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January 21, 2020 by Faith Dickerson et al | Medscape

Schizophrenia Is Associated With an Aberrant Immune Response to Epstein–Barr Virus

Faith Dickerson; Lorraine Jones-Brando; Glen Ford; Giulio Genovese; Cassie Stallings; Andrea Origoni; Colm O’Dushlaine; Emily Katsafanas; Kevin Sweeney; Sunil Khushalani; Robert Yolken. Schizophr Bull. 2019;45(5):1112-1119.

Abstract

Background: Epstein–Barr virus (EBV) is a highly prevalent human herpesvirus capable of infecting the central nervous system and establishing persistent infection.

Methods: We employed solid phase immunoassay techniques to measure immunoglobulin G (IgG) class antibodies to EBV virions and defined proteins in 432 individuals with schizophrenia and 311 individuals without a history of a psychiatric disorder. Western blot testing was performed to document reactivity to specific EBV proteins. Polygenic risk for schizophrenia was calculated from genome sequencing arrays. Levels of antibodies between the groups were compared by multivariate analyses incorporating clinical, genetic, and demographic measures.

Results: Individuals with schizophrenia had marked elevations in the levels of antibodies to EBV virions as compared to the control population. Further analyses indicated increased levels of reactivity to EBV-viral capsid antibody (VCA) but not to EBV nuclear antigen-1 (EBNA-1) or to other human herpesviruses. Western blot analysis confirmed increased reactivity to VCA proteins in the group of individuals with schizophrenia and documented a lack of increased levels of antibodies to EBNA-1. Genetic analyses indicated an additive effect of increased levels of antibodies to EBV virions and genetic susceptibility to schizophrenia, with individuals with elevated levels of both type of markers having a greater than 8.5-fold odds of a schizophrenia diagnosis.

Conclusions: Individuals with schizophrenia have increased levels of antibodies to some but not all EBV proteins indicating an aberrant response to EBV infection. This aberrant response may contribute to the immunopathology of schizophrenia and related disorders.

Introduction

Schizophrenia is a serious neuropsychiatric disorder of uncertain etiology with a lifetime prevalence of approximately 1% in the United States. While schizophrenia has clear genetic underpinnings, currently known genes explain only a portion of disease risk.[1,2] In addition to genetic factors, environmental exposures have been identified as increasing risk for the disease. Environmental factors associated with increased risk of schizophrenia include winter-spring birth, urban birth, maternal preeclampsia, and perinatal and postnatal infections.[3–6] The potential role of infections in the etiopathogenesis of schizophrenia is supported by the associations between schizophrenia risk and genes which encode HLA and other factors which control the immune response to infectious agents.[1,7]

Epstein–Barr virus (EBV), also known as human herpesvirus 4, is a member of the Herpesviridae family. EBV is a lymphotropic virus that produces latent infections with immunomodulatory effects.[8,9] Primary infection with EBV is often associated with self-limited fever and adenopathy. Following acute infection, the virus persists in host B and T lymphocytes, monocytes, and epithelial cells; asymptomatic salivary viral shedding leads to onward transmission.[10] EBV can establish latency in many body sites including the brain where reactivation can be associated with encephalitis[11] and brain-specific immune responses.[12] The immune response to EBV infection can be monitored by the measurement of levels of antibody directed at antigens derived from virions as well as specific EBV proteins. Commonly measured anti-EBV antibodies include: anti-early antigen (EA) that arises early in the course of infection and decreases after 3–6 months; viral capsid antibody (VCA) that also arises early in infection but persists for extended periods of time; anti-EB nuclear antigen (EBV NA or EBNA) that does not arise until late in infection but does persist for extended periods of time[13,14] (supplementary figure 4). The response to additional EBV proteins can be measured by western blotting techniques[15] to further define the immune response to infection.

Supplemental Figure 4. Classic time course of anti-EBV antibodies. Following acute infection, IgG class anti-EBV antibodies arise first to early antigens (EA) followed closely by viral capsid antigens (VCA). Anti-EBV nuclear antigen (NA) arises slowly over a period of months until reaching a plateau in the chronic infection stage. Antibodies not measured in the current report are labeled in grey. Modified from Wong, G. 1999. http://virology-online.com/viruses/EBV.htm

Classic time course of anti-EBV antibodies. Following acute infection, IgG class anti-EBV antibodies arise first to early antigens (EA) followed closely by viral capsid antigens (VCA). Anti-EBV nuclear antigen (NA) arises slowly over a period of months until reaching a plateau in the chronic infection stage. Antibodies not measured in the current report are labeled in grey. Modified from Wong, G. 1999. http://virology-online.com/viruses/EBV.htm

EBV infections have been associated with a number of autoimmune disorders including multiple sclerosis, systemic lupus, autoimmune encephalitis, and fibromyalgia.[16,17] In many cases the immune response to EBV in individuals with these disorders is aberrant and differs from the immune response noted in otherwise healthy individuals in terms of the response to EA, VCA, and EBNA antigens.[18,19]

Many individuals with EBV-associated disorders have psychiatric symptoms during the course of their illness. For example, in the disorder systemic lupus, cognitive dysfunction is reported to occur in more than 80% of patients and psychosis in more than 20%.[20] Individuals with multiple sclerosis also have relatively high rates of cognitive impairment[21] and psychiatric symptoms[22] including psychosis.[23] However, there have been few studies of EBV exposure in individuals with schizophrenia. We thus measured the levels of antibodies to EBV virions and defined EBV proteins in a cohort of individuals with schizophrenia and compared these to levels in a group of control individuals without a history of psychiatric symptoms.

Methods

Study Population

The study population consisted of 743 individuals: 432 with a schizophrenia diagnosis and 311 without a history of psychiatric disorder. The details of the recruitment and evaluation of individuals in these populations have been previously described.[24] Individuals with schizophrenia met the following criteria: age between 18 and 65 inclusive, diagnosis of schizophrenia or schizoaffective disorder meeting criteria in the Diagnostic and Statistical Manual of Mental Disorder Fourth Edition (DSM-IV); currently receiving antipsychotic medications. Individuals in the nonpsychiatric control sample met the criteria: age between 18 and 60 inclusive, absence of a current or past psychiatric disorder as confirmed by screening with the Structured Clinical Interview for DSM-IV Axis I Disorders – Non-patient Edition (SCID-I/NP). Participants were assessed for the deficit syndrome, a putative schizophrenia subtype comprised of individuals with schizophrenia who have primary and enduring negative symptoms such as restricted affect and diminished social drive.[25] Additional details of the methods for recruitment and evaluation are presented in the supplementary materials and methods.

The studies were approved by the Institutional Review Boards of the Sheppard Pratt Health System and the Johns Hopkins Medical Institutions following established guidelines. All participants provided written informed consent after the study procedures were explained.

Antibody Measurements

Immunoglobulin G (IgG) antibodies to EBV antigens derived from intact virions were measured by means of solid phase enzyme immunoassay. Details of the procedure are provided in the supplementary materials and methods. IgG antibodies to EBV-VCA and EBV nuclear antigen-1 (EBNA-1) were measured by solid phase immunoassay employing commercially available assay kits (IBL America).[26] IgG antibodies to other human herpesviruses were measured by similar procedures.[26,27] Complete baseline sample sets consisting of N = 743 individual blood samples were available for all immunoassay tests.

Quantitative western blot assays were performed using methods presented in the supplementary materials and depicted in supplementary figure 5. Samples were available for western blot from 257 individuals, 150 of whom were individuals with schizophrenia and 107 controls. The individuals from whom samples were available for western blot did not differ significantly from the overall study population in any demographic or clinical variable.

Supplemental Figure 5. Using the “normalized intensity” option of the software displays the intensity curve of the entire test strip (shown above the test strip) in which any background interferences have been removed. The positions of the subset of 12 viral proteins specified by the software are shown above the curve. The software measures the intensity for each band and then determines the corresponding arbitrary intensity units as shown in the table of results below the strip. The intensities are then compared to those of the kit calibrator/control strip and a determination for each band as + Positive, (+) Borderline, or ○ Negative is made by the software. Anl: Positioning mark used by the software for proper alignment of each strip; Co: Control band for ensuring that the procedure was performed correctly, i.e., human IgX was applied followed by the alkaline phosphatase-labeled anti-human IgX and then the enzyme substrate; M, G, A: conjugate control that displays which specific human immunoglobulin, IgM, IgG, or IgA, has been tested on the strip. See Supplementary Methods for protocol details.

Using the “normalized intensity” option of the software displays the intensity curve of the entire test strip (shown above the test strip) in which any background interferences have been removed. The positions of the subset of 12 viral proteins specified by the software are shown above the curve. The software measures the intensity for each band and then determines the corresponding arbitrary intensity units as shown in the table of results below the strip. The intensities are then compared to those of the kit calibrator/control strip and a determination for each band as + Positive, (+) Borderline, or ○ Negative is made by the software. Anl: Positioning mark used by the software for proper alignment of each strip; Co: Control band for ensuring that the procedure was performed correctly, i.e., human IgX was applied followed by the alkaline phosphatase-labeled anti-human IgX and then the enzyme substrate; M, G, A: conjugate control that displays which specific human immunoglobulin, IgM, IgG, or IgA, has been tested on the strip. See Supplementary Methods for protocol details.

Genetic Analyses

DNA was extracted from whole blood and analyzed for genetic polymorphisms using the Illumina array. Polygenic risk score was calculated using a P-value cutoff of P < .05 for inclusion of individual polymorphisms used to calculate the polygenic risk score. Details of the methods used are provided in the supplementary material.

Statistical Analyses

The results of the assays were compared between the individuals with schizophrenia and controls employing parametric and nonparametric regression models.28 Details of the statistical methods are presented in the supplementary material. In light of the performance of 3 sets of immunoassay measurements (EBV virions, EBV VCA, EBNA-1), a critical value of P < .05/3 = .016 was employed to indicate statistical significance for assays using these measures. A value of .016 ≤ P ≤ .05 was considered to represent a trend.

esults

The demographic and clinical characteristics of the 743 individuals in the study, 432 individuals with schizophrenia and 311 nonpsychiatric controls, are presented in Table 1. Within the schizophrenia group, participants had the following diagnoses per DSM-IV criteria: schizophreniform disorder (n = 17, 4%); paranoid subtype (n = 51, 12%); undifferentiated subtype (n = 126, 29%); other schizophrenia subtype (n = 10, 2%); schizoaffective disorder (n = 228, 53%). A total of 124 (29%) of the schizophrenia participants met the criteria for the deficit syndrome. The following antipsychotic medications were the most commonly received at the time of the study assessment by the persons in the schizophrenia group: risperidone (n = 115, 27%); olanzapine (n = 76, 18%); clozapine (n = 68, 16%); ziprasidone (n = 26, 6%). The schizophrenia participants also received additional types of psychotropic medications including antidepressants (n = 169, 39%) and valproate (n = 87, 20%).

Initial analyses were performed to compare the quantitative levels of antibodies between the diagnostic groups. As shown in figure 1 there were significantly elevated levels of IgG antibodies to EBV virions in the schizophrenia group vs the control group (effect size = 0.356; 95% CI 0.168, 0.543; P < .0002). This association was confirmed by analysis employing a nonparametric interquartile regression analysis (effect size = 0.467; 95% CI 0.240, 0.693; P < .0001). There was also a trend towards increased levels of IgG antibodies to VCA in the schizophrenia group (effect size = 0.197; 95% CI 0.025, 0.370; P = .025). However, the levels of antibodies to EBNA-1 did not differ significantly between the groups. Histograms of the distribution of values for these EBV antibodies in individuals with schizophrenia and controls are shown in supplementary figures 1–3 and the prevalence of antibodies in the case and control population are displayed in Supplementary Table 1. We also measured antibodies to the other human herpesviruses HSV-1, HSV-2, CMV, VZV, and HHV-6. There were no significant differences between the schizophrenia and the control group in any of these antibody levels. There was a trend toward decreased levels of antibodies to CMV in individuals with schizophrenia (effect size= −0.192; 95% CI −0.357, 0.026; P = .023).

Figure 1. Effect sizes of immunoglobulin G (IgG) antibody reactivity to Epstein–Barr virus (EBV) and other human herpesviruses by solid phase immunoassays in individuals with schizophrenia as compared to controls calculated using logistic regression models. **P < .001; #P < .05.

Supplemental Figure 1. Histogram distribution of antibodies to EBV Virions in Individuals with Schizophrenia and Controls. Effect size = .356; 95% CI= .168 – .543; p<.0002 adjusted for age, gender, race, maternal education and cigarette smoking.

Supplemental Figure 2. Histogram distribution of antibodies to EBV Viral Capsid Antigen (VCA) in Individuals with Schizophrenia and Controls. Effect size =.197; 95% CI= .025 – .370; p=.025 adjusted for age, gender, race, maternal education, and cigarette smoking.

 

Supplemental Figure 3. Histogram distribution of antibodies to EBV Nuclear Antigen 1 (ENV NA1 , EBNA-1 ) in Individuals with Schizophrenia and Controls. The differences between the populations was not statistically significant.

We also examined the odds ratios associated with elevated levels of antibodies defined by values greater than pre-defined percentile levels of the control group adjusted for age, sex, race, cigarette smoking, and maternal education. As depicted in figure 2 we found increased odds of elevated antibodies to EBV virions in the schizophrenia group relative to cutoffs greater than the 50th (OR = 1.71; 95% CI 1.18, 2.48; P = .005), the 75th (OR = 2.22; 95% CI 1.50, 3.28; P < .001) and the 90th (OR = 2.31; 95% CI 1.39, 3.84; P = .001) percentile of the levels of antibodies in the controls. Further, we found a trend to increased odds of antibodies to VCA in the schizophrenia group vs the control group for cutoffs greater than the 50th (OR = 1.45; 95% CI 1.00, 2.08; P = .048), and the 75th (OR = 1.54;, 95% CI 1.04, 2.28; P = .032) percentile, as well as a significant effect at the 90th (OR= 2.03; 95% CI 1.23, 3.37; P = .007) percentile of the levels of the antibodies in the controls. There were no significant differences in the odds associated with increased levels of antibodies to EBNA-1.

Figure 2. Odds ratios of immunoglobulin G (IgG) anti-EBV antibody levels in schizophrenia as compared to controls by percentile values of IgG antibodies to Epstein–Barr virus (EBV) virions, EBV Viral Capsid Antigen (VCA) and EBV nuclear antigen (NA). The odds ratios were calculated by the use of logistic regression models **P < .001, *P < .012, #P < .05.

Odds ratios of immunoglobulin G (IgG) anti-EBV antibody levels in schizophrenia as compared to controls by percentile values of IgG antibodies to Epstein–Barr virus (EBV) virions, EBV Viral Capsid Antigen (VCA) and EBV nuclear antigen (NA). The odds ratios were calculated by the use of logistic regression models **P < .001, *P < .012, #P < .05.

We further measured the reactivity of samples towards EBV proteins employing a quantitative western blot assay system. As shown in figure 3, there was a significant increase related to schizophrenia diagnosis in the levels of antibodies to VCA p33 (effect size = 0.363; 95% CI 0.113, 0.614; P < .005), VCA p22 (effect size = 0.326; 95% CI 0.085, 0.568; P < .008), VCA p41 (effect size = 0.392; 95% CI 0.088, 0.696; P < .012).and viral protein p27 (effect size = 0.507; 95% CI 0.116, 0.898; P < .011). There was also a trend towards increased levels in the schizophrenia group associated with antibodies to VCA p65 (effect size = 0.381; 95% CI 0.033, 0.729; P = .032) as well as the early antigen EA-D p43 (effect size = 0.290; 95% CI 0.015, 0.565; P < .039). There were no schizophrenia-associated increases in antibodies to the other antigens including EBNA-1 p79 and the other early antigens.

Figure 3. Effect sizes of immunoglobulin G (IgG) antibody reactivity to individual Epstein–Barr virus (EBV) proteins as measured by quantitative western blot comparing reactivity in individuals with schizophrenia and controls. The effect sizes were calculated using logistic regression models employing *P < .012; #P < .05.

Effect sizes of immunoglobulin G (IgG) antibody reactivity to individual Epstein–Barr virus (EBV) proteins as measured by quantitative western blot comparing reactivity in individuals with schizophrenia and controls. The effect sizes were calculated using logistic regression models employing *P < .012; #P < .05.

We examined the bivariate relationship between antibodies to EBV virions and clinical and demographic variables within the group of individuals with schizophrenia. In terms of basic demographic variables, the levels of antibodies to EBV virions were positively associated with increased age (correlation coefficient = .023; 95% CI .020, .038; P < .0001), female sex (F = 9.93, P < .0.0017), lower levels of maternal education (correlation coefficient = −.070; 95% CI −.115, .024; P < .003), cigarette smoking (F = 8.31, P < .0042) and non-Caucasian race (F = 15.75, P < .0001) but not with participant educational level, age of onset, illness duration, or birth outside of the United States or Canada (all P > .1).

We employed regression models to examine the relationship between levels of antibodies to EBV virions and clinical variables as described in the supplementary methods employing age, sex, race, cigarette smoking and maternal education. We found that levels of antibodies to EBV virions were significantly associated with the presence of deficit syndrome (effect size = 0.363; 95% CI 0.111, 0.616; P < .005) and the administration of the medication valproate (effect size = 0.399; 95% CI 0.119, 0.680; P < .005). There were no significant associations with the PANSS symptom score, RBANS cognitive score, BMI, or other medications (all P > .05).

We also examined the interaction between EBV antibodies and the genetic risk for schizophrenia as measured by the polygenic risk score employing regression models adjusted for age, sex, race, cigarette smoking, and maternal education. The polygenic risk score was associated with an increased risk of schizophrenia in the study population (effect size = 0.498; 95% CI 0.245, 0.751; P < .001) but was not significantly associated with the level of anti-EBV virion, anti-EBV VCA, or anti-EBNA-1 antibodies (P > .1). Analyzed in terms of percentiles, a polygenic risk score of ≥50th percentile was associated with a schizophrenia diagnosis with an odds ratio of 2.18 (95% CI 1.27, 3.74; P < .005) and a polygenic risk score of ≥75th percentile was associated with a schizophrenia diagnosis with an odds ratio of 1.61 (95% CI 0.904, 2.88; P > .1).

There was an apparent additive effect of odds ratios associated with the polygenic risk score and EBV virion antibodies. The odds ratio for schizophrenia diagnosis associated with having both EBV virion antibody levels and the schizophrenia polygenic risk score ≥50th percentile was 3.41 (95% CI 1.47, 7.95; P < .004 adjusted for age, sex, race, maternal education, cigarette smoking, and genotyping array). The odds ratio for schizophrenia diagnosis associated with having both EBV virion antibody levels and the schizophrenia polygenic risk score greater than the 75th percentile was 8.86 (95% CI 2.59, 30.37; P < .001). There was no significant statistical interaction between the levels of EBV virion antibody and the polygenic risk score in relation to their association with schizophrenia (P > .1).

Follow-up samples were obtained and analyzed from 183 individuals: 131 individuals with schizophrenia and 52 controls. The median interval between the initial and follow-up was 182 days (interquartile range 112–578 d). The individual levels of EBV antibodies to virions VCA and EBNA-1 at the first and last sampling periods were highly correlated (effect size = 0.88, 0.76, and 0.84, respectively). None of the differences between the levels measured at the first and second sampling were statistically significant (P > .05 adjusted for age, sex, race, diagnosis, maternal education and time interval between first and second sampling).

Discussion

We found that individuals with schizophrenia have increased levels of IgG antibodies to EBV virions as compared to a control group. The differences were independent of demographic variables that are known to affect EBV exposure such as age, sex, race, and socioeconomic status as measured by maternal education. We also measured antibodies to specific viral proteins. These studies indicated that the individuals with schizophrenia had an aberrant immune response to EBV in that there were elevated levels of antibodies to EBV VCA but not to EBNA-1. It is likely that most of the participants in our study have undergone primary EBV infection at some time in the past as evidenced by the presence of low levels of antibody to EBV early antigen (EA). Generally, individuals with past infections to EBV have increased levels of IgG class antibodies to both VCA and EBNA-1 antigens.[13] The levels of EBV antibodies in the study populations are relatively stable over extended periods of time since the levels did not change significantly in the individuals for whom follow-up samples were available. This finding suggests that the aberrant immune response is unlikely to be related to timing of sampling but is relatively stable within an individual over time; additional longitudinal studies should be done to assess the stability of the markers over time. Antibodies to other herpesviruses including HSV-1, HSV-2, CMV, VZV, and HHV-6 were not significantly increased in the same population of individuals with schizophrenia. This finding indicates that the increased levels of antibodies to EBV virions found in the individuals are not reflective of a heightened immune response to human herpesvirus but is specific to EBV within this group of infectious agents.

As noted above, healthy individuals generally develop and maintain approximately equal levels of antibodies to both EBV VCA and EBNA proteins following the resolution of acute infection. The finding of increased levels of VCA but not EBNA-1 antibodies in individuals with schizophrenia is thus reflective of an aberrant immune response to EBV infection. Aberrant responses to EBV have been described in a range of EBV-associated disorders including autoimmune diseases such as multiple sclerosis[29] and systemic lupus as well as neoplastic conditions such as nasopharyngeal carcinoma.[30]

Increased levels of antibodies to EBV virion and VCA proteins were not associated with cognitive functioning or symptom scores but were associated with an increased prevalence of the deficit syndrome. EBV virion antibodies were also significantly increased in individuals who were receiving the mood-stabilizing medication valproate. This finding, which should be verified in larger samples, is of interest in light of the immunomodulatory effects of this medication which are likely related to its effects on histone deacetylation.[31] Valproate has also been used in the treatment of EBV-associated tumors based on its ability to modulate EBV gene expression.[31] Increased levels of EBV antibodies were also marginally associated with an increased prevalence of cigarette smoking.

The pattern of reactivity to EBV proteins in the study cohort was further analyzed by performing western blot analyses directed at IgG antibodies directed at 12 EBV proteins. These analyses confirmed significant reactivity to VCA proteins and other EBV proteins as well as the lack of reactivity to EBNA-1. Of interest is the significant increased reactivity to p27 in individuals with schizophrenia as compared to controls. The material provided by the manufacturer of the quantitative immunoblot system that we employed states that the viral strain used to produce the blot strips is P3HR1. Although we have not yet confirmed the identity of this 27kDa protein present on the blot strips, it is likely that this p27 represents the truncated form of the EBNA leader protein that Garibal et al[32] reported to be produced by P3HR1 EBV strain but not the predominant, wild-type strains, suggesting that individuals with schizophrenia might have been differentially exposed to an EBV variant with a mutation similar to this strain.

Primary EBV infection generally occurs in adolescence following viral transmission facilitated by oral contact. Infection in adolescents is often manifested by a syndrome of fever, pharyngitis, lymphadenopathy, and splenomegaly generally referred to as infectious mononucleosis. Most cases of infectious mononucleosis are self-limited and are followed by increases in antibodies to VCA and EBNA proteins. The timing of primary EBV infection is of interest in terms of the first manifestations of schizophrenia which also often occur in adolescence. It is of note in this regard that Khandaker et al found that previous exposure to EBV as measured by VCA antibodies was associated with subsequent psychotic experiences in adolescence.[33]

Our studies indicate that the source of EBV antigen in the test immunoassay is important in evaluating the association between EBV exposure and a schizophrenia diagnosis. It is thus of interest that DeWitte et al did not find an association between levels of antibodies to EBV and a schizophrenia diagnosis.[34] However, as they measured EBNA antibodies (DeWitte L, personal communication) as markers of EBV exposure, their findings are consistent with ours. The consistency of our results with other studies that have measured EBV antibodies in individuals with schizophrenia[35–37] is difficult to evaluate without additional information relating to the immune responses to defined EBV proteins.

The reasons for altered levels of antibodies to EBV proteins and specific EBV proteins are not known with certainty. Possible mechanisms for this association include ones relating to the virus and to the host response. In terms of the virus, a differential response to infection might be related to variation in the timing of primary EBV exposure, the genomic composition and pathogenicity of the infecting EBV, and possible re-exposure to differing strains of EBV.[38] Virological analysis of samples obtained from individuals in prospective studies will be required to address these possibilities. An altered immune response to EBV infection could also be based on host factors such as genetics or other environmental factors. In terms of genetic factors, we found an additive effect of increased EBV antibodies and increased genetic risk with a combined odds ratio of 8.86 for individuals with values ≥75th percentile of both measures. However, there was no statistically significant association interaction between the levels of EBV virion antibodies and the polygenic risk scores suggesting that the risks associated with genetic susceptibility and increased levels of EBV virion antibodies are independent. However larger sample sizes might be employed to detect low levels of interaction. Furthermore, additional genetic studies are required to define the genetic contribution to the aberrant response to EBV infection found in individuals with schizophrenia. The possibility use of combinations of immune and genetic markers for the diagnosis and management of schizophrenia is an important area for future study. Antigen-specific antibodies to additional herpesviruses and other viruses might also be examined with the goal of incorporating them into this approach.

In terms of other environmental factors, it is of note that we found an association between levels of antibodies to EBV virion proteins and cigarette smoking in individuals with schizophrenia. An interaction between EBV exposure and cigarette smoking has also been noted in other EBV-associated disorders such as multiple sclerosis, possibly based on the many immunomodulatory effects of cigarette smoking.[39] As for the above factors, the timing of the interaction between cigarette smoking and EBV exposure in terms of risk for schizophrenia should be addressed in prospective studies. The possible effects of lymphotropic viruses such as EBV and other environmental factors which can affect B-cell activity on the immunopathology of schizophrenia should be the subject of future investigations.

The neurobiological mechanisms by which increased levels of EBV virion antibodies might be associated with schizophrenia are not entirely known. One potential scenario is that individuals with aberrant EBV immune responses underwent previous replication of EBV within the central nervous system. This possibility is consistent with reports of psychosis in individuals undergoing EBV encephalitis and the finding of increased levels of EBV VCA antibodies in the CSF of some individuals with psychiatric disorders.[40–45] It is also possible that psychiatric symptoms are related to neuro-inflammatory effects on the brain including alterations in neurotransmitter receptor interactions, as has been found in a range of autoimmune disorders which affect the brain.[46]

In conclusion, this study indicates that many individuals with schizophrenia have an aberrant immune response to EBV. There are a number of therapeutic interventions available for the modulation of EBV infection including anti-viral medications and pharmacological compounds which can modulate the immune response.[47] An increased understanding of the role of EBV infection might thus lead to novel methods for the prevention and treatment of schizophrenia.

Sidebar

Study Population

All participants met the criteria for schizophrenia1 and the following additional criteria: absence of current substance abuse or dependence over the past one month, and of any history of intravenous substance abuse; absence of mental retardation; absence of clinically significant medical disorder that would affect cognitive performance such as epilepsy, history of encephalitis or head trauma, or any other reported neurological disorder of the central nervous system that had resulted in past or current treatment. The participants with schizophrenia were recruited from psychiatric programs affiliated with Sheppard Pratt Health System and at other outpatient treatment sites in central Maryland initially for a study on the association between antibodies to infectious agents and schizophrenia 2. The control individuals without a history of psychiatric disorder were recruited from posted announcements at local health care facilities and universities in the same geographic area as the sites where the individuals with schizophrenia were drawn.

At the initial visit, demographic and background information was obtained by interview. A review of systems was conducted including a past history of autoimmune disorders. Body Mass Index (BMI) was calculated based on height and measured weight. Participants were also asked about current cigarette smoking status. All participants were individually administered the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS)3. The schizophrenia individuals were interviewed and rated on the Positive and Negative Syndrome Scale (PANSS) 4 and their medication data were recorded from the clinical chart. Deficit syndrome, a putative schizophrenia subtype comprised of individuals with schizophrenia who have primary and enduring negative symptoms such as restricted affect and diminished social drive 5, was measured based on PANSS scores reflecting a distinctive combination of high negative symptom scores and an absence of dysphoria 6.

Follow-up blood samples were also available from 183 individuals, 131 of whom were individuals with schizophrenia and 52 controls. The follow-up samples from the schizophrenia individuals were obtained as part of their participation in subsequent clinical trials. The follow-up samples from the non-psychiatric controls were obtained as part of the planned follow-up evaluations. The median interval between the initial and follow-up was 182 days (interquartile range 112–578 days).

Measurement of IgG Antibodies to EBV Virions

Microtiter plates from Thermo Fisher (Immulon 2) were coated with 100 μL of Epstein Barr Virus Antigen Lysate (Meridian Catalog 7420); the EBV virus was diluted to 4 μg/mL in 0.05 M Potassium Carbonate buffer, pH 9.6. Plates were stored at 4°C for 16–20 hours and subsequently washed with high purity water. Plates were then blocked with 200 μL Non-Mammalian blocker (ImmunoO4, Catalog NMPCC diluted 1:10 in 5% sucrose) for two hours at room temperature and stored desiccated. On the day of testing Blood samples were diluted 1:300 in chaotrope buffer (0.25% non-fat dry milk; 1% BSA, 50 mM Tris pH 8.1, 0.5% Triton X 100, 0.5% Tween 20; 0.5 M sodium chloride; 0.2% casein). Diluted serum (100 μL) was added to each well and allowed to react at room temperature for four (4) hours. After four (4) washings with PBS/0.05%Tween 20, 100 μL of HRP goat anti-human IgG (KPL Catalog 074–1006) diluted 1:5000 in Super Shield (Immun04, Catalog HRPZR) was added to each well. The diluted conjugate was incubated at room temperature for two hours and then washed with PBS-Tween. ABTS substrate (Moss Catalog ABTS-1000) was added to each well and after 20 minutes the color reaction was read at 405 nm in a BioTek microplate reader.

Western Blot Analysis

Western blot analysis was performed employing kits purchased from Euroimmun, Luebeck, Germany following the manufacturer’s instructions. Total IgG reactivity was also quantitated by measuring reactivity to a control band included on each blot. This analytic system consists of kits containing 16 nitrocellulose blot strips onto which proteins from EBV viral particles have been transferred following separation by SDS polyacrylamide gel electrophoresis. Each kit contains a control/calibrator strip against which analysis software (EuroLineScan, Euroimmun) compares each test strip. Briefly, each blot strip was incubated with a 1:50 dilution of a human serum sample, washed and then incubated with alkaline-phosphatase-conjugated goat anti-human IgG. Following washing, strips were exposed to enzyme substrate solution (NBT/BCIP) and the reaction stopped after ten minutes. Strips were allowed to air dry, scanned on a flatbed scanner and the resulting images analyzed using EuroLineScan software. This software performs a digital evaluation of each test strip thus allowing quantitative evaluation of the western blot results. The software determined numerical antigen/antibody reactivity band intensities for each of twelve EBV antigens (Supplementary Table S1) on the strip. The band intensities for each EBV antigen were compared between cases and controls.

Genetic Analyses

Genotyping was performed using the Infinium Omni 2.5 (https://www.illumina.com/products/by-type/microarray-kits/infinium-omni25-8.html), the Illumina Global Screening Array https://www.illumina.com/products/by-type/microarray-kits/infinium-global-screening.html), or the Infinium PsychArray BeadChip (http://www.illumina.com/products/psycharray.html). Genotype array data was analyzed using plink 1.97 and principal components were computed with GCTA using a freely available protocol (see https://github.com/freeseek/kgp2anc) using methods which have been previously described.8,9. The protocol listed in the supplement (https://github.com/freeseek/kgp2anc) includes the following quality control measures:1) Removal of variants with minor allele frequency less than 0.5%; 2) Removal of variants with missingness above 2%; 3) Removal of variants with excess heterozygosity (p<1e-6); 4) Removal of autosomal variants associating with sex (p<1e-6). Further removal of variants in long-range linkage disequilibrium regions* was performed for principal component analysis as previously described10.

Genotypes were phased using referenced based phasing 11 and imputed genotype dosages were computed using Minimac 12 and the 1000 Genomes project phase 3 reference panel 13. The schizophrenia polygenic score was computed using available summary statistics 14 retaining markers with an association p-value p<0.05 and using plink 1.9 to compute the score from genotype dosages. Computed scores were further adjusted against the first 10 principal components to avoid potential stratification issues. Sufficient DNA samples were available from 377 individuals, 235 of whom were individuals with schizophrenia and 142 were control individuals without a psychiatric disorder.

Assay Standardization. To allow for comparison across assays, the values measured at the completion of the solid phase assays and western blot assays were converted to normalized scores whereby the control samples on each microplate had a mean value of 1.0 and a standard deviation of 1.0 as described in reference 24 in the main text.

Determinations of Antibody Prevalence. Standard samples corresponding to low levels of reactivity to the target virus were run in with each assay in at least 2 replicated per reaction microplate. An individual was considered to be exposed to the target antigen is the sample gave a reaction value which was at last 80% of the mean value of the standard samples

Additional Statistical Methods

The levels of antibodies to EBV virions and defined EBV proteins in individuals with schizophrenia and non-psychiatric controls were compared using linear regression models employing age, sex, race, cigarette smoking, and maternal education as covariates with maternal education serving as an indication of socio-economic status. The resulting coefficients were used as estimates of effect sizes. Significant relationships between antibodies and schizophrenia diagnosis determined by linear regression models were confirmed by the use of non-parametric bootstrapped quantile regression models examining the 50th quantile and employing the same covariates.15 Groups were also compared employing logistic regression models to calculate the odds of elevated antibody levels in the schizophrenia group compared to the levels in the control group. For these analyses, elevated levels of antibodies were defined as antibody levels that were ≥50th, ≥75th, and ≥90th percentile of the levels of the controls. Percentile cutoffs were employed to allow for the calculation of individual and combined adjusted odds ratios as well as to account for possible non-normal distribution of some of the data. We further examined the relationship between genetic susceptibility to schizophrenia and EBV antibodies to virions employing linear and logistic regression models in which EBV antibodies to virions and the schizophrenia polygenic risk score were examined as independent and as interactive variables. For these regressions, an additional variable was added relating to the type of sequencing array used for the determinations.

The association between the antibody levels at baseline and at follow-up was assessed by means of mixed effects models employing the covariates age, sex, race, maternal education, follow-up duration, and schizophrenia vs. control group status.

Within the schizophrenia group, basic demographic and clinical data such as age, sex, race, place of birth, cigarette smoking, and education were related to levels of antibodies using bivariate associations. The association between EBV antibody levels and additional demographic, clinical, and medication variables were also analyzed using regression models employing age, sex, race, cigarette smoking, and maternal education as covariates.

The results of the Western Blot analyses were analyzed using linear regression models with the covariates of age, sex, race, cigarette smoking, and maternal education as well as reactivity to the control band described above.

In light of the performance of three sets of immunoassay measurements (EBV Virions, EBV VCA, EBNA-1) a critical value of p<.05/3 = .016 was employed to indicate statistical significance for assays using these measures. A value of .016 ≤ p ≤ .05 was considered to represent a trend. All statistical analyses were performed with STATA version 15, College Station, Texas

Supplemental References

  1. First M, Gibbon, M, Spitzer, RL, Williams, JBW. User’s Guide for the SCID-I, Structured Clinical Interview for DSM IV Axis I Disorders. . New York, NY: Biometrics Research; 1996.
  2. Dickerson FB, Boronow JJ, Stallings C, Origoni AE, Ruslanova I, Yolken RH. Association of serum antibodies to herpes simplex virus 1 with cognitive deficits in individuals with schizophrenia. Archives of general psychiatry May 2003;60(5):466–472.
  3. Randolph C, Tierney MC, Mohr E, Chase TN. The Repeatable Battery for the Assessment of Neuropsychological Status (RBANS): preliminary clinical validity. J Clin Exp Neuropsychol Jun 1998;20(3):310–319.
  4. Kay SR, Fiszbein A, Opler LA. The positive and negative syndrome scale (PANSS) for schizophrenia. Schizophrenia bulletin 1987;13(2):261–276.
  5. Kirkpatrick B, Buchanan RW, McKenney PD, Alphs LD, Carpenter WT, Jr. The Schedule for the Deficit syndrome: an instrument for research in schizophrenia. Psychiatry research Nov 1989;30(2):119–123.
  6. Kirkpatrick B, Buchanan RW, Breier A, Carpenter WT, Jr. Case identification and stability of the deficit syndrome of schizophrenia. Psychiatry research Apr 1993;47(1):47–56.
  7. Chang CC, Chow CC, Tellier LC, Vattikuti S, Purcell SM, Lee JJ. Second-generation PLINK: rising to the challenge of larger and richer datasets. Gigascience 2015;4:7.
  8. Teo YY, Inouye M, Small KS, Gwilliam R, Deloukas P, Kwiatkowski DP, Clark TG. A genotype calling algorithm for the Illumina BeadArray platform. Bioinformatics (Oxford, England) Oct 15 2007;23(20):2741–2746.
  9. Korn JM, Kuruvilla FG, McCarroll SA, et al. Integrated genotype calling and association analysis of SNPs, common copy number polymorphisms and rare CNVs. Nature genetics Oct 2008;40(10):1253–1260.
  10. Price AL, Weale ME, Patterson N, et al. Long-range LD can confound genome scans in admixed populations. American journal of human genetics Jul 2008;83(1):132–135; author reply 135–139.
  11. Loh PR, Danecek P, Palamara PF, et al. Reference-based phasing using the Haplotype Reference Consortium panel. Nat Genet Nov 2016;48(11):1443–1448.
  12. Das S, Forer L, Schonherr S, et al. Next-generation genotype imputation service and methods. Nat Genet Oct 2016;48(10):1284–1287.
  13. Genomes Project C, Auton A, Brooks LD, et al. A global reference for human genetic variation. Nature Oct 1 2015;526(7571):68–74.
  14. Pardinas AF, Holmans P, Pocklington AJ, et al. Common schizophrenia alleles are enriched in mutation-intolerant genes and in regions under strong background selection. Nat Genet Mar 2018;50(3):381–389.
  15. Koenker R. Quantile Regression. New York: Cambridge University Press; 2005.

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