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Rubella persistence in epidermal keratinocytes and granuloma M2 macrophages in patients with primary immunodeficiencies

2016; Elsevier BV; Volume: 138; Issue: 5 Linguagem: Inglês

10.1016/j.jaci.2016.06.030

1097-6825

Ludmila Perelygina, Stanley A. Plotkin, Pierre Russo, Timo Hautala, Francisco A. Bonilla, Hans D. Ochs, Avni Y. Joshi, John M. Routes, Kiran Patel, Claudia Wehr, Joseph P. Icenogle, Kathleen E. Sullivan,

Mast cells and histamine

Discuss this article on the JACI Journal Club blog: www.jaci-online.blogspot.com. Cutaneous granulomas are a well-recognized pathologic feature in patients with various primary immunodeficiency diseases (PIDs) and may be self-limited or can progress to a persisting granulomatous disorder.1Sillevis Smitt J.H. Kuijpers T.W. Cutaneous manifestations of primary immunodeficiency.Curr Opin Pediatr. 2013; 25: 492-497Crossref PubMed Scopus (32) Google Scholar, 2Nanda A. Al-Herz W. Al-Sabah H. Al-Ajmi H. Noninfectious cutaneous granulomas in primary immunodeficiency disorders: report from a national registry.Am J Dermatopathol. 2014; 36: 832-837Crossref PubMed Scopus (11) Google Scholar Rubella virus (RV) vaccine strain RA27/3 has been recently detected in disseminated cutaneous granulomas of 2 patients with ataxia telangiectasia (AT) and a patient with Simpson-Golabi-Behmel syndrome (who had combined immunodeficiency [CID]).3Bodemer C. Sauvage V. Mahlaoui N. Cheval J. Couderc T. Leclerc-Mercier S. et al.Live rubella virus vaccine long-term persistence as an antigenic trigger of cutaneous granulomas in patients with primary immunodeficiency.Clin Microbiol Infect. 2014; 20: O656-O663Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar However, a more detailed study of a larger series of granuloma cases in patients with different PIDs was required to confirm and extend this observation. Patients with cutaneous granulomas and with diverse PIDs were selected from the United States Immune Deficiency Network registry. Additional cases (cases 1 and 11) were recruited from the Clinical Immunology Society immune deficiency Listserv. Presence of RV in granuloma-containing tissue samples (Fig 1, A-C) was examined by immunofluorescence staining with 2 different RV capsid-specific antibodies (see this article's Methods section in the Online Repository at www.jacionline.org) by a reader blinded to the diagnosis. Seven out of 14 patients (50%) exhibited positive RV antigen staining (Table I), whereas the tissue samples of 5 non-PID granuloma patients were negative.Table IInformation on patients with PIDCaseCountryImmune deficiencyAge at biopsy (y)Current status∗Vaccination status and age of vaccination for rubella were indicated in parentheses, if known.Biopsy description (all patients were negative for mycobacteria)Other sites1†Indicates rubella antigen positive.FinlandCID21, 24, & 28Alive (MMR, 5 and 9 y)Dermis with strong lymphocytic infiltrate, granulomatous inflammation. Some Langerhan giant cells. Over time, progressive necrosisThree skin biopsies were all positive2†Indicates rubella antigen positive.USACID11Alive (MMR)Epidermal hyperplasia, dermal edema, suppurative granulomatous inflammation3†Indicates rubella antigen positive.USACHH8Deceased (MMR 15 mo)Skin and subcutaneous tissue incorporating predominantly dermal lymphohistiocytic infiltrates with occasional multinucleated giant cells, extending into the subcutaneous fat, and with inflammation focally extending into the epidermis4†Indicates rubella antigen positive.USAAT9DeceasedNecrotizing granulomatous inflammation5†Indicates rubella antigen positive.USAAT1Alive (MMR 1 y)The dermis contains chronic inflammatory infiltrate. The deep dermis/subcutaneous tissue is markedly abnormal. The tissue is replaced by large and small granulomas. The granulomas are necrotizing. Outlines of fat cells are seen within pink amorphous matrix, which includes necrotic nucleoli6†Indicates rubella antigen positive.USAAT5DeceasedNecrotic vasculitis with neutrophilic infiltrates at some locations and necrotizing granulomatous inflammation of the dermis at other locations. Bone biopsy showed histiocytic infiltrate with early myelofibrosisMultiple skin sites, not all positive but bone biopsy positive7†Indicates rubella antigen positive.USAAT7AliveNoninfectious granulomatous process8USAMWS3Alive (MMR)Marked hyperkeratosis, parakeratosus with focal scale, crust, and follicular plugging. Epidermal disruption, dermal lymphoplasmacytic infiltrate with prominent dermal granulomas with central necrosisMultiple skin sites9USACVID28AliveSuperficial and deep dermal diffuse granulomatous infiltrate composed of giant cells and epithelial cellsGranulomas in 2 skin biopsies both negative10USACVID47AliveNoncaseating dermal granulomatous inflammation with eosinophils11GermanyXLA38Alive (MMR)Perivascular T-cell infiltrates, with oligoclonality. PAS-positive (PAS reaction) particles in the subepidermal region12USANEMO10AliveSubacute spongiotic dermatitis with focal parakeratosis, mild acanthosis, mild spongiosis with superficial dermal lymphohistiohistic infiltrate and absence of eccrine glands. CD3, CD5, and CD7 staining is seen on the lymphocytes. CD68 and CD163 decorate admixed histiocytes13USAAT10AliveWidespread collagen necrobiosis associated with granulomatous inflammation14USAAT3Alive (MMR)Sarcoidal granulomatous dermatitis with many associated CD8-positive lymphocytes, which focally obscure the dermoepidermal junction. The overlying epidermis is acanthotic with compact hyperkeratosis, parakeratosis, and plugged infundibula. Collections of epithelioid histiocytes, some of which are multinucleated, that palisade around central foci of fibrin and mucinCHH, Cartilage hair hypoplasia; CVID, common variable immunodeficiency; MWS, Marden-Walker syndrome; NEMO, nuclear factor kappa B essential modulator deficiency; PAS, periodic acid–Schiff; XLA, X-linked agammaglobulinemia.∗ Vaccination status and age of vaccination for rubella were indicated in parentheses, if known.† Indicates rubella antigen positive. Open table in a new tab CHH, Cartilage hair hypoplasia; CVID, common variable immunodeficiency; MWS, Marden-Walker syndrome; NEMO, nuclear factor kappa B essential modulator deficiency; PAS, periodic acid–Schiff; XLA, X-linked agammaglobulinemia. Staining intensity varied substantially between patients and did not correlate with the severity of granulomatous disease. RV immunostaining was typically observed in both epidermis and granulomas in dermis (Fig 1, D-F); however, staining only in granulomas (case 3) was seen (Table I). Multiple granulomas within a sample contained RV antigen with typically a few positive cells in the middle except cases 2 and 6, in which virtually all cells in the granulomas were positive. RV was found exclusively in patients with CIDs: CID cause unknown (n = 2), AT (n = 4), and cartilage hair hypoplasia (n = 1). The immune deficiencies in which granulomas were not found to be positive for rubella were common variable immune deficiency (n = 2), AT (n = 2), X-linked agammaglobulinemia (n = 1), Marden-Walker syndrome (n = 1), and nuclear factor kappa B essential modulator (n = 1). None of the nonimmune deficient samples was positive. RV-positive cells in granulomas were positive for CD14 and CD68, markers of monocyte/macrophage cell lineage, and CD206 and CD163, activation markers for M2 macrophages, but negative for iNOS, an M1 macrophage marker (see this article's Methods section; see Table E1 in this article's Online Repository at www.jacionline.org; Fig 1, G-H). Endothelial cells (vWF+), T cells (CD3+), B cells (CD20+), dermal Langerhans (CD1a+), and dendritic (CD11c+) cells were negative for RV antigen (see this article's Results section in the Online Repository at www.jacionline.org; Table E1). These results demonstrate that M2 macrophages were the cell type harboring RV antigen in granulomas. There was a high production of cytokeratin in many RV-positive keratinocytes, suggesting that RV replication in keratinocytes can lead to dysregulation of keratin synthesis (Fig 1, I). Overexpression of keratin is known to alter the architecture of the epidermis and impact healing of ulcers.4Takahashi K. Folmer J. Coulombe P.A. Increased expression of keratin 16 causes anomalies in cytoarchitecture and keratinization in transgenic mouse skin.J Cell Biol. 1994; 127: 505-520Crossref PubMed Scopus (89) Google Scholar The RV-immunostaining patterns were unchanged in the 3 skin samples obtained within a 7-year period (case 1), indicating long-term persistence of RV antigen. Antigen persistence is a hallmark of granulomas. Biopsy specimens collected from different body sites contained both RV-positive and RV-negative specimens (case 3), indicating focal distribution of RV antigen-positive cells in patient tissues. In addition to the skin samples from case 6, a bone periosteum tissue (collected 5 years later) contained RV-positive M2 macrophages in the granulomas. PCR fragments covering the entire genome were amplified and sequenced from 1 patient. Phylogenetic analysis revealed that rubella virus of genotype 1a was present in the patient skin (RVs/Oulu.FIN/22.15/PID; see Fig E1 in this article's Online Repository at www.jacionline.org). The sequence was similar (97.4% identity) to that of the RA27/3 vaccine virus. In RVs/Oulu.FIN/22.15/PID, 2 out of 7 RA27/3-specific amino acid residues had reverted to the wild type (see this article's Results section; see Table E2 in this article's Online Repository at www.jacionline.org). There were 69 amino acid substitutions in RVs/Oulu.FIN/22.15/PID compared with RA27/3; 52 of them were not found in wild-type RV genomes (see Table E3 in this article's Online Repository at www.jacionline.org). Most neutralizing epitopes, which are located in the E1 protein, were conserved including the immunodominant epitope E1214-233, whereas each of 3 known CD8+ T-cell epitopes (largely predicted to be A2 binding), all located in the capsid, contained single mutations (see this article's Results section; see Table E4 in this article's Online Repository at www.jacionline.org), suggesting a role for CD8+ T-cell–selective pressure in viral evolution during chronic RA27/3 infection. Similar to the previous report,3Bodemer C. Sauvage V. Mahlaoui N. Cheval J. Couderc T. Leclerc-Mercier S. et al.Live rubella virus vaccine long-term persistence as an antigenic trigger of cutaneous granulomas in patients with primary immunodeficiency.Clin Microbiol Infect. 2014; 20: O656-O663Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar AT was the most common single diagnosis among RV-positive patients. We have also identified additional PIDs (cartilage hair hypoplasia and CID) in which RV was found in granulomas. Thus, current data have clearly shown an association between defects in T-cell immunity, granulomas, and RV. One of the important findings of this study is the identification of RV-positive cells in granulomas as M2 macrophages. Proinflammatory M1 macrophages play an essential role in eliminating pathogens, whereas anti-inflammatory M2 macrophages are crucial for maintaining tissue homeostasis. RV can infect most cell types and can persistently infect a macrophage-like cell line,5Sarmiento R.E. Tirado R. Gomez B. Reinfection-induced increase of rubella persistently infected cells in a macrophage-like cell line.Virus Res. 1997; 50: 15-22Crossref PubMed Scopus (6) Google Scholar but it is currently unknown whether this occurs in vivo. Another novel finding is persisting RV antigen in epidermal keratinocytes, in all epidermal layers except the basal layer. In contrast, in acute postnatal rubella cases, rubella antigen was found only in the deep dermis (cell types not defined) in skin biopsies from rubella rashes, whereas the epidermis was negative.6Takahashi H. Umino Y. Sato T.A. Kohama T. Ikeda Y. Iijima M. et al.Detection and comparison of viral antigens in measles and rubella rashes.Clin Infect Dis. 1996; 22: 36-39Crossref PubMed Scopus (17) Google Scholar Wild-type RV can establish persistent infections and cause disease in immunologically normal individuals if infection occurs in immune-privileged sites, for example, Fuchs' uveitis or fetal development. A role for vaccine virus in Fuchs’ uveitis is also suspected.7Islam S.M. El-Sheikh H.F. Tabbara K.F. Anterior uveitis following combined vaccination for measles, mumps and rubella (MMR): a report of two cases.Acta Ophthalmol Scand. 2000; 78: 590-592Crossref PubMed Scopus (41) Google Scholar, 8Kreps E.O. Derveaux T. De Keyser F. Kestelyn P. Fuchs' uveitis syndrome: no longer a syndrome?.Ocul Immunol Inflamm. 2016; 24: 348-357Crossref PubMed Scopus (31) Google Scholar Granulomas have been reported only once in CRS.9Hancock M.P. Huntley C.C. Sever J.L. Congenital rubella syndrome with immunoglobulin disorder.J Pediatr. 1968; 72: 636-645Abstract Full Text PDF PubMed Scopus (38) Google Scholar We hypothesize that the immune deficiency allows persistence of the attenuated virus, polarization of macrophages to M2 occurs, compromising viral clearance, mutations accrue, and complete viral escape occurs upon acquisition of CD8 epitope mutations. As the virus persists, damage to the keratinocytes occurs and ulcers appear. One patient exhibited persistence over 23 years, defined by sequential biopsies. Our finding that mutations occurred in cytotoxic T lymphocytes epitopes is consistent with CD8+ T-cell–selective forces playing a dominant role. Whether RV is the cause of the granulomas or a passenger in the presence of absent cellular immune responses to the virus can be determined only if antiviral therapy results in resolution of the granulomas. The inability to isolate live virus from well-preserved skin tissue was unexpected because RV can generally be isolated from the skin with similar amounts of RNA. One conceivable explanation for our failure to recover infectious RV would be if the persisting virus is defective, analogous to the defective measles viruses in subacute sclerosing panencephalitis cases. In summary, our results demonstrate that RV can establish chronic infection in M2 macrophages and keratinocytes in patients with diverse T-cell deficiencies. Our study suggests that individuals with cellular immune deficiencies may be at risk for persistent RV infections that stimulate nonprotective immune response associated with chronic M2-type granuloma formation. We thank Dr Min-hsin Chen for providing genomic sequences of clinical RV strains for phylogenetic analysis, Drs Laura S. Finn and Sejal Shah for expert pathologic support, and Dr Mikko Seppänen for critical insights. We thank, remember, and honor the patients who have contributed. Patients with cutaneous granulomas and with diverse PIDs were selected from the USIDNET registry. Additional cases (cases 1 and 11) were recruited from the CIS immune deficiency Listserv. Hospitals participating in the study submitted archival patient material to the Children's Hospital of Philadelphia, where specimens were anonymized and then sent to the Rubella Global Specialized Laboratory (Centers for Disease Control and Prevention [CDC], Atlanta, Ga) to be tested blindly. Slides were cut from archived formalin-fixed, paraffin-embedded (FFPE) tissue samples, which were routinely collected for diagnosis according to standards of practice. Tissue sections of skin biopsies were taken from 19 patients with granulomatous diseases: 14 patients with PID and 5 patients without PID (pyoderma gangrenosum, cutaneous Crohn disease, nonspecific granulomatous dermatitis, histiocytosis, and granulomatous inflammation of the face). FFPE skin tissues from nongranuloma cases were used as negative control material. Fresh-frozen skin tissue was received and tested for RV by the CDC Laboratory as a part of reference/surveillance responsibilities. RV detection analysis for this case was conducted for the purpose of public health response and was not considered to be human research. Archived FFPE specimens from all patients were tested anonymously, with a nondisclosure agreement, which was determined to be ethically acceptable by the Internal Review Board at the CDC. For immunofluorescence studies, 3- to 4-μm tissue sections were deparaffinized in Histoclear II (National Diagnostics, Atlanta, Ga) and rehydrated using a series of ethanol washes. Epitope retrieval was performed by heating deparaffinized tissue sections in citrate buffer (10 mM sodium citrate, pH 6.0, 0.05% Tween 20) for 20 minutes at 98°C and cooling down in the same buffer for 20 minutes. After this and each subsequent immunostaining step, the sections were washed 3 times, 5 minutes each, in 1× PBS. Tissues were permeabilized with 0.05% Tween-20 in 1× PBS for 45 minutes at room temperature. Nonspecific binding of fluorescent dyes to tissues was reduced by incubation with Image-iT FX signal enhancer (Thermo Fisher Scientific, Waltham, Mass) for 30 minutes at room temperature. Nonspecific antibody-binding sites were blocked with BlockAid solution (Thermo Fisher Scientific) for 1 hour at room temperature. Tissue sections were then incubated with either mouse monoclonal anti-RV capsid antibody (Abcam, Cambridge, United Kingdom) or a mixture of capsid antibody with each of rabbit cell-type–specific mAb or polyclonal antibody (diluted with BlockAid to the working concentration) at 4°C overnight. Types of antibodies, their suppliers, and working dilutions are listed in Table E5. Incubation with the secondary antibody, goat anti-mouse IgG Alexa Flour-555, and anti-rabbit IgG Alexa Flour-488 (Molecular Probes, Waltham, Mass) diluted 1:1000 in BlockAid was carried out for 1 hour at room temperature followed by counterstaining with 4′-6-diamidino-2-phenylindole, dihydrochloride (Invitrogen, Carlsbad, Calif). Autofluorescence was blocked by incubating sections in 0.3% Sudan black solution in 70% ethanol for 10 minutes at room temperature followed by a brief rinse in 70% ethanol and 3 PBS washes (Fig E2). Cells were mounted with fluorescence- mounting medium (Dako, Carpinteria, Calif). Images were acquired with a Zeiss fluorescent microscope using AxioVision software (Carl Zeiss, Thornwood, NY). Specificity of RV capsid immunostaining in patient tissues was confirmed by probing sequential tissue sections with the different RV-specific antibodies, either mouse anticapsid RV mAb (produced by CDC core facility), rabbit polyclonal antibody against purified RV virions (produced by CDC core facility), or RV capsid antibody (manufactured by Abcam). The RV antibodies were preabsorbed with either human proteins (mixture of A459 and HUVEC cell lysates) (Fig E3) or purified RV virions (Fig E4; results for only RV polyclonal antibody are shown). Sections from an FFPE block containing a mixture of uninfected and RV-infected A549 human lung carcinoma cells (ATCC#CCL-185) served as a positive control in each immunofluorescence assay. In addition, negative controls were run in parallel in each assay and consisted of sequential tissue sections of case patients incubated with mAb to measles virus nucleoprotein or mengovirus RNA polymerase. Negative tissue control was also performed in each assay and consisted of a noncase FFPE skin section incubated with the RV antibody. Each test was repeated at least once. Interpretation of IF assay results included determination of the location of the RV-positive signal in a tissue, the type of cells infected, and the intensity of staining. Skin biopsy tissue from case 1 was cut into small pieces and then homogenized in high glucose Dulbecco's modified Eagle medium (Invitrogen) containing 5% FBS (Atlanta Biologicals, Lawrenceville, Ga) supplemented with 50 μg/mL gentamicin (Invitrogen) with Bead Beater (Sigma, Valencia, Calif) tissue homogenizer using 1.5-mm zirconium beads (Sigma Aldrich, St Louis, Mo). The tissue homogenate was then inoculated onto monolayers of WI-38 human fetal fibroblasts (Coriell Cell Repository, Camden, NJ) and Vero cells (ATCC #CCL-81). Both media and cells were passaged 3 times weekly. Real-time RT-PCR for RV RNA and infectivity titration on Vero cells were performed to monitor the infected cultures for the presence of RV in the culture media. After each passage, the cells were seeded into chamber slides, fixed with methanol, and then subjected to indirect immunofluorescence for RV structural proteins and in situ hybridization for RV genomic RNA as described elsewhere.E1Perelygina L. Zheng Q. Metcalfe M. Icenogle J. Persistent infection of human fetal endothelial cells with rubella virus.PLoS One. 2013; 8: e73014Crossref PubMed Scopus (20) Google Scholar, E2Perelygina L. Adebayo A. Metcalfe M. Icenogle J. Differences in establishment of persistence of vaccine and wild type rubella viruses in fetal endothelial cells.PLoS One. 2015; 10: e0133267Crossref Scopus (8) Google Scholar RNA was isolated from a frozen skin sample using an RNeasy Fibrous Tissue Mini Kit (Qiagen, Valencia, Calif) according to the manufacturer's instructions. Primers and conditions for real-time RT-PCR for measles, mumps, and RVs, genotyping RT-PCR, a detailed strategy for a whole-genome sequencing, and phylogenetic analyses have been described.E1Perelygina L. Zheng Q. Metcalfe M. Icenogle J. Persistent infection of human fetal endothelial cells with rubella virus.PLoS One. 2013; 8: e73014Crossref PubMed Scopus (20) Google Scholar, E3Abernathy E. Chen M.H. Bera J. Shrivastava S. Kirkness E. Zheng Q. et al.Analysis of whole genome sequences of 16 strains of rubella virus from the United States, 1961–2009.Virol J. 2013; 10: 32Crossref PubMed Scopus (23) Google Scholar, E4Namuwulya P. Abernathy E. Bukenya H. Bwogi J. Tushabe P. Birungi M. et al.Phylogenetic analysis of rubella viruses identified in Uganda, 2003-2012.J Med Virol. 2014; 86: 2107-2213Crossref PubMed Scopus (19) Google Scholar, E5Hummel K.B. Lowe L. Bellini W.J. Rota P.A. Development of quantitative gene-specific real-time RT-PCR assays for the detection of measles virus in clinical specimens.J Virol Methods. 2006; 132: 166-173Crossref PubMed Scopus (130) Google Scholar, E6Rota J.S. Rosen J.B. Doll M.K. McNall R.J. McGrew M. Williams N. et al.Comparison of the sensitivity of laboratory diagnostic methods from a well-characterized outbreak of mumps in New York city in 2009.Clin Vaccine Immunol. 2013; 20: 391-396Crossref PubMed Scopus (59) Google ScholarFig E2RV-positive control slides containing a mixture of 4 human tissues and RV-infected A459 cell double immunostained with capsid mAb and pan-keratin polyclonal antibody and counterstained with DAPI. The slides were either untreated (A) or treated (B) with 0.3% Sudan black solution before mounting. Note the reduction in autofluorescence after Sudan black treatment (Fig E2, B). DAPI, 4′-6-Diamidino-2-phenylindole, dihydrochloride.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3Histological immunofluorescent staining of positive control slides with either rabbit RV polyclonal antibody (CDC) (A), mouse capsid mAb (Abcam) (B), or mouse capsid mAb (CDC) (C), which were preabsorbed with human proteins. Anti-rabbit IgG Alexa Flour-555 (Fig E3, A), anti-mouse IgG Alexa Flour-555 (Fig E3, B), or Alexa Flour-488 (Fig E3, C) served as secondary antibody; nuclei were counterstained with DAPI. DAPI, 4′-6-Diamidino-2-phenylindole, dihydrochloride.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E4Specificity of rabbit RV polyclonal antibody (CDC). Immunofluorescent staining of Vero cells infected with rubella virus (A and B) with either RV polyclonal or RV polyclonal preabsorbed with purified RV virions. C, Mock-infected Vero cells. Note the lack of specific staining with the antibody preabsorbed with the specific antigen.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table E1Characterization of RV-positive cells in skin tissues of patients with PID by double immunostaining with cell-type–specific antibodiesAntigenCell typeCostaining with capsidCD1aLangerhans cellsNCD3T cellsNCD11cMyeloid dendritic cellsNCD20B cellsNvWFEndothelial cellsNCD14Monocytes/macrophagesYCD68Macrophages, dendritic cellsYCD163M2-activated macrophagesYCD206M2-activated macrophagesYiNOSM1-activated macrophagesNPan-cytokeratinKeratinocytesY Open table in a new tab Table E2Vaccine-specific amino acids (aa) in RV proteinsGenomesp150 aap90 aaCapsid aaE1 aa35411209931848210RA27/3RVSVTTHOther RVKAP or LIASYRVs/Oulu.FIN/22.15/PIDRVSVATYBoldface with underline indicates reversions to wild-type aa in the patient's virus. Open table in a new tab Table E3Amino acid substitutions in RVs/Oulu.Fin/22.15/PIDNo.ProteinAmino acidNotesPositionRA27/3RVs/Oulu.Fin/22.15/PIDOther 75 viruses1p150203VA∗Boldface with underline denotes amino acids, which are unique for RVs/Oulu.Fin/22.15/PID.V2251DHD, E3427GDD, G4464RCR, H5488ATA6507RPR, GQ-domain (aa 497-717), includes hypervariable region (HVR, aa 697-803)7551AVA, V, T8700AVA, T9718ATA, S, V10728PLP, S, L11730PSP, S12740SPS, P, L, M, A, T13746PLP14751AVA, V15757PTP16797PSP, S, T, A17799SLP, S, T, A18821LILADP-ribose binding, domain (aa 814-966)19883HYH20996PLP211014RCH, P, RProtease domain (aa 1000-1300)221017SPS, P, L231025DND, E241117VAM, V, I25p9067RCRHelicase domain (aa 19-308)26115IVI27582NSNRdRp catalytic domain (aa 569-680)28694HYH29703PSP30C18TAAVaccine-specific aa31118PSP32119RHR33125PSP34141LPL, P35163EVE36166VTV37228AVA, T38237TMT39262TIT, I40267TIT41295VLV, A42E251HYH, Y43109AVA44111SPS, A, T, F45112TMT, I46116TAT, A, P47134GDG48136LSL49165YFY50211VAV51218TIT, I, A52223SLS53230ATA54235LFL55266AVA56E134AVA5757VAV, L, I5870PSP5984FLF, L6095YHY61210HYYVaccine-specific aa62280ITI63329VIV64415PSP65429TIT66437QEQ67440AVA, V68444ATA69445ASA∗ Boldface with underline denotes amino acids, which are unique for RVs/Oulu.Fin/22.15/PID. Open table in a new tab Table E4Changes in the antigenic structure of RV antigens of RVs/Oulu.FIN/22.15/PID relative to RA27/3 vaccineEpitopeEpitope sequence in vaccine virusSubstitutions in patients' virusNeutralizing B-cell epitopes E1214-233∗Immunodominant epitope.QQSRWGLGSPNCHGPDWASPNone E1245-251LVGATPENone E1260-266ADDPLLRNone E1274-285VWVTPVIGSQARI280→TCD8+ T-cell epitopes C9-22MEDLQKALETQSRAT18→A C11-29DLQKALETQSRALRAELAAT18→A C264-272RIETRSARHT267→I∗ Immunodominant epitope. Open table in a new tab Table E5Primary antibodies used for the immunofluorescence assayAntigenTypeVendorCatalog no.DilutionCD1a [EP3622]Rb mAbAbcamab1083091:200CD3 [SP7]Rb mAbAbcamab166691:200CD11c [EP1347Y]Rb mAbAbcamab526321:200CD20 [EP459Y]Rb mAbAbcamab782371:200vWFRb PabSigmaF35201:500CD14 [EPR3653]Rb mAbAbcamab18999151:100CD68Rb PabAbcamab1250471:100CD163 [EPR14643-36]Rb mAbAbcamAb1899151:500CD206Rb PabAbcamab646931:200iNOSRb PabNovusNBP1-337801:200Pan-cytokeratinRb PabAbcamab93771:500Rubella capsidMs mAbAbcamab347491:500Rubella capsidMs mAbCDCNA1:500Rubella virionsRb PabCDCNA1:2000Measles NP [83KKII]Ms mAbMilliporeMAB8906-KC1:500Mengo 3DPol [3B7]Ms mAbSanta Cruzsc-656331:500NA, Not available/applicable; Pab, polyclonal antibody. Open table in a new tab Boldface with underline indicates reversions to wild-type aa in the patient's virus. NA, Not available/applicable; Pab, polyclonal antibody.