples were observed by conventional fluorescent microscopy (DP73, Olympus, Tokyo) and confocal laser microscopy (FV3000, Olympus Tokyo) and images stored as photos were analyzed by two investigators and judged independently in a double-blind manner.

Specificity examination on FFPE tissues using IHC
Pancreatic sections of FFPE tissue were processed as first antibody with isotype-matched control rabbit IgG (1 : 1000) (X0903, DAKO) or pre-immune rabbit serum (1:1600), or in the absence of primary antibody to confirm the specificity of immunostaining.
Non-diabetic human FFPE tissues including non-diabetic human autopsied donor's pancreas, thyroid glands, duodenum, liver, spleen, kidney, and colon were stained using antiserum against 2A pro antigen.

4. Ethics
Written, informed consent was obtained from the next of kin of the autopsied patients. The ethical committees of the Toranomon Hospital approved all procedures (No: 948, Date: 2017.5.23).

3-1.
Titers of antiserum against CVB1 2A pro by ELISAThe interaction between antiserum against CVB1 2A pro and the peptide used for immunization without carrier protein KHL was confirmed by ELISA, showing high specific binding (Fig. 2). of CVB1-infected Vero-cells for 24 hours after infection and fixed by 5% formaldehyde-PBS (Fig. 3a-i). The number of cultured cells stained for 2A pro and VP1 were higher in 3.0 MOI of CVB1 infected cells than 0.1 MOI of CVB1 infected cells (Fig. 3a, b, d, e). Mock cultured cells did not show positive staining for both 2A pro and VP1 (Fig. 3c, f). Confocal microscopy showed positive 2A pro in cytosol of cultured cells and VP1 were stained perinuclear area in same cells in which VP1 located peri-nuclear areas and 2A pro located peripheral cytosolic areas (Fig. 3g, h, i).   Pancreatic islets were positively stained for 2A pro in EV-induced FT1DM (Fig.4 a). Pre-absorption with 2A pro peptide at 10 g/ml abolished positive immunostaining (Fig. 4b). Pancreases from in 14 non-diabetic controls whose islets were negative for VP1 by IHC showed negative staining for 2A pro and VP1(data not shown). Staining of tissues from other organs than pancreases were all negative for 2A pro and VP1 (data not shown).

Discussion
We demonstrated that a polyclonal antibody against CVB1 2A pro protein produced specific staining in acetone-fixed Vero-cells infected by CVB1 and the FFPE pancreatic tissues of EV-induced FT1DM. This represents an important diagnostic tool of EV IHC for FFPE tissues because there is no antiserum against CVB1 2A Pro for IHC staining of FFPE tissues [6]. In addition, it has been reported that the monoclonal antibody (5D8/1) (Dako) notoriously cross reacts with other protein epitopes in the pancreas [7] and requires optimized IHC conditions [8,9]. Moreover, discordant results have been obtained between IHC and RT-PCR in acuteonset type 1 diabetic organs [14]. In general, polyclonal antibodies remain stable in various environments including alterations in pH, salt concentration, and dilution ranges associated with reduced nonspecific staining [15]. Our serum against CVB1 2A pro (ET2112) is polyclonal and is likely to utilize multiple epitopes that may be associated with other enterovirus subtypes, which are potentially associated with type 1 diabetes. Polyclonal antibodies are required more rigorous validation than monoclonal antibodies because they are raised against native proteins or fragment of proteins [15]. Polyclonal antibodies will be recognized multiple epitopes [15]. Some of these risks can be eliminated by absorption with immunogens used immunization of short peptide antigens.
For this purpose, we synthetized short peptide of Coxsackie virus B1 2A pro peptide were used for antigen (Fig. 1). Furthermore, we excluded possible non-specific reaction with non-diabetic organ tissues and ascertained that antibody ET2112 dosed not react with other organ tissues.
It is generally assumed that persistent EV infection of the pancreas occurs with truncated viral RNA [16,17] that lack active replication during the preclinical period of type 1 diabetes. EV-2A pro is generated during active replication of EV [2,6]. Thus, EV-2A pro may be a useful marker of active EV replication in FFPE tissues, as observed in cases positive for EV-2A pro in the pancreas of FT1DM. An unresolved issue for EV infection in type 1 diabetes pancreas during the preclinical phase includes whether IHC with antibody raised against CVB1 2A pro protein is capable of discriminating between the inactive/dormant or active replicating states. In addition, further studies on pathogenetic roles of 2A pro of EV, which potentially cleaves islet proteins and beta cell damage and subsequent autoimmunity similar to chronic myocarditis [4] await to be clarified.
Author Contributions: E. J., T. K, T. F, A. T. and S. Y. conducted to immunohistochemical staining and to the analysis of the immune-stained sample data and discussions. K.N. and S.N. prepared samples of Vero cells infected with CBV1and discussed. S.N. contributed to discussions and reviewing the manuscript. S. Y. contributed to the planning and discussions and edited the manuscript. E. J. contributed to the analysis of the data, discussions, and reviewed the manuscript. T. K. planned the study, sampled autopsied pancreases, analyzed data, and wrote and edited the manuscript. All authors approved the final version for publication. T. K. is the guarantor of this work and takes responsibility for the integrity of the data and the accuracy of the data analysis.