Autoimmune encephalitides (AE) comprise a group of inflammatory brain diseases characterized by prominent neuropsychiatric symptoms and the detection of serum IgG antibodies specific for antigens expressed by neuronal cells [1]. Aside from tumors, viral infections are thought to be potential triggers of antibody-mediated AE. Indeed, herpesvirus infection and reactivation are associated with the development or exacerbations of several autoimmune diseases including those confined to the CNS such as MS [2]. Previous studies reported that AE can develop in up to one-third of patients with a history with of herpes simplex encephalitis (HSE) [3-5]. Here, we profiled serum antibody responses to ubiquitous human herpesviruses in a cohort of immunotherapy-naïve patients with AE without a history for HSE compared to healthy controls matched by age and gender (Table 1).

We used ELISA to assess IgG responses specific for HSV-1 viral lysates, human cytomegalovirus (HCMV) viral lysates, the immunogenic C-terminus of the latent EBV nuclear antigen-1 (EBNA1), EBV-derived capsid antigen (EBV-VCA), and the HSV-1 encoded gp C. The nonparametric Mann–Whitney U rank sum test was performed to compare antibody levels between cohorts.

The magnitude of antibody responses toward HSV-1 viral lysates was significantly increased in immunotherapy-naïve AE patients (n = 42) compared to healthy donors (n = 25) matched by age and gender (Fig. 1A). In contrast, IgG responses to HCMV-derived viral antigens, EBV capsid antigen and EBNA1 were unchanged in patients with AE (Fig. 1A). Enhanced immune responses to HSV-1 were specifically observed in patients with AE associated with antibodies targeting neuronal surface antigens including the leucine-rich glioma inactivated 1 (LGI1) protein, the contactin-associated protein-like 2, and the N-methyl-d-aspartate receptor (NMDAR) (Fig. 1). These patients also showed the highest seropositivity rate for HSV-1 compared to healthy blood donors (Fig. 1A).

Since viral lysates can be enriched for nonviral antigen derived from mammalian cells used to generate lysates which, we additionally tested IgG antibody responses to recombinant HSV-1 gp C in patients (n = 42) and controls (n = 25) using an independent ELISA system. IgG responses specific for HSV-1 gp C were significantly increased in patients with AE associated with antibodies targeting neuronal surface antigens (Fig. 1B) with higher seroprevalence rates for HSV-1 compared to healthy controls.

The finding that only patients with AE associated with neuronal surface antigens show higher HSV-1-specific immune responses might be a reflection of the sample size. However, only AE associated with antibodies targeting neuronal cell surface antigens, as opposed to intracellular antigens, have been described to develop following HSE [3-5]. These include develop AE associated with antibodies targeting the NMDA receptor [3], the gamma-aminobutyric acid b receptor [4] or associated with voltage-gated calcium channels [5]. Our data support an association of HSV-1 infection and the development of AE even in absence of a history for HSE. Immunotolerance mechanisms might more efficiently protect from the development of immune responses toward ubiquitously expressed intracellular proteins such as GAD-65 in patients with AE associated with intracellular antigen.

A recent study found evidence for intrathecal synthesis of HSV-1-specific IgG in some patients with NMDAR and LGI1 encephalitis, but did not report on systemic antiviral immune responses [6]. Although patients with AE show significantly higher IgG levels to HSV-1 as assessed by two independent assays in our study, there is clearly an overlap of reactivities between patients and controls. Such an overlap has also observed for elevated antiviral immune responses with a more established link to autoimmune diseases, such as EBV nuclear antigen-1 antibody levels in patients with MS compared to controls [7]. Higher levels of anti-HSV-1 IgG levels imply increased exposure of viral antigen to the immune system. After primary replication in epithelial cells, the human alphaherpesvirus HSV-1 enters peripheral neurons, travels to the cell body by retrograde axonal transport and releases viral DNA into the nucleus to establish latency [8]. Reactivation from neurons can infect epithelial cells in the mucosa or skin, resulting in a vesicular rash. None of the patients had clinical evidence for HSV-1 reactivation. Smaller scale studies, however, indicate that asymptomatic reactivation of herpesviruses in general and specifically of HSV-1 is well documented and might occur more frequently than symptomatic reactivations [9, 10]. The mechanisms responsible for elevated HSV-1-specific immune responses in AE remain to be clarified.

There is experimental evidence for a role of HSV-1 infection in triggering autoimmune corneal injury through molecular mimicry following herpetic stromal keratitis [11]. The aforementioned study identified an epitope within a capsid portal protein of HSV-1 which is cross-recognized by T cells specific for a corneal antigen [11]. HSV-1 infection could contribute to the development of antibody-mediated AE by exposure of high amounts of normally sequestered antigens through lysis of infected neuronal cells, molecular mimicry with the synthesis of viral proteins that resemble cellular molecules, or

aberrant expression of histocompatibility molecules, thereby promoting the presentation of neuronal autoantigen [2].

The size of cohorts and the number of patients diagnosed with a specific antibody-defined entity, due to the rarity of AE, is a limitation of our study. This initial assessment, however, provides incentive to conduct larger studies to further elucidate the potential role of HSV-1 infection and HSV-1 elicited immune responses in AE (Supporting information 1).

Ethics approval statement

All samples were taken with written informed consent and Hospital Ethics Committee approval (registration nos. 2010-262-f-S, 2011-665-f-S, 2013-350-f-S, 2014-068-f-S and 2016-053-f-S), in accordance with the Declaration of Helsinki in its contemporary form.

Patient consent statement

All patients provided written informed consent.

Author contribution

J.D.L. contributed to the conception and design of the study. O.C., C.S., C.C.G., N.M., J.K., S.K., H.W., and J.D.L. contributed to the acquisition and analysis of data. O.C., H.W., and J.D.L. contributed to the drafting of the text and preparing the figures. All authors critically revised the manuscript for its intellectual content.

Conflict of interest

O.C., C.S., C.C.G., and J.K. have no conflict of interest. N. M. has received honoraria for lecturing and travel expenses for attending meetings from Biogen Idec, GlaxoSmith Kline, Teva, Novartis Pharma, Bayer Healthcare, Genzyme, Alexion Pharmaceuticals, Fresenius Medical Care, Diamed, UCB Pharma, and BIAL, have received royalties for consulting from UCB Pharma, and Alexion Pharmaceuticals and has received financial research support from Euroimmun, Fresenius Medical Care, Diamed, Alexion Pharmaceuticals, and Novartis Pharma. S.K. receives research funding from Biogen and has received speaker honoraria from Esai. H.W. received speaker fees, travel support, and/or served on advisory boards by Alexion, Amicus Therapeuticus, Biogen, Biologix, Bristol Myers Squibb, Cognomed, EMD Serono, F. Hoffmann-La Roche Ltd., Gemeinnützige Hertie-Stiftung, Genzyme, Idorsia, Immunic, Janssen., Medison Merck, Novartis, Roche Pharma AG, Sanofi, the Swiss Multiple Sclerosis Society TEVA, UCB, and WebMD Global. His research is supported by the German Ministry for Education and Research (BMBF), Deutsche Forschungsgesellschaft (DFG), Deutsche Myasthenie Gesellschaft e.V., Alexion, Amicus Therapeutics Inc., Argenx, Biogen, CSL Behring, F. Hoffmann - La Roche, Genzyme, Merck KgaA, Novartis Pharma, Roche Pharma, and UCB Biopharma. J.D.L. received speaker fees, research support, travel support, and/or served on advisory boards by Abbvie, Argenx, Alexion, Biogen, Merck, Novartis, Roche, Sanofi.