Human endogenous retrovirus type W envelope expression in blood and brain cells provides new insights into multiple sclerosis disease

Abstract

Background: The envelope protein from multiple sclerosis (MS) associated retroviral element (MSRV), a member of the Human Endogenous Retroviral family 'W' (HERV-W), induces dysimmunity and inflammation.

Objective: The objective of this study was to confirm and specify the association between HERV-W/MSRV envelope (Env) expression and MS.

Methods: 103 MS, 199 healthy controls (HC) and controls with other neurological diseases (28), chronic infections (30) or autoimmunity (30) were analysed with an immunoassay detecting Env in serum. Env RNA or DNA copy numbers in peripheral blood mononuclear cells (PBMC) were determined by a quantitative polymerase chain reaction (PCR). Env was detected by immunohistology in the brains of patients with MS with three specific monoclonals.

Results: Env antigen was detected in a serum of 73% of patients with MS with similar prevalence in all clinical forms, and not in chronic infection, systemic lupus, most other neurological diseases and healthy donors (p<0.01). Cases with chronic inflammatory demyelinating polyneuropathy (5/8) and rare HC (4/103) were positive. RNA expression in PBMC and DNA copy numbers were significantly elevated in patients with MS versus HC (p<0.001). In patients with MS, DNA copy numbers were significantly increased in chronic progressive MS (secondary progressive MS vs relapsing-remitting MS (RRMS) p<0.001; primary progressive MS vs RRMS -<0.02). Env protein was evidenced in macrophages within MS brain lesions with particular concentrations around vascular elements.

Conclusion: The association between MS disease and the MSRV-type HERV-W element now appears quite strong, as evidenced ex-vivo from serum and PBMC with post-mortem confirmation in brain lesions. Chronic progressive MS, RRMS and clinically isolated syndrome show different ELISA (Enzyme-Linked Immunosorbent Assay) and/or PCR profiles suggestive of an increase with disease evolution, and amplicon sequencing confirms the association with particular HERV-W elements.

Conflict of interest statement

Conflict of interest statement: Hervé Perron, Raphaël Faucard and Alois Lang are employees of GeNeuro; Corinne Bernard was a PhD student at the Claude Bernard University, Lyon-France, and has been financially supported by GeNeuro; Marta Garcia-Montojo was on a post-Doctoral position within Geneuro-Innovation R&D laboratories. Ilias Stefas is founder and employee of APOH Technologies; Francisco Veas is founder of APOH Technologies and employee of Université du Languedoc Montpellier-France. Babs Fabriek is an employee of TNO, The Netherlands. Claire Gerdil is an employee of “Etablissement Français du Sang”. Other authors from the European collaborative network are employees or have a position in non-profit academic research organisations and/or University hospitals only, as indicated in the first page; all tubes, disposables, logistics fees and compensation for Institution expenses linked to blood sampling and data records were paid or refunded by Geneuro to the Institutions only. Thus, Raphaëlle Germi, Roberta Mancuso, Marina Saresella, Sébastien Marcel, Alain Creange, Rosella Cavaretta, Domenico Caputo, Giannina Arru, Stefano Sotgiu, Klemens Ruprecht, Peter Rieckmann, Jack Van Horssen, Paul Van Der Valk, Pablo Villoslada, Michel Chofflon, Joseph Boucraut, Jean Pelletier, and Hans-Peter Hartung have no conflict of interest.

Figures

Figure 1.

Human Endogenous Retroviral family ‘W’ (HERV-W) envelope (Env) detection. (A) HERV-W Env antigenemia in multiple sclerosis (MS) and clinically isolated syndrome (CIS) serum compared to normal population; (B) HERV-W Env antigenemia in serum of different MS clinical forms. Arrows with ‘p’ values represent the statistical significance of the observed difference between patients versus blood donors (Mann–Whitney rank sum test); NS=Not Significant (p>0.05). Y-axis: the value is expressed as the ratio of the measured ELISA (Enzyme-Linked Immunosorbent Assay) value to the cut-off value from the normal population of healthy blood donors (mean+2SD of healthy controls, HC); values >1 are positive. PPMS: primary progressive MS; RLU: relative light unit; RRMS: relapsing–remitting MS; SPMS: secondary Progressive MS.

Figure 2.

Human Endogenous Retroviral family ‘W’ (HERV-W) envelope (Env) (multiple sclerosis associated retroviral element (MSRV) type) RNA expression. (A) MSRV Env RNA expression in MS peripheral blood mononuclear cells (PBMC) compared to normal population; (B) MSRV Env RNA expression in MS PBMC of different MS clinical forms; (C) MSRV Env RNA expression in MS PBMC compared to normal population (results from internal replication study in bold). Arrows with ‘p’ values represent the statistical significance of the observed difference between patients versus healthy blood donors (Mann–Whitney rank sum test); NS=Not Significant (p>0.05). Y-axis: the results are presented as the ratio between the measured copy number and the threshold value determined from the mean value plus twice the standard deviation of copy numbers from all healthy controls (HC) (mean+2SD of HC). Results with higher copy number than the threshold of the normal individuals have values above ‘1’. PPMS: primary progressive MS; RRMS: relapsing–remitting MS; SPMS: secondary progressive MS.

Figure 3.

Human Endogenous Retroviral family ‘W’ (HERV-W) envelope (Env) (multiple sclerosis associated retroviral element (MSRV) type) DNA copy number variation. (A) MSRV Env DNA copy number in MS peripheral blood mononuclear cells (PBMC) compared to normal population; (B) MSRV Env DNA copy number in MS PBMC of different MS clinical forms; (C) MSRV Env DNA copy number in MS PBMC compared to normal population (results from internal replication study in bold). Arrows with ‘p’ values represent the statistical significance of the observed difference between patients versus blood donors (Mann–Whitney rank sum test); NS=Not Significant (p>0.05).Y axis: the results are presented as the ratio between the measured copy number and the threshold value determined from the mean value plus twice the standard deviation of copy numbers from all healthy controls (HC) (mean+2SD of HC). Results with higher copy number than the threshold of the normal individuals have values above ‘1’. PPMS: primary progressive MS; RRMS: relapsing–remitting MS; SPMS: secondary progressive MS.

Figure 4.

Alignment tree of multiple sclerosis (MS) RNA and DNA clones with reference multiple sclerosis associated retroviral element (MSRV), Human Endogenous Retroviral family ‘W’ (HERV-W) (ERVWE1 and ERVWE2) sequences and distant HERV-K envelope (Env) sequence. ClustalW (v1.83) multiple alignment parameters: open gap penalty=10.0; extend gap penalty=5.0; delay divergent=40%. Transitions: weighted. The calculated distance between branched clones is indicated by the horizontal length separating them (reference length unit represented at the bottom), or by the numbers on the lines (when not proportional). The total distance between two clones is the addition of these measures.

Figure 5.

Alignment tree of healthy controls (HC) RNA and DNA clones with reference multiple sclerosis associated retroviral element (MSRV), Human Endogenous Retroviral family ‘W’ (HERV-W) (ERVWE1 and ERVWE2) sequences and distant HERV-K envelope (Env) sequence. ClustalW (v1.83) multiple alignment parameters: open gap penalty=10.0; extend gap penalty=5.0; delay divergent=40%. Transitions: weighted. The calculated distance between branched clones is indicated by the horizontal length separating them (reference length unit represented at the bottom), or by the numbers on the lines (when not proportional). The total distance between two clones is the addition of these measures.

Figure 6.

Multiple sclerosis (MS) brain immunohistology evidencing Human Endogenous Retroviral family ‘W’ (HERV-W)/MS associated retroviral element (MSRV) envelope (Env) protein in MS brain lesions. (A) Normal appearing white matter (magnification ×10); (B) early active lesion with numerous Env-positive cells stained in brown (magnification ×40); (C) edge of active lesion, showing less numerous Env-positive cells stained in brown (magnification ×40); (D) detail of (B), magnifying a vascular element within the lesion with perivascular Env-positive cells stained in brown. These cells present morphological features and perivascular dissemination of macrophages. Immunohistological staining is representative of anti-HERV-W Env specific staining in different sections. Brown cells represent positive cells, significantly labelled by anti-Env monoclonals (examples with GN-mAb_03 are presented in this figure).

Figure 7.

Confirmation of envelope (Env) expression in perivascular macrophages by double Immunostaining. Sections were selected from a lesion area including a vascular element with evidence of perivascular macrophage cuffs infiltrating the parenchyma, as in Figure 6D. (A) Perivascular macrophages labelled with anti-Env GN-mAb_05 mouse monoclonal (1:100) and revealed in green with fluorescent labelled secondary antibodies; (B) immunoactive macrophage labelled with anti-MHC class II LN3 antibody (1:50) and revealed in red with fluorescent labelled secondary antibodies (same slide); (C) merge of (A) and (B) immunolabelling evidencing the co-localization of both anti-Env and anti-MHC II antigens in macrophage cells from a perivascular cuff, in the same sample from an active multiple sclerosis lesion. In cells where the two antigens are co-expressed, orange, yellow or light-brown colour is seen when the two antigens have superimposed location with varying proportion, thus modulating the merged colours. Few cells also seem to express a single antigen, with single colour.

Figure 8.

Global scenario of an immunopathogenic and neuroinflammatory pathway involving Human Endogenous Retroviral family ‘W’ (HERV-W) envelope (Env) expression as a pivotal element between infectious virus co-factors and the neuroimmune pathogenic cascade.