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. 2021 Jan 7;16(1):e0245077. doi: 10.1371/journal.pone.0245077

Immune alterations in subacute sclerosing panencephalitis reflect an incompetent response to eliminate the measles virus

Sibel P Yentür 1, Veysi Demirbilek 2, Candan Gurses 3,¤a, Safa Baris 4, Umit Kuru 5, Semih Ayta 6, Zuhal Yapici 3, Suzan Adin-Cinar 7, Serap Uysal 8, Gulden Celik Yilmaz 9,¤b, Emel Onal 10, Ozlem Cokar 6, Güher Saruhan-Direskeneli 1,*
Editor: Edgar Meinl11
PMCID: PMC7790413  PMID: 33411786

Abstract

In subacute sclerosing panencephalitis (SSPE) the persistence of measles virus (MeV) may be related to the altered immune response. In this study, cytokine responses of lymphocytes and monocytes were evaluated in SSPE compared to controls with non-inflammatory (NICON) and inflammatory (ICON) diseases. Patients with SSPE (n = 120), 78 patients with ICON and 63 patients with NICON were included in this study. Phenotypes of peripheral blood mononuclear cells (PBMC) have been analyzed by flow cytometry. CD3 and CD28, and S. aureus Cowan strain I (SAC) stimulated and unstimulated cells were cultured and IL-2, IL-10, IFN-γ, IL-12p40, IL-12p70 and IL-23 were detected in supernatants by ELISA. MeV peptides were used for MeV-specific stimulation and IFN-γ secretion of PBMC was measured by ELISPOT. Spontaneous and stimulated secretions of IL-10 were lower in SSPE compared to both control groups. T cell stimulation induced lower IFN-γ production than ICON group, but higher IL-2 than NICON group in SSPE. Stimulated PBMC produced lower IL-12p70 in SSPE and had decreased CD46 on the cell surface, suggesting the interaction with the virus. IFN-γ responses against MeV peptides were not prominent and similar to NICON patients. The immune response did not reveal an inflammatory activity to eliminate the virus in SSPE patients. Even IL-10 production was diminished implicating that the response is self-limited in controlling the disease.

Introduction

Subacute sclerosing panencephalitis (SSPE) is a progressive disease of the central nervous system (CNS) affecting mainly children and early adolescents. It is a rare and late complication of measles virus (MeV) infection with fatal outcome. Typically SSPE patients have a history of primary measles infection at an unusually young age followed by a latent period of 6 to 8 years. The incidence of SSPE worldwide is estimated 1 per million [1]. In a recent epidemiological study of Istanbul, the incidence was found as 2 per million with a girl dominance [2].

Both alterations in the host immune system and changes in the MeV have been the subject of investigations on the pathogenesis of SSPE. In early studies, antibodies against nucleocapsid and matrix proteins of the virus were detected in the serum and cerebrospinal fluid (CSF) of patients. Proliferative T cell responses were found comparable to healthy individuals [3]. However, virus-specific cytotoxic activity was impaired while NK cell cytotoxicity was preserved [4]. Suppression of Th1 cytokine production was reported by demonstrating defective IFN-γ response of peripheral blood mononuclear cells (PBMC) to MeV in SSPE patients with severe disease progression [5]. Elevated IL-12p70+p40 and CXCL10 levels in CSF without an accompanying IFN–γ increase compared with other inflammatory and non-inflammatory disease controls were reported [6]. Higher serum IL-2 concentrations and suppressed Th2 cytokines (IL-4, IL-6 and IL-10) were also demonstrated [7]. Lower IL-12 secretion in response to MeV vaccine and PPD, and IFN-γ and IL-10 productions to PPD have also provided evidence for alterations in immune response regulation in a previous study [8]. More recently, activated IL-12/IFN-γ and the IL-23/IL-17/IL-22 pathways were reported in SSPE patients [9].

The virus is anticipated to induce alterations in the immune response of infected cells to MeV. As a costimulatory molecule of the immune system, signaling lymphocyte activating molecule (SLAMF1/SLAM/CD150) is a common receptor for MeV and expressed mainly on activated lymphocytes [10–12]. In MeV infection, SLAM expression is reduced on host cells [13]. On the other hand, CD46 has been shown to act as a receptor mainly for a limited number of MeV strains [14]. The binding of CD46 by MeV has reduced IL-12 production of monocytes thereby regulating the immune response against the virus [15].

In the present study, the immune response to MeV in SSPE patients has been investigated by cytokine secretions of PBMC in response to non-specific and specific stimulations. Possible regulatory changes on the immune response have been evaluated mainly by cytokine measurements.

Materials and methods

Patients and controls

The study was approved by the institutional ethics committee of Istanbul University Istanbul Medical Faculty and written informed consent was taken from the patients and from the parents of the children according to the Declaration of Helsinki. Patients with SSPE (n = 120) were enrolled to the study between the years 2003–2017. Seventy-five of them were male and 45 were female (Median age: 9 years (1–34 years)). All patients fulfilling the criteria for the diagnosis of SSPE are included in this prospective study: Typical clinical presentations (myoclonus, head drops, hemiplegia, deafness, severe mental and behavioral changes, dementia, visual and speech involvement), EEG findings, measles antibody titers in the CSF [1, 16, 17]. Additionally, all 108 patients tested had oligoclonal IgG bands; in 17.6% of patients presented pattern 2, while in 82.4% patients patterns 3 and 4 with concomitant IgG bands in the serum were detected. Only the patients whose parents did not consent are excluded. The patients were referred to the specialized neurology centers of the referral hospitals from Marmara Region and also from Black Sea, East and Southeast Anatolia regions of Turkey. According to the limited information available from the patients, only 56.7% of patients had a natural history of measles disease occurring between 2 months and 9 years of age and 46.7% of the patients had a known history of measles vaccination (Table 1).

Table 1. Characteristics of donors.

Group N (M/F) Age (years) Disease onset age Measles vaccination Measles Age of infection (months)
SSPE 120 (75/45) 9 (1–34) 9 (3–19) 46.7% 56.7% 18 (2–108)
ICON 78 (41/37) 8 (1–24) 6 (2–15) 43.6% 7.7%
NICON 63 (43/20) 11 (1.5–38) 6.5 (1–16) 47.6% 6.4%
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SSPE: Subacute sclerosing panencephalitis patients, ICON: Controls with inflammatory diseases, NICON: Controls with non-inflammatory diseases. Ages were presented as median values, and minimum and maximum values were given in parentheses. M: male, F: female.

Blood from 141 totally unrelated donors were used as controls: 78 of them had inflammatory diseases (ICON) such as multiple sclerosis, asthma, bronchitis, Miller-Fisher syndrome, type I diabetes, tonsillitis, upper respiratory tract infection and viral infection (37 female and 41 male, median age: 8 years (1–24 years)). As another control group, 63 patients (20 female and 43 male, median age: 11 years (1.5–38 years)) with non-inflammatory diseases (NICON) like afebrile convulsion, anemia, headache, leg pain, fatigue, joint pain, epilepsy, X-linked adenoleucodystrophy, nesioblastosis, vomiting and urticeria were included in this study (Table 1). SSPE patients and control donors were not on immunomodulatory treatment.

Due to the scarcity of the blood obtained from the donors, not all measurements have been performed in all donors. Measles antibodies (IgG) were detected with ELISA kit (2326000, Trinity Biotech, Ireland and ESR102G, Serion, Institut Virion, Germany) in all CSF samples.

Phenotypic staining and reagents

PBMC of donors were isolated from EDTA anti-coagulated blood samples by ficoll density gradient centrifugation. Cells were surface stained with fluorochrome-conjugated mouse anti-human CD3-FITC and -APC (IgG1, A07746, Beckman Coulter, France and C7225, Dako Cytomation, Denmark), CD8-APC (IgG1, C7227, Dako Cytomation, Denmark), CD4-APC and -FITC (IgG1, C7226 and F0766 Dako Cytomation, Denmark), CD19-PC5 (IgG1, A07771, Beckman Coulter, France), CD14-FITC and -PC5 (IgG2a, F0844, Dako Cytomation, Denmark and A07765, Beckman Coulter, France), CD45-FITC/CD14-PE (IgG1/IgG2a, Catalog Nr: 873.032.050, Diaclone, France), CD46-PE (IgG2a, 197–050, Ancell, USA), CD150-PE (IgG1, 12–1509, eBioscience), PD-1-PE (IgG1, 557946, BD Pharmingen, USA) and isotype control antibodies (BD Biosciences Pharmingen, USA and X0950, X0933, X0968, Dako Cytomation, Denmark and A07798, Beckman Coulter, France). Phenotypes have been analyzed by flow cytometry (FACSCalibur, Becton-Dickinson) and compared between groups in respective cell gates.

Cell stimulation and measurements of proliferation and cytokines

To stimulate the T cells, flat-bottomed 96-well plates (TPP, Switzerland) were coated overnight at 4°C with anti-CD3 (10 μg/ml, 854.010.000, Diaclone, France) and anti-CD28 (5 μg/ml, 177–020, Ancell, USA) or with IgG1 isotypic control (5 μg/ml, 857.070.000, Diaclone, France) antibodies. PBMC were seeded as 200.000 cells/well in triplicates. In another set of donors, S. aureus Cowan strain I (SAC, Pansorbin, 507858, Calbiochem, USA) was added to a final dilution of 1:10000. After 72 hours of incubation at 37°C in 5% CO2 with culture medium containing RPMI-1640 (R0883, Sigma, USA), 10% FBS (10082139, Gibco, USA), 100 IU/100 μg/ml penicillin/streptomycin (P4333, Sigma, USA) and 2 mM L-glutamine (25030081, Gibco, USA), supernatants were collected and fresh medium was added. Proliferation was detected by [3H] thymidine incorporation (0.5 μCi/well, 20 Ci/mmol, ART 178C, American Radiolabeled Chemicals, USA) after overnight incubation in culture. Supernatants were stored at -80°C and used for the detection of IL-2, IL-10, IFN-γ (KHC0022, CHC1323, CHC1233, Biosource, USA), IL-12p40 and IL-12p70 (551116 and 559258, BD Biosciences, USA) and IL-23 (BMS2023, Bender MedSystems, Austria) by ELISA according to manufacturer’s protocols.

Peptides from hemagglutinin (H, 30–38), matrix (M, 211–219), C protein (C, 84–92), two of nucleoprotein (N1, 210–218 and N2, 340–348) and the pool of these peptides (MeVp) were used to MeV specific stimulations [18]. All peptides and the MeVp were used as 10 μM (NMI Peptides, Germany). IFN-γ secretion of PBMC was detected with ELISPOT (3420-2AW-Plus, MabTech, Sweden and 874.000.005 Diaclone, France) according to manufacturer’s guidelines. PHA (5 μg/ml, Sigma) was used as positive control and spot counts were presented as spots/200000 cells (CTL Europe GmbH).

Statistical analysis

Statistical analyses were performed by non-parametric tests (Anova and Mann-Whitney U tests) for comparisons between groups using SPSS. Results were presented as median values. A p value < 0.05 was regarded as significant.

Results

Cytokine responses in SSPE

When we evaluated the distributions of T and B cells, and monocytes in the peripheral blood of SSPE patients with two different age-matched control groups with (ICON) or without inflammatory diseases (NICON), the proportion of CD3+ T cells was slightly decreased in SSPE patients compared only to NICON (64.7% vs. 68.9%, p = 0.02), confirming our previous findings [19]. No other alterations were observed in the distribution of CD4+ and CD8+ T cells, CD19+ B cells and CD14+ monocytes in all groups (Table 2).

Table 2. Distribution of CD3+, CD4+ and CD8+ T cells, CD19+ B cells and CD14+ cells among PBMC in the study groups.

CD3+ cells CD4+ cells CD8+ cells CD19+ cells CD14+ cells
SSPE 64.7* 32.4 26.1 11.6 73.9
(15.5–82.7) (11.4–62.8) (9.0–45.0) (1.7–25.7) (2.4–99.1)
N 50 90 89 27 62
ICON 66.6 34.3 25.2 10.2 76.0
(0.4–82.5) (11.8–53.7) (7.9–47.9) (2.6–17.5) (12.1–98.8)
N 44 64 66 22 55
NICON 68.9* 36.3 27.1 7.0 71.8
(29.2–80.5) (12.5–59.2) (15.7–52.8) (3.8–16.0) (5.5–96.0)
N 22 33 36 9 26
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Results are presented as median values, and minimum and maximum values are given in parenthesis. SSPE: Subacute sclerosing panencephalitis patients, ICON: Controls with inflammatory diseases, NICON: Controls with non-inflammatory diseases.

*p = 0.02.

Spontaneous cytokine secretion

To analyze in vivo stimulated state of PBMC, we firstly measured spontaneous secretion of IL-2, IFN-γ, IL-12 and IL-10 in cell culture. Among all measured cytokines, only IL-10 levels were significantly lower in SSPE compared with both ICON and NICON groups (4.1 vs. 29.8 and 44.5 pg/ml; p = 0.014 and p = 0.002) (Fig 1).

Fig 1. Spontaneous cytokine secretion of PBMC.

Fig 1

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Spontaneous in vitro IL-2, IL-10, IFN-γ, IL-12p40 and IL-12p70 secretion of PBMC from subacute sclerosing panencephalitis patients (SSPE, n = 29), controls with inflammatory diseases (ICON, n = 13) and with non-inflammatory diseases (NICON, n = 16) are shown. Horizontal lines depict median values.

T cell receptor mediated stimulation by CD3 and CD28

To evaluate the response of T cells by stimulation via T cell receptor, PBMC were incubated with anti-CD3 and anti-CD28 antibodies for 72 hours and cytokine productions were compared between the groups. With stimulation, IL-10 secretion was still lower in patients than in ICON and NICON groups (5.4 vs. 40.2 and 39.5 pg/ml; p = 0.002 and p = 0.012). Similarly, T cell stimulation induced lower levels of IFN-γ production compared with ICON group only (3.8 vs. 201 pg/ml, p = 0.023). However, IL-2 secretion of T cells was higher than that of NICON group (18.7 vs. 0.0 pg/ml; p = 0.005) (Fig 2).

Fig 2. CD3 and CD28 induced cytokine secretion of PBMC.

Fig 2

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CD3 and CD28 induced productions of IL-2, IL-10, IFN-γ in subacute sclerosing panencephalitis patients (SSPE, n = 29), controls with inflammatory diseases (ICON, n = 13) and with non-inflammatory diseases (NICON, n = 16) are shown. Horizontal lines depict median values.

As a costimulatory molecule possibly effecting the cytokine production and a receptor for the MeV, we measured the expression of SLAM on CD4+, CD8+ T cells and B cells. SLAM on CD4+ T cells was slightly higher in SSPE and ICON groups without reaching statistical significance. On CD19+ B cells, SLAM was mainly increased in ICON group (25.29% p = 0.033 and p = 0.04). Relatively higher levels of SLAM in SSPE patients (14.6%) were not significantly different from NICON group (2.1%) (Fig 3, S1 Fig).

Fig 3. Signaling Lymphocyte Activating Molecule (SLAM) expression on T and B lymphocytes.

Fig 3

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SLAM expression on CD4+, CD8+ T cells and CD19+ B cells of subacute sclerosing panencephalitis patients (SSPE, n = 44, n = 43 and n = 26), controls with inflammatory diseases (ICON, n = 35, n = 34 and n = 18) and with non-inflammatory diseases (NICON, n = 12 n = 13 and n = 9) are shown. Horizontal lines depict median values.

Despite the differences in cytokine productions, no significant changes between the groups were observed in proliferative responses of T cells to CD3 and CD28 stimulation (S2 Fig).

Stimulation with SAC

Cytokine production by PBMC was also studied by SAC stimulation to cover a broader group of cell responses [20, 21]. In SSPE patients, SAC induced lower IL-12p70 production compared with ICON and NICON groups (5.3 vs. 10.5 and 9.7 pg/ml, p = 0.043 and p = 0.013). Higher IL-12p40 secretion was detected in ICON patients compared with NICON group (283.4 vs. 101.4 pg/ml, p = 0.011). No other differences in IL-10, IL-23 and IFN-γ productions were detected between the groups in response to SAC (Fig 4).

Fig 4. SAC induced cytokine productions.

Fig 4

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IFN-γ, IL-10, IL-23, IL-12p40 and IL-12p70 productions of SAC stimulated PBMC in subacute sclerosing panencephalitis patients (SSPE, n = 26), controls with inflammatory diseases (ICON, n = 6) and with non-inflammatory diseases (NICON, n = 25) are shown. Horizontal lines depict median values.

As the main source of IL-12 is monocytes, the reduced IL-12 production may have been induced by interaction of the virus with cellular receptors/molecules on monocytes [15]. Therefore, we screened the possible MeV receptor, CD46 on monocytes (CD14+). CD46 was lower in SSPE patients than NICON (63.0% vs. 74.4%; p = 0.033), but not than the ICON group (69.3%) (Fig 5, S3 Fig).

Fig 5. CD46 expression on CD14+ monocytes.

Fig 5

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CD46 on CD14+ cell populations was analyzed in subacute sclerosing panencephalitis patients (SSPE, n = 46), controls with inflammatory diseases (ICON, n = 40) and with non-inflammatory diseases (NICON, n = 23). Horizontal lines depict median values.

Antigen-specific T cell stimulation

As the etiological agent of SSPE, the specific response against MeV has also been screened and compared with diseased controls. We have evaluated the antigen specific IFN-γ responses against immunodominant MeV peptides which were described previously [18]. SSPE patients had similar reactivity to N1, N2, M, H and C peptides of MeV and MeV peptide pool as in NICON patients. Interestingly, IFN-γ responses to all peptides and peptide pool were reduced in ICON group compared to SSPE, and NICON groups (Fig 6).

Fig 6. MeV peptide induced IFN-γ production of PBMC.

Fig 6

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IFN-γ secretion without and with MeV peptide (C, H, N1, N2, M and the pool) induction of PBMC in subacute sclerosing panencephalitis patients (SSPE, n = 26), controls with inflammatory diseases (ICON, n = 13) and with non-inflammatory diseases (NICON, n = 15) are shown. Horizontal lines depict median values of IFN-γ secreting cells per 200000 PMBC.

Expression of programmed cell death-1 (PD-1) on CD8+ T lymphocytes

As a possible mechanism of viral persistence of MeV in SSPE, we have looked at the exhaustion of T cells by analyzing the expression of PD-1 on CD8+ T cells. PD-1 is a member of the CD28 superfamily that delivers negative signals upon interaction with its ligands. However, CD8+ PD-1+ cells were not different between groups in PBMC (S4 Fig).

Discussion

The pathogenesis of the persistence and the late complication of MeV infection in the CNS have not been elucidated. In this study, the immune alterations in the SSPE patients have been compared with two different age-matched control groups, namely patients with other inflammatory- or non-inflammatory-diseases. The immune response against MeV-specific and non-specific stimuli was evaluated in these groups. Changes were detected in cytokine secretion in SSPE compared with both control groups. Notably, cytokine production pattern of SSPE patients was relatively similar to that of NICON group. Thus, the findings of the present study emphasized the moderate immune response in the patients.

MeV infection induces profound and prolonged abnormalities in cellular immune responses in infected hosts causing an immunosuppression [22]. The mechanistic models underlying this suppression have been proposed as altered cytokine profiles, bystander lymphocyte apoptosis and lymphocyte infection and depletion [23]. The immunosuppression associated with MeV could underlay the viral persistence and the development of SSPE [4, 8]. The findings of this study did not provide evidence for an active immunosuppressive mechanism similar to acute measles infection in SSPE development.

In previous evaluations of the immune response, mainly CD4+ T cells, B cells as well as IL-1β, IL-2, IL-6, TNF-α, lymphotoxin and IFN-γ have been detected in brain specimens from SSPE patients, which demonstrated cellular infiltrates, demyelination, gliosis and inflammatory activation [24–27]. Detection of higher IL-10 levels in both CSF [6, 28], and serum of SSPE patients [29], supported a suppressive environment in this disease. In another study, CSF IL-4 and IL-6 concentrations were lower, whereas serum IL-2 concentration was higher in SSPE patients implicating the dominance of Th1 over Th2-type cytokines particularly at the early inflammatory response in SSPE [7]. On the other hand, preserved IL-10 production by PBMC of SSPE patients was reported, whereas defective IFN-γ secretion has been related to the worse progression of disease in response to MeV vaccine [5]. In our previous study, proliferation, as well as IFN-γ, IL-12 and IL-10 productions in response to MeV vaccine were not different from controls, although the response to purified protein derivate (PPD) was impaired in SSPE patients [8]. The decrease in regulatory T cell phenotype and the lower inhibitory NK receptors on CD8+ T cells, and higher activating NK receptors on NK cells indicated also an active state of the immune response possibly caused by the chronic stimulation with viral antigens [19]. A recent report demonstrated increased productions of IL-12, IL-23, IL-17 and IL-22 and higher frequencies of IL-17 and IFN-γ producing cells in response to MeV peptide stimulation in SSPE pointing at both Th1 and Th17 responses [9]. In the present study, production of IL-10 was lower in SSPE compared with both control groups spontaneously and after T cell stimulation. Supporting an immune activity potential, IL-2 response of T cells was higher than in NICON group, whereas IFN-γ production was not as high as in other inflammatory diseases. The MeV peptide specific IFN-γ responses were also similar to NICON patients and not higher. This inefficient IFN-γ response in our cohort is not in accordance with previous findings, but in accordance with the lower IL-12 production. This discrepancy may be related to the selection of our patients with late stages or to the small numbers of patients in different experimental settings in these studies.

Similar to measles infection, a reduction of T cells in SSPE samples was observed in the present and previous studies [19, 30]. This observation may be related to the lymphopenia caused by MeV during the infection. MeV infected monocytes induce apoptosis in uninfected T cells which also may contribute to the pathogenesis of MeV-induced immunosuppression [31].

IL-10 is considered a prototypical anti-inflammatory cytokine, which significantly contributes to the maintenance and reestablishment of immune homeostasis. However, with its pleiotropic roles, it can also promote immune responses by supporting B cell and CD8+ T cell activation. In infectious diseases, it is considered as a master regulator of immunity, as it can mainly help to ameliorate the excessive Th1, Th2 and CD8+ T cell responses in infections [32]. Functionally, reduced production of IL-10 with IFN-γ in response to general T cell stimulation in SSPE may highlight the absence of a highly pro-inflammatory state of the disease. On the other hand, polymorphic features of IL-10 gene may have an effect on susceptibility to SSPE and the decreased production may be related to the low-producer alleles of IL-10 [21], which has not been investigated in SSPE.

The MeV cell entry receptor, SLAM is classified as a costimulatory molecule that favors lymphocyte proliferation, Ig synthesis, and secretion of IFN-γ [33–35]. Therefore, the interaction of MeV with SLAM could have effects on those immune cells leading to virus-mediated activation. In a previous study, SLAM expression was also demonstrated to be increased in lymphocytes, monocytes and brain tissues of two SSPE patients [36]. However, in the present study SLAM on T or B cells was not increased significantly in SSPE.

On the other hand, MeV has been shown specifically to ablate IL-12 production by monocyte/macrophages in vitro through binding to CD46 [15]. Reduced IL-12 production of monocytic cells was also accompanied by the down-regulation of CD46 from the surface of cells infected with vaccine strains of MeV in vitro [37, 38]. Lower expression of CD46 in lesions of SSPE brains suggested also an interaction of CD46 with a SSPE-specific MeV strain [14]. In SSPE patients, the lower frequencies of CD46+ monocytes compared with NICON group in this study also implicated a related interaction of the virus with these cells. The presence of viral RNA has been documented in monocytes during acute measles infection [39]. Although not confirmed in SSPE patients, persistence of viral RNA has been demonstrated in PBMC and lymph nodes of the monkeys experimentally infected with MeV after months [40]. Possibly a low number of MeV present in the body and inducing the stable antibody response may use CD46 to enter the monocytes and internalize these molecules persistently. Moreover, reduced IL-12 production may be related to CD46-downregulation as well as effected by complement and phagocytic receptors on monocytes [41]. As IL-12 is critical for the development of cell mediated immunity and a potent inducer of IFN-γ from T and NK cells, the development of SSPE can pursue in these patients.

On the other hand, a relatively strong immune response generated in the CNS is evident by the anti-MeV antibodies and oligoclonal IgG bands in the CSF and in the serum of majority of SSPE patients. Probably this antibody response is induced by persistent virus in the CNS and with the leakage of the antibodies, some viral antigens can also reach the periphery and cause the subtle changes detected in this study, but not effective in eliminating virus or controlling replication in the CNS. Demonstration of persistent viral RNA in lymphoid cells would have contributed to explain dysfunctional immune response [40].

The study has certain limitations mainly due to the nature of this rare disease. Findings presented here were derived from data obtained throughout many years, which has been a caveat for prospective planning of the experiments in the study. Additionally, the comparisons with two different diseased-control groups have been somehow problematic, as the diseases of the controls were heterogeneous and the donors have been difficult to assign to the respective groups. The two control groups have been composed of children with known diseases with or without inflammatory features.

Conclusions

In SSPE patients, T cells produced lower levels of IL-10 and IFN-γ, but were inducible to produce IL-2, consistent with an altered immune response of T cells, not competent enough to eliminate the virus in SSPE. Monocytic cells of the patients revealed reduced IL-12 production and CD46 surface expression implicating the effect of CD46 binding in SSPE similar to some MeV strains. Reduced production of IL-10 in combination with reduced IFN-γ points at inefficiency of effector functions of T cells. These observations in SSPE pointed at an attenuated inflammatory pattern at a chronic phase of the disease.

Supporting information

S1 Fig. Detection of SLAM on CD19+ B lymphocytes.

SLAM expression on CD19+ cells in a subacute sclerosing panencephalitis patient (SSPE) and in controls with inflammatory diseases (ICON) or non-inflammatory diseases (NICON) are shown.

(TIF)

Click here for additional data file. (112.9KB, tif)
S2 Fig. Proliferative responses of T cells to CD3 and CD28 stimulation.

Spontaneous and CD3+CD28 induced proliferative responses of T cells in subacute sclerosing panencephalitis (SSPE) patients and in controls with inflammatory diseases (ICON) and non-inflammatory diseases (NICON) are shown. Cpm: Counts per minute, SI: Stimulation index (SI = Induced cpm / Spontaneous cpm).

(TIF)

Click here for additional data file. (154.5KB, tif)
S3 Fig. Detection of CD46 on monocytes.

CD14+CD46+ monocytes in a subacute sclerosing panencephalitis patient (SSPE), controls with inflammatory diseases (ICON) and with non-inflammatory diseases (NICON) are shown.

(TIF)

Click here for additional data file. (46.6KB, tif)
S4 Fig. PD-1 on CD8+ T cells.

PD-1 on CD8+ T cells in a subacute sclerosing panencephalitis patient (SSPE), controls with inflammatory diseases (ICON) and with non-inflammatory diseases (NICON) are shown. Horizontal lines depict median values.

(TIF)

Click here for additional data file. (83.3KB, tif)
S1 Data

(PDF)

Click here for additional data file. (199KB, pdf)

Acknowledgments

We are grateful to patients and their parents, and to Dr. Erdem Tüzün for critically reading the manuscript.

Data Availability

All relevant data are within the manuscript.

Funding Statement

The study is supported by Istanbul University Research Fund (BAP: ACIP3363-ACIP- 22421-T291).

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Decision Letter 0

Edgar Meinl

15 Sep 2020

PONE-D-20-23751

Immune alterations in subacute sclerosing panencephalitis reflect an incompetent response to eliminate the measles virus

PLOS ONE

Dear Dr. Saruhan-Direskeneli,

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i) what type of consent you obtained (for instance, written or verbal, and if verbal, how it was documented and witnessed), and

ii) whether informed consent was also obtained from adults in the study.

In addition, we note that you refer to patients as "boys" and "girls" however some patients are not minors. Please revise your manuscript to refer to these patients as "male" and "female".

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a) provide more detailed criteria for the diagnosis of SSPE

b) provide the source, name, and catalog numbers of the ELISA kits for detecting measles

c) provide the source, catalog numbers, and dilutions for all primary/secondary/isotype control antibodies used in the study.

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This study is supported by Istanbul University Research Fund.'

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #2: Yes

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Reviewer #1: The authors investigated the immune response in patients with SSPE in comparison to two well age-matched control groups consisting of inflammatory (ICON) and non-inflammatory (NICON) diseases. Taking into account that SSPE is a rare disease the number of patients is very high.

The authors used established methods (FACS, ELISA, Elispot assays, 3H thymidine incorporation). PBMCs of patients and controls were investigated ex vivo or after different stimulations in vitro – unspecific (antibodies, SAC) or MeV specific (viral peptides).

The results demonstrate, that PBMC of SSPE patients produced lower levels of IL-10 and IFN-g after stimulation with anti-CD3 and anti CD28, but were inducible to produce IL-2. After SAC stimulation PBMC of SSPE patients showed reduced IL-12p70 production and CD 14+ monocytes demonstrate lower CD46 surface expression. In Elispot assays spontaneous IFN-g production and antigen stimulated IFN-g production was elevated in SSPE patients and NICON compared with ICON.

The authors concluded, that T cells of SSPE patients demonstrate an altered immune response that is not sufficient to eliminate the virus. In monocytes reduced IL-12 production and CD46 surface expression implicate the effect of CD46 binding in SSPE similar to MeV infection.

Major points:

1. The authors report that in a recent epidemiological study of Istanbul a girl dominance was found. In their SSPE cohort there is a dominance of boys. Is this just by chance? From which part of the country or from which country were the patients recruited?

2. Different clinical stages of SSPS are known. For reference see e.g. Jabbour J, et al., SSPE-clinical staging, course, and frequency. Arch Neurol. 1975;32(7):493–494. 24 or Gutierrez J, et al., Dev Med Child Neurol. 2010 Oct;52(10):901-7. It would be interesting to know in which clinical state the patients had been at the time of blood sampling.

3. How do the authors interpret the reduction of T cells in SSPE samples?

4. The authors report “Monocyte stimulation with SAC”. In the legend of Fig. 4 they report “… SAC stimulated PBMC…”. If they don´t select monocytes before stimulation, the headline of this section and the legend of figure 4 should be modified. In addition, the authors should mention that SAC does not stimulate only monocytes in their cell culture.

5. At the beginning of the discussion the authors claim “… no evidence was found for immunosuppressive mechanisms as a determining factor in SSPE development.” The authors may explain more detailed the reasons for this statement or omit it.

6. The authors report a reduced production of IL-10 in the present study, but mention a production of IL-10 as in controls in their previous study. Do the authors have an explanation for this?

7. IL-10 is a cytokine with strong immunosuppressive properties. However, there are also publications demonstrating immunostimulation by IL-10, e.g. Il-10 enhance the capacity of resting CD4+ lymphocytes to produce cytokines. The authors may discuss this aspect as well and not solely the immunosuppressive properties of IL-10.

8. In the section “Antigen-specific T cell stimulation” the authors report “Interestingly, Ifn-g responses to all peptides and peptide pool were reduced in ICON group compared to SSPE (…) and NICON groups (…) …”. The authors may comment on this finding in the discussion, especially as they show an IFN-g response in PBMCs of ICON after unspecific stimulation (Fig. 2).

9. In the discussion the authors report “…SLAM expression was relatively higher in all cell subgroups both in SSPE and other inflammatory diseases compared with donors without inflammation…”. Fig. 3 demonstrate significant differences only for B-cells. This statement has to be modified.

10. The authors report that “… the donors have been difficult to assign to the respective groups.” Please explain the reasons.

Minor points:

1. Determination of CD46 and SLAM should be illustrated with an example showing a FACS analysis of a patient and two controls, e.g. as supplementary figures.

2. In the section “Antigen-specific T cell stimulation” there is a list of p-values. It is unclear to which peptide stimulation a given p-value belongs. The authors should present peptide stimulation and corresponding p-value in a supplementary table or omit the p-values in the text, as they are given in Fig. 6.

3. The citation format of two references in the discussion does not comply with the journal style.

Reviewer #2: In this study, authors aimed to test the cytokine profile of lymphocytes and monocytes obtained from the blood of SSPE patients to gain more insight into the immunopathogenesis of disease. SSPE is a latent brain infection that occurs many years after measles infection and it is a fatal disease. It is an important health problem in some of the developing countries. It is relatively frequent in Turkey, and a high number of SSPE patients were tested in this study. As SSPE is very rare, this study has a potential to contribute to the related literature. However, some points need to be cleared:

Minor revisions:

- Exclusion criteria for all groups should be mentioned.

- How would the authors interpret the finding that IL10 secretion is lower compared to controls? Is this a result of the latent brain infection, or is this more like a risk factor for the disease as a result of genetic factors etc? A comment on that in the discussion would be useful.

- What is the possible functional outcome of the finding that IL12 is decreased in monocytes? A comment on that in the discussion would be useful.

- The authors stated that “In SSPE patients, the lower frequencies ofCD46+ monocytes compared with NICON group in this study also implicated a related interaction of the virus with these cells.” However, SSPE is known as a latent brain infection and active involvement of monocytes in the periphery would be a surprising finding which may refer to ongoing MV activity in peripheral organs. Can the authors make this point clearer in discussion?

- In the conclusion, authors state that their findings pointed at an attenuated inflammatory pattern at a chronic phase of SSPE. Considering that SSPE is a brain-restricted latent infection, what may be the reason behind the attenuated inflammatory pattern seen in the peripheral blood cells? Discussion of this point would be useful.

**********

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Reviewer #1: No

Reviewer #2: Yes: Atay Vural

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PLoS One. 2021 Jan 7;16(1):e0245077. doi: 10.1371/journal.pone.0245077.r002

Author response to Decision Letter 0

2 Nov 2020

To comply with PLOS ONE submission guidelines, we have added the description of the methodology in more detail and the supplementary information in supplementary figures (1-4) as requested. We also removed the funding information from the manuscript, which should be included in the manuscript as below:

The study is supported by Istanbul University Research Fund (BAP: ACIP3363-ACIP-22421-T291).

Responses to Reviewers

Reviewer #1: The authors investigated the immune response in patients with SSPE in comparison to two well age-matched control groups consisting of inflammatory (ICON) and non-inflammatory (NICON) diseases. Taking into account that SSPE is a rare disease the number of patients is very high.

The authors used established methods (FACS, ELISA, Elispot assays, 3H thymidine incorporation). PBMCs of patients and controls were investigated ex vivo or after different stimulations in vitro – unspecific (antibodies, SAC) or MeV specific (viral peptides).

The results demonstrate, that PBMC of SSPE patients produced lower levels of IL-10 and IFN-g after stimulation with anti-CD3 and anti CD28, but were inducible to produce IL-2. After SAC stimulation PBMC of SSPE patients showed reduced IL-12p70 production and CD 14+ monocytes demonstrate lower CD46 surface expression. In Elispot assays spontaneous IFN-g production and antigen stimulated IFN-g production was elevated in SSPE patients and NICON compared with ICON.

The authors concluded, that T cells of SSPE patients demonstrate an altered immune response that is not sufficient to eliminate the virus. In monocytes reduced IL-12 production and CD46 surface expression implicate the effect of CD46 binding in SSPE similar to MeV infection.

Major points:

1. The authors report that in a recent epidemiological study of Istanbul a girl dominance was found. In their SSPE cohort there is a dominance of boys. Is this just by chance? From which part of the country or from which country were the patients recruited?

As pointed out by the reviewer, in our epidemiological study, the distribution of sexes was in favor of the women, whereas our cohort was not in agreement with this observation. The reason for this discrepancy may be related to the natures of the studies. The epidemiology was planned for the determination the incidence in Istanbul area in two years, whereas in our study the patients were collected throughout 14 years in the same area. Some information about the referral system of the patients is also added to the material and methods section.

2. Different clinical stages of SSPS are known. For reference see e.g. Jabbour J, et al., SSPE-clinical staging, course, and frequency. Arch Neurol. 1975;32(7):493–494. 24 or Gutierrez J, et al., Dev Med Child Neurol. 2010 Oct;52(10):901-7. It would be interesting to know in which clinical state the patients had been at the time of blood sampling.

We agree with the reviewer. Unfortunately the information related to the clinical stages of the patients was only available in a small number of patients (n: 44). As the patients were referred from several centers, we could not access their files later on. Patients with known stages were not representing the whole cohort, so that we did not include this information.

3. How do the authors interpret the reduction of T cells in SSPE samples?

As pointed at by the reviewer, a reduction of T cells in SSPE samples was observed in our previous study (reference 19) and in other studies (reference 30) as well. This observation may be related to the lymphopenia caused by MeV during the infection. MeV infected monocytes induce apoptosis in uninfected T cells which also may contribute to the pathogenesis of MeV-induced immunosuppression (reference 31). We have added this issue to the discussion.

4. The authors report “Monocyte stimulation with SAC”. In the legend of Fig. 4 they report “… SAC stimulated PBMC…”. If they don´t select monocytes before stimulation, the headline of this section and the legend of figure 4 should be modified. In addition, the authors should mention that SAC does not stimulate only monocytes in their cell culture.

As the reviewer highlighted, the assumption that the reduced IL-12 production is from monocytes was based on the fact that SAC would not be able to induce IL-12 by any other cells type in PBMC. The only differentially produced cytokine induced by SAC was IL-12, so that the monocytes have been accounted for this result. But we agree that the finding is based on an indirect finding and is an interpretation of the data. We have changed the text accordingly.

5. At the beginning of the discussion the authors claim “… no evidence was found for immunosuppressive mechanisms as a determining factor in SSPE development.” The authors may explain more detailed the reasons for this statement or omit it.

This sentence was meant as a general evaluation of the study and emphasized that the disease was not accompanied by an immunosuppression as observed in measles infection. We have changed this section now accordingly.

6. The authors report a reduced production of IL-10 in the present study, but mention a production of IL-10 as in controls in their previous study. Do the authors have an explanation for this?

We agree with the reviewer that the results of two studies are not in accordance for the spontaneous IL-10 response of the patients. The control group in our previous study was consisted of healthy adults whereas the controls in this study are age-matched diseased controls. The variance between cytokine measurements may be related to this parameter.

7. IL-10 is a cytokine with strong immunosuppressive properties. However, there are also publications demonstrating immunostimulation by IL-10, e.g. Il-10 enhance the capacity of resting CD4+ lymphocytes to produce cytokines. The authors may discuss this aspect as well and not solely the immunosuppressive properties of IL-10.

We agree with the reviewer that IL-10 has pleiotropic roles. However, in infectious diseases, apparently it mainly acts as regulator in a context dependent manner. As the production of IL-10 was reduced in our patient group, we have interpreted these finding as a dysregulation of regulatory response and added a comment to the discussion.

8. In the section “Antigen-specific T cell stimulation” the authors report “Interestingly, Ifn-g responses to all peptides and peptide pool were reduced in ICON group compared to SSPE (…) and NICON groups (…) …”. The authors may comment on this finding in the discussion, especially as they show an IFN-g response in PBMCs of ICON after unspecific stimulation (Fig. 2).

We agree with the reviewer about this comment on the unexpected findings. We consider that the IFN-g response to CD3+CD28 in ICON group is produced by a broad range of T cells as the stimulation is not specific in this case. On the other hand, the peptide specific IFN� production in ICON group may be compromised particularly for the specific responses.

9. In the discussion the authors report “…SLAM expression was relatively higher in all cell subgroups both in SSPE and other inflammatory diseases compared with donors without inflammation…”. Fig. 3 demonstrate significant differences only for B-cells. This statement has to be modified.

The differences of SLAM we observed on CD4, CD8 and CD19 + cells were not significantly different and the only increase was detected on B cells of ICON group. That is why we did not mention the activation state of PMBC in the results section and in the discussion as a comment only. Now we have omitted this statement from the discussion, as it was not based on significant differences.

10. The authors report that “… the donors have been difficult to assign to the respective groups.” Please explain the reasons.

As some of the diseases we included in the controls are not easily assignable to inflammatory or no-inflammatory subgroups. Due to the subtle inflammatory changes in these disease states, we were not sure to include these patients in non-inflammatory cases (X-linked adenoleucodystrophy, nesioblastosis). We have added the some of the diagnosis in these groups.

Minor points:

1. Determination of CD46 and SLAM should be illustrated with an example showing a FACS analysis of a patient and two controls, e.g. as supplementary figures.

FACS analysis of SLAM and CD46 staining is now added as supplementary figures as requested (S1 Fig and S3 Fig).

2. In the section “Antigen-specific T cell stimulation” there is a list of p-values. It is unclear to which peptide stimulation a given p-value belongs. The authors should present peptide stimulation and corresponding p-value in a supplementary table or omit the p-values in the text, as they are given in Fig. 6.

We have omitted the p values from the text.

3. The citation format of two references in the discussion does not comply with the journal style.

We have corrected them.

 

Reviewer #2: In this study, authors aimed to test the cytokine profile of lymphocytes and monocytes obtained from the blood of SSPE patients to gain more insight into the immunopathogenesis of disease. SSPE is a latent brain infection that occurs many years after measles infection and it is a fatal disease. It is an important health problem in some of the developing countries. It is relatively frequent in Turkey, and a high number of SSPE patients were tested in this study. As SSPE is very rare, this study has a potential to contribute to the related literature. However, some points need to be cleared:

Minor revisions:

1. Exclusion criteria for all groups should be mentioned.

Based on the diagnosis of the patients, we did not exclude any patients with SSPE in the study group. Only patients whose parents did not agree to consent were excluded. All other patients who were not diagnosed with SSPE were included in the control groups according to the disease with or without apparent inflammation. We added this information to the manuscript.

2. How would the authors interpret the finding that IL10 secretion is lower compared to controls? Is this a result of the latent brain infection, or is this more like a risk factor for the disease as a result of genetic factors etc? A comment on that in the discussion would be useful.

We have added a comment to the discussion related to lower IL-10 production in SSPE. We considered the reduced production of IL-10 in combination with reduced IFN-� production as an inefficiency of effector functions of T cells. An exhausted state which we could not demonstrate would be the good explanation for that.

3. What is the possible functional outcome of the finding that IL12 is decreased in monocytes? A comment on that in the discussion would be useful.

As IL-12 is a key inducer of Th1 responses, the decreased activity of Th1 type cells reflected by the lower IFN-� production could be the consequence of this finding. We have added a comment to the discussion.

4. The authors stated that “In SSPE patients, the lower frequencies ofCD46+ monocytes compared with NICON group in this study also implicated a related interaction of the virus with these cells.” However, SSPE is known as a latent brain infection and active involvement of monocytes in the periphery would be a surprising finding which may refer to ongoing MV activity in peripheral organs. Can the authors make this point clearer in discussion?

Although this explanation is not evidence based, we have added some speculative considerations to the discussion about these data. Unfortunately we do not have a good explanation for the development in SSPE in these terms.

5. In the conclusion, authors state that their findings pointed at an attenuated inflammatory pattern at a chronic phase of SSPE. Considering that SSPE is a brain-restricted latent infection, what may be the reason behind the attenuated inflammatory pattern seen in the peripheral blood cells? Discussion of this point would be useful.

With the given data, SSPE is considered as a result of reactivation of a latent infection after many years. The RNA from the measles virus has been isolated in the brain, eyes, and spinal cord in patients with SSPE and persistence of MeV was confirmed in PBMC even after years. An immune response is generated against the virus and a strong antiviral immune response is induced as evidenced by the unusually high levels of antibody in serum and CSF. But it is not effective in eliminating virus or controlling replication in the CNS. During persistence, viral RNA in PBMCs and lymphoid tissue is detected in B, T lymphocytes and monocytes. Persistent RNA in lymphoid cells may contribute to immune response dysfunction by altering the ability of cells to proliferate in response to immune signaling (reference 39).

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Decision Letter 1

Edgar Meinl

23 Nov 2020

PONE-D-20-23751R1

Immune alterations in subacute sclerosing panencephalitis reflect an incompetent response to eliminate the measles virus

PLOS ONE

Dear Dr. Saruhan-Direskeneli,

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Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: The authors improved the paper in general by providing some corrections and further explanations. However, some points would still be better to be improved:

Point #1: If there were no exclusion criteria, were there any patients or controls who have a concomitant acute/chronic infection (other than SSPE), or autoimmune disorder at the time of blood sampling?

Points #2 and #3: The authors provided appropriate answers.

Point #4: The lower frequencies ofCD46+ monocytes compared with NICON group in the current study is an interesting finding and as the authors state “may implicate a related interaction of the virus with these cells.” Can the authors provide data regarding the presence or absence of MeV RNA in monocytes from SSPE patients?

Point #5:

In response to the point #5, the authors commented that “With the given data, SSPE is considered as a result of reactivation of a latent infection after many years. The RNA from the measles virus has been isolated in the brain, eyes, and spinal cord in patients with SSPE and persistence of MeV was confirmed in PBMC even after years…. During persistence, viral RNA in PBMCs and lymphoid tissue is detected in B, T lymphocytes and monocytes. Persistent RNA in lymphoid cells may contribute to immune response dysfunction by altering the ability of cells to proliferate in response to immune signaling…”

Reference #39 given as the source for this information is a review article and from its references it is seen that persistence of MeV was shown in Macaque Monkeys, HIV-infected children, or shortly after acute infection, but not after many years, or in children with SSPE. However, this is an important and interesting point. Could the authors provide more specific literature, or better, could they provide data regarding the presence or absence of MeV RNA in lymphocytes and also in monocytes, in SSPE children vs controls?

In addition, the authors stated that “An immune response is generated against the virus and a strong antiviral immune response is induced as evidenced by the unusually high levels of antibody in serum and CSF. But it is not effective in eliminating virus or controlling replication in the CNS.”

This is again an interesting and important point. According to the authors’ hypothesis, persistence of MeV and slow replication of the virus in the peripheral lymphoid organs and blood can be a source for the latent brain infection. And generation of a higher titer of anti-measles antibodies would show the presence of the viral persistence, but would be ineffective to eliminate the virus. Can the authors provide the specific literature showing the higher titer of anti-measles IgG in SSPE patients compared to controls? Or better, could they provide the data as an addition to the current study?

**********

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Reviewer #2: No

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PLoS One. 2021 Jan 7;16(1):e0245077. doi: 10.1371/journal.pone.0245077.r004

Author response to Decision Letter 1

16 Dec 2020

PONE-D-20-23751R2

Responses to the reviewer

Point #1: If there were no exclusion criteria, were there any patients or controls who have a concomitant acute/chronic infection (other than SSPE), or autoimmune disorder at the time of blood sampling?

There were no SSPE patients with other disorders according to the assessment of the following clinician. We have added a remark about the problem of data acquisition from the patients in the methods section.

Point #4: The lower frequencies of CD46+ monocytes compared with NICON group in the current study is an interesting finding and as the authors state “may implicate a related interaction of the virus with these cells.” Can the authors provide data regarding the presence or absence of MeV RNA in monocytes from SSPE patients?

In this study, although we intended to identify the virus in the patient material in the beginning, we were not able to assess the MeV RNA in the samples of SSPE patients. However, the presence of viral RNA has been documented in monocytes during acute measles infection (Reference 39). MeV infection has been also shown to suppress IL-12 secretion from monocytes (Reference 15). Assuming that the virus used CD46 to enter the monocytes, we claimed that the decreased IL-12 production and CD46 expression could fit well with this picture. Unfortunately, there is no evidence of the viral persistence in the peripheral blood cells, including monocytes, shown formally in SSPE. We have changed the section accordingly.

Point #5:

In response to the point #5, the authors commented that “With the given data, SSPE is considered as a result of reactivation of a latent infection after many years. The RNA from the measles virus has been isolated in the brain, eyes, and spinal cord in patients with SSPE and persistence of MeV was confirmed in PBMC even after years…. During persistence, viral RNA in PBMCs and lymphoid tissue is detected in B, T lymphocytes and monocytes. Persistent RNA in lymphoid cells may contribute to immune response dysfunction by altering the ability of cells to proliferate in response to immune signaling…”

Reference #39 given as the source for this information is a review article and from its references it is seen that persistence of MeV was shown in Macaque Monkeys, HIV-infected children, or shortly after acute infection, but not after many years, or in children with SSPE. However, this is an important and interesting point. Could the authors provide more specific literature, or better, could they provide data regarding the presence or absence of MeV RNA in lymphocytes and also in monocytes, in SSPE children vs controls?

The hypothesis that “the persistence of MeV RNA may contribute to the late development of the slowly progressive disease subacute sclerosing panencephalitis in children infected at a young age” (reference 22) has been proposed by the authors studying measles infection. We also found this approach somehow suitable to our findings. As pointed at by the reviewer, there are no data on the presence of the viral RNA in any peripheral cell or tissue in SSPE. However, based on the proposed similarities between the monkey model and the humans in terms of measles (Measles virus infection in rhesus macaques: Altered immune responses and comparison of the virulence of six different virus strains. J Infect Dis. 1999;180:950–958), we assumed that this explanation can also be applied to our data. Now we have changed the section accordingly. Unfortunately, we do not have any data about the presence of MeV RNA in any cell group of SSPE patients in this study group.

In addition, the authors stated that “An immune response is generated against the virus and a strong antiviral immune response is induced as evidenced by the unusually high levels of antibody in serum and CSF. But it is not effective in eliminating virus or controlling replication in the CNS.”

This is again an interesting and important point. According to the authors’ hypothesis, persistence of MeV and slow replication of the virus in the peripheral lymphoid organs and blood can be a source for the latent brain infection. And generation of a higher titer of anti-measles antibodies would show the presence of the viral persistence, but would be ineffective to eliminate the virus. Can the authors provide the specific literature showing the higher titer of anti-measles IgG in SSPE patients compared to controls? Or better, could they provide the data as an addition to the current study?

We thank the reviewer for raising this point. Unfortunately, we were not precise in our wording: what we know about the antibody response in SSPE is that anti-MeV antibodies are present in the CSF of patients and this is utilized even as a diagnostic finding for SSPE (Reference 16 and 17). The clinical diagnosis of SSPE is confirmed by the finding of increased levels of anti-measles virus anti- body in serum and CSF with an elevated CSF-to- serum ratio in antibody level. Actually, most of the time the antibodies are also detected in the plasma. In our own experience, we have screened the oligoclonal IgG bands in 108 of these study patients. The positivity of IgG bands in the CSF was 100% with a few atypical patterns and all OCB positive patients had also anti-MeV antibodies in the CSF. In 82.4 % of the patients, serum samples also contained accompanying and presumably leaked IgG bands (pattern 3 in 79.6%, pattern 4 in 2.8%). Although these antibodies were not tested for their specificity for MeV, there are data supporting that these antibodies are MeV specific. Our interpretation of the peripheral anti-viral antibody was based on the oligoclonal bands in the patients. The findings only allow to explain the immune response in periphery induced by leaked antigens and antibodies from the CNS. We have also changed the section in the discussion accordingly.

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Decision Letter 2

Edgar Meinl

22 Dec 2020

Immune alterations in subacute sclerosing panencephalitis reflect an incompetent response to eliminate the measles virus

PONE-D-20-23751R2

Dear Dr. Saruhan-Direskeneli,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Edgar Meinl, M.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

**********

6. Review Comments to the Author

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Reviewer #2: (No Response)

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Reviewer #2: No

Acceptance letter

Edgar Meinl

29 Dec 2020

PONE-D-20-23751R2

Immune alterations in subacute sclerosing panencephalitis reflect an incompetent response to eliminate the measles virus

Dear Dr. Saruhan-Direskeneli:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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on behalf of

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Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Fig. Detection of SLAM on CD19+ B lymphocytes.

    SLAM expression on CD19+ cells in a subacute sclerosing panencephalitis patient (SSPE) and in controls with inflammatory diseases (ICON) or non-inflammatory diseases (NICON) are shown.

    (TIF)

    Click here for additional data file. (112.9KB, tif)
    S2 Fig. Proliferative responses of T cells to CD3 and CD28 stimulation.

    Spontaneous and CD3+CD28 induced proliferative responses of T cells in subacute sclerosing panencephalitis (SSPE) patients and in controls with inflammatory diseases (ICON) and non-inflammatory diseases (NICON) are shown. Cpm: Counts per minute, SI: Stimulation index (SI = Induced cpm / Spontaneous cpm).

    (TIF)

    Click here for additional data file. (154.5KB, tif)
    S3 Fig. Detection of CD46 on monocytes.

    CD14+CD46+ monocytes in a subacute sclerosing panencephalitis patient (SSPE), controls with inflammatory diseases (ICON) and with non-inflammatory diseases (NICON) are shown.

    (TIF)

    Click here for additional data file. (46.6KB, tif)
    S4 Fig. PD-1 on CD8+ T cells.

    PD-1 on CD8+ T cells in a subacute sclerosing panencephalitis patient (SSPE), controls with inflammatory diseases (ICON) and with non-inflammatory diseases (NICON) are shown. Horizontal lines depict median values.

    (TIF)

    Click here for additional data file. (83.3KB, tif)
    S1 Data

    (PDF)

    Click here for additional data file. (199KB, pdf)
    Attachment

    Submitted filename: Plos review.docx

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    Attachment

    Submitted filename: RtR-281020.docx

    Click here for additional data file. (34.2KB, docx)
    Attachment

    Submitted filename: RtR-151220.docx

    Click here for additional data file. (21.1KB, docx)

    Data Availability Statement

    All relevant data are within the manuscript.

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