SARS-CoV-2 awakens ancient retroviral genes and the expression of proinflammatory HERV-W envelope protein in COVID-19 patients

Summary Patients with COVID-19 may develop abnormal inflammatory response, followed in some cases by severe disease and long-lasting syndromes. We show here that in vitro exposure to SARS-CoV-2 activates the expression of the human endogenous retrovirus (HERV) HERV-W proinflammatory envelope protein (ENV) in peripheral blood mononuclear cells from a subset of healthy donors, in ACE2 receptor and infection-independent manner. Plasma and/or sera of 221 COVID-19 patients from different cohorts, infected with successive SARS-CoV-2 variants including the Omicron, had detectable HERV-W ENV, which correlated with ENV expression in T lymphocytes and peaked with the disease severity. HERV-W ENV was also found in postmortem tissues of lungs, heart, gastrointestinal tract, brain olfactory bulb, and nasal mucosa from COVID-19 patients. Altogether, these results demonstrate that SARS-CoV-2 could induce HERV-W envelope protein expression and suggest its involvement in the immunopathogenesis of certain COVID-19-associated syndromes and thereby its relevance in the development of personalized treatment of patients.


INTRODUCTION
The human ''coronavirus disease 2019''  caused by the ''severe acute respiratory syndrome coronavirus 2'' (SARS-CoV-2) continues to cause high morbidity and mortality, with uncertain but recurrent evolution with the emergence of viral variants. 1 Numerous long-lasting, post-infectious symptoms or syndromes are commonly observed among patients who had COVID-19, from benign to severe forms. 2 Moreover, beyond a dominant respiratory tract tropism, extra-pulmonary COVID-19 forms are more frequent and diverse than initially expected. 3 The dysregulation of innate and adaptive immunity has been recognized to play a critical role in the clinical outcome of COVID-19 patients. Severe evolution of COVID-19 is thought to be driven by hyperactivated innate immunity, 4 in addition to adaptive immune defects often resulting in lymphopenia and neutrophils/lymphocytes imbalance. 5 A deficient interferon response has also been shown to favor or result from SARS-CoV-2 infection. 6,7 Multifaceted immunological dysregulations are underlying hyper-immune reactions such as the ''cytokine storm'' syndrome, the multisystem inflammatory syndrome in children, and inflammation-driven thromboembolic events, as well as neurological and various other manifestations. 7-10 The present COVID-19 pandemic has thus raised many questions about the pathophysiological mechanisms that could explain numerous symptoms and syndromes associated with SARS-CoV-2 infection.
Certain infectious agents have been shown to activate pathological processes via receptor-coupled signaling pathways, by impairing the epigenetic control and/or by directly activating endogenous retroviral elements (human endogenous retroviruses [HERVs]) present in the human genome. 11 HERVs represent about 8% of human chromosomal sequences and comprise about 22 families independently acquired Figure 1. Expression of HERV-W ENV or HERV-K ENV RNA and HERV-W ENV protein in PBMC from healthy blood donors (HBDs) exposed to SARS-CoV-2 in vitro (A) The level of HERV-W ENV and HERV-K ENV mRNA in PBMC cultures from HBD, exposed to SARS-CoV-2 (MOI:0.1) or mock-treated (culture medium), was analyzed by RT-qPCR. The graph presents the mean results from triplicate (7 out 11 donors) or single cultures (4 out 11 donors) at 2h post-exposure. For each healthy blood donor, a color was assigned in order to be able to compare the induction of HERV-W ENV and HERV-K ENV according to the cultures of PBMC from the ll OPEN ACCESS iScience 26, 106604, May 19, 2023 3 iScience Article We next analyzed whether HERV RNA expression was followed by HERV protein production. PBMC cultures of independent HBD, inoculated with SARS-CoV-2 or mock-inoculated, were analyzed for the presence of envelope proteins of HERV-W, HERV-K, or SARS-CoV-2 N by immunofluorescence (IF) ( Figures 1B  and 1C). HERV-W ENV expression was donor dependent as observed in 3 out of 8 HBDs PBMC cultures inoculated with SARS-CoV-2. It was abundantly detected in donor #7, moderately in #12, and in isolated cells of #9, whereas it was not detected in others like donor #11 ( Figures 1B and 1C). Nevertheless, in HERV-W ENV-positive cells of ''responders'', a marked cellular expression was seen at high magnification ( Figure 1C). Conversely, neither HERV-K ENV (Figure 1Bi) nor SARS-CoV-2 antigens ( Figures 1B and 1C) were detected in any of the analyzed conditions. Finally, neither HERV-W ENV nor HERV-K ENV protein was detected in control cultures without exposure to SARS-CoV-2 ( Figures 1B and 1C). In SARS-CoV-2-infected Vero E6 cells, used as a positive control, a clear cellular expression of SARS-CoV-2 N and spike proteins ( Figure S1A), along with an increased SARS-CoV-2 N mRNA load, was confirmed ( Figure S1B). The specificity of the anti-HERV-W and -K ENV antibodies had been established in previous studies, 15,35 and that of secondary antibodies was also verified on PBMC from studied HBD ( Figure S1C).
To compare HERV RNA kinetics in PBMC exposed to SARS-CoV-2 from different individuals, the PBMCs of 4 representative HBDs were analyzed ( Figure S1D). Results showed that the levels of both HERV-W ENV and HERV-K ENV RNA decreased at 19h and/or 24h post-inoculation after an earlier peak in cells from ''HERVactivating'' donors. In parallel, in the same cell cultures, SARS-CoV-2 N RNA kinetics showed abundant RNA load only after viral inoculation in all inoculated samples, with a significant decrease at 19h post-exposure, confirming the absence of viral replication in PBMC ( Figure S1E).

Exposure to SARS-CoV-2 triggers HERV-W envelope production in a T cell subset of healthy individuals
HERV-W ENV protein expression has been previously observed in CD3 + T cells of COVID-19 patients. 27 We therefore analyzed whether direct exposure to SARS-CoV-2 could induce HERV-W ENV in T lymphocytes from healthy donors as well. For this purpose, HERV-W ENV expression in PBMC cultures from HBD, with or without exposure to SARS-CoV-2, was analyzed using cytofluorometry analysis on permeabilized cells. As illustrated in Figure S2A, the gating strategy identified CD3 + T lymphocytes in non-infected (NI) cultures. CD3 + cells comprised CD3 high T cells, physiologically representing naive/non-activated cells, and CD3 + cells with increased size, normally representing activated T cells, further decreasing CD3 level at their surface and corresponding to the CD3 low subpopulation. 36,37 To discriminate CD14 + monocytes from larger CD3 low T cells, we eliminated CD14 + cells by specifically gating CD14 À cells. After exposure to SARS-CoV-2, CD3 + cells showed HERV-W ENV cell surface expression. Double labeling with anti-CD3 and anti-HERV-W ENV antibodies revealed that a significant proportion of CD3 low T cells were positive for HERV-W ENV both at 24h and 72h after exposure to SARS-CoV-2 ( Figures 1D and S2A, CD3 + top panels), compared to CD3 high T cells ( Figures 1D and S2A; CD3 + bottom panels), while mock-infected cultures were negative (Figures 1D and S1A, CD3 + top and bottom panels). Globally, minimal fluorescence associated with HERV-W ENV detection (compatible with technical background noise) was observed with CD3 + T cells from mock-inoculated PBMC, whereas a significant increase was characterized in SARS-CoV-2-exposed PBMC and mainly observed in CD3 low T cells ( Figure 1D).   Figure S1). The percentage of HERV-W ENV-positive cells within CD3 high and CD3 low T cell subpopulations was determined by cytofluorometry and presented with histograms (mean from 3 donors GSD). NI: mockinfected cells. Statistical analysis was performed as described in STAR Methods (*p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001). iScience Article Figure 2. Induction of HERV-W ENV and HERV-K ENV mRNA after exposure to SARS-CoV-2 spike trimer, followed by HERV-W ENV protein expression in PBMC from healthy blood donors (A) PBMCs from 4 HBDs were exposed to 0.5 mg/mL of active trimer spike recombinant protein in parallel to mock control (buffer), and HERV-W ENV (Ai) and HERV-K ENV (Aii) mRNA levels were assessed at 2, 15, and 24h post-inoculation (pi) by RT-qPCR. A recombinant trimeric spike protein corresponding to the spike expressed on initially circulating SARS-CoV-2 isolate 39 was added into the culture medium of PBMC from HBD ( Figure 2). PBMC RNA was collected at 2h, 15h, and 24h post-inoculation and analyzed by RT-qPCR for HERV-W and HERV-K envelope gene expression. Donor #31 showed increased RNA levels for both HERV-W and HERV-K ENV at 2h postinoculation; those from donor #30 peaked at 15h, while RNA levels from both donors #32 and #33 immediately (R2h) dropped below the baseline of non-exposed cells (Figure 2A), a pattern similar to the one observed in HBD PBMC ''non-responding'' to infectious SARS-CoV-2 ( Figure S1D). Interestingly, donor #30 was the only one tested positive for anti-SARS-CoV-2 serum antibodies, which did not prevent HERV transcriptional activation by this spike trimer but coincided with a delayed peak of HERV RNA.
We next analyzed whether the exposure to recombinant spike could induce interleukin 6 (IL-6) secretion from PBMC from HBDs. Initially, we tested a large range of concentrations and found that the induction of IL-6 was observed from 100 ng of spike protein in all tested donors ( Figure 2B). IL-6 kinetics analysis was performed on HBD PBMC tested for HERV expression using 500 ng of the spike protein. Results showed a significantly increased IL-6 production starting from 15h after exposure in PBMC culture supernatants of all donors, including HERV ''non-responding'' ones ( Figure 2C). IL-6 production was not linked with possible endotoxin contamination as the treatment with the endotoxin inhibitor polymyxin B did not diminish the spike-induced IL-6 secretion ( Figure 2D). Thus, data demonstrated that spike-induced HERV activation occurs very early after exposure, before the release of IL-6 and only in a subgroup of healthy PBMC donors, therefore independently from IL-6 production.
HERV-W ENV protein production was confirmed by IF analysis at 72h in cultured PBMC of two donors inoculated with spike trimers (500 ng/mL) and not in mock-control cultures ( Figure 2E). HERV-W ENV-positive cells were detected as previously seen with the infectious virus. Interestingly, the absence of SARS-CoV-2 entry receptor (angiotensin converting enzyme-2, ACE2) expression in PBMC ( Figure S2B) suggests an interaction of the spike protein with another receptor. Finally, results were not influenced by the cell viability, which did not vary significantly within the analyzed culture period (%5 days; Figure S2C).
HERV-W ENV protein is expressed at the surface of T lymphocytes from COVID-19 patients and correlates with the detection of soluble HERV-W ENV hexameric protein in plasma  (D) To exclude an effect of endotoxin possibly present in the recombinant protein buffer, the PBMCs of 3 HBDs were exposed either to 10 ng/mL of LPS or to 2 mg/mL of recombinant spike, in combination or not with 12.5 mg/mL of polymyxin B (PMB). IL-6 was then quantified in the culture supernatants by ELISA. The volume of buffer equivalent to 2 mg/mL of spike protein was used as a negative control.
(E) PBMCs from healthy blood donors were exposed during 24h with 0.5 mg/mL of recombinant spike trimer. Results obtained on 2 responding donors [(# 18 (Eii) and #21 (Eiii)] are presented. Untreated PBMCs from the responding donor #18 (Ei). (Ei-Eii) HERV-W ENV was detected in few cells of cultures exposed to the spike trimer (geen staining). Higher magnifications of HERV-W ENV-positive cells are presented in white squares. DAPI was used to stain nuclei (in blue). Bars c-e: 50 mm. Statistical analysis was performed as described in STAR Methods (*p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001). The proportion of HERV-W ENV-positive cells was analyzed in PBMCs from 57 patients with COVID-19 (patients #1 to #27 and #192 to #221) compared to 31 HBDs ( Figure 3A). As for previous analyses, the gating strategy selected CD14 À cells to specifically analyze CD3 + T lymphocytes and CD14 + to investigate monocytes ( Figure S2D). A highly significant difference of HERV-W ENV expression on CD3 + T cells was observed between COVID-19 patients and HBD controls. The percentage of HERV-W ENV-positive CD3 high T cells was low but significantly higher when compared to HBD (p < 0.0001). HERV-W ENV-positive CD3 low T cells of COVID-19 individuals were numerous and significantly elevated compared to HBD (p = 0.0003). Interestingly, while no difference between HBD and COVID-19 patients was observed in CD14 + monocytes stained for HERV-W ENV, a significant expression was also observed in CD19 + B cells from COVID-19 patients, compared to HBD (p < 0.0001) ( Figure 3A).
The same PBMCs from four series of patients recruited during pandemic waves with successive variants were next studied in the cohort of 51 COVID-19 patients (patients #1 to #27 and #192 to #221 excepted patients without variant identification cf. ''Lyon'' cohort clinical data; https://doi.org/10.17632/ 3v4hfxv4w8.1) split into subgroups corresponding to infections with Wuhan, Alpha, Delta, and Omicron variants of SARS-CoV-2 ( Figure 3B). In all cases, regardless of the SARS-CoV-2 variant, CD3 + /HERV-W ENV + T lymphocytes were significantly detected in COVID-19 patients, i.e., values above the threshold of the background signal noise varying with cell type, represented by the ''mean+2SD'' of values from HBD, homogeneously distributed and grouped below this threshold. However, despite a significant difference on CD3 high /HERV-W ENV + cells, a lower proportion had CD3 low /HERV-W ENV + lymphocytes among the patients infected with the Omicron variant. The Omicron series included patients hospitalized with less severe forms, whereas most patients with other variants were admitted into intensive care units. Interestingly, patients infected with Omicron and Delta variants presented the highest and most significant proportion of CD19 + /HERV-W ENV + B lymphocytes.
HERV-W ENV soluble protein, which presents a hexameric structure, 15 was further quantified by immunocapillary analysis in the plasma of 8 HBDs and 21 COVID-19 patients (patients #28 to #48 from ''Lyon'' cohort described in https://doi.org/10.17632/3v4hfxv4w8.1) presenting different levels of severity. The prevalence of HERV-W ENV soluble protein was significantly elevated in plasma from COVID-19 patients (11/21), compared to HBDs (0/8) ( Figure 3C). Conversely, HERV-K ENV antigen was not detected in any plasma sample ( Figure S3A). As shown in Figure S3C, the specificity of these detections was also validated with an isotype control antibody, which gave non-detectable signal in plasma samples from COVID-19 patients.
The levels of HERV-W ENV soluble hexameric protein in plasma of COVID-19 patients correlated with the percentage of HERV-W ENV-positive T-CD3 + cells, as determined by cytofluorometry in the same blood samples (patients #1 to #27 and 8 HBD) ( Figure 3D). The correlation observed between HERV-W ENV released in plasma and its expression on the membrane of both CD3 high and CD3 low T lymphocytes was statistically significant, while no correlation was found for CD14 + monocytes and CD19 + B cells. Thus, in addition to the previously reported correlation between HERV-W ENV expression quantified by RT-qPCR and the expression determined by cytofluorometry in CD3 + T lymphocytes from COVID-19  Figure S2D. Statistical analysis was performed as described in STAR Methods (*p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001). iScience Article patients, 27 these results provide another orthogonal validation of HERV-W ENV protein detection by two unrelated techniques in COVID-19 blood samples.

The prevalence of soluble HERV-W ENV in plasma from COVID-19 patients correlates with blood biomarkers and disease severity
Analyses of results from available blood analyses from hospitalized patients (''Lyon'' cohort, patients #28 to #48) revealed a significant difference between polymorphonuclear neutrophils counts among patients with positive HERV-W ENV detection in serum and those among HERV-W-negative COVID-19 patients ( Figure 4A). Interestingly, all neutrophil counts in HERV-W-positive patients (11/11) were above the physiological level. With an opposite distribution below the normal range, HERV-W-positive patients all had lymphopenia ( Figure 4A). No difference was observed for other white blood cells, as exemplified with basophils ( Figure 4A).
Furthermore, the ratio between neutrophils and lymphocytes counts (N/L), previously suggested to be a biomarker of COVID-19 severity, 5 was significantly increased in more symptomatic COVID-19 cases positive for HERV-W ENV. This is illustrated with serum samples obtained from hospitalized patients (see clinical data of patients #28 to #48 from ''Lyon'' cohort, https://doi.org/10.17632/3v4hfxv4w8.1) with more and longer evolution, when compared with the cohort of early-diagnosed outpatients (symptomatic patients #49 to #191 from ''Zaragoza'' cohort, p = 0.0013) ( Figure 4B). These results also highlight the difference in the disease clinical status of patients between the two cohorts, with early diagnosed versus cases later hospitalized.
Thus, to study the production of HERV-W ENV patients at early COVID-19 diagnosis, compared to patients with other diseases and healthy controls, the soluble HERV-W was quantified in sera from a cohort consisting of two successive sampling series of SARS-CoV-2 PCR-positive cases with heterogeneous clinical presentation (patients #49 to #191 from ''Zaragoza'' cohort, described in https://doi.org/10.17632/3v4hfxv4w8. 1). A significant difference was still found between these COVID-19 patients compared to HBD or to other diseases ( Figure 4C). Among these 143 early SARS-CoV-2 PCR-positive cases, 21% were positive for HERV-W ENV, unlike the 44 HBD, all negative for HERV-W. HERV-W ENV antigen was not detected in 43 sera from non-COVID-19 other diseases either.
Finally, patients were classified into mild, moderate, and severe groups using the clinical scale for COVID-19, recommended by the National Institute of Health of the United States (USA) guidelines (https://www. covid19treatmentguidelines.nih.gov/overview/clinical-spectrum/). The mean titers of HERV-W ENV in plasma (patients #28 to #48 from ''Lyon'' cohort) progressively increased with disease severity, and HERV-W ENV was detected in all cases with severe forms ( Figure 4D). The comparison between the ''severe'' group and healthy controls was statistically significant despite low numbers of analyzed patients in this group. The analysis of cytofluorometry data obtained from patients classified using the same clinical scale is presented in Figure 4E (patients #1 to #27 and #192 to #221 from ''Lyon'' cohort, described in ll OPEN ACCESS iScience Article https://doi.org/10.17632/3v4hfxv4w8.1). The same strongly significant HERV-W ENV expression is observed in CD3 high T cells from mild cases but also increased from mild to severe cases in CD3 low T cells. HERV-W ENV detection was significant in CD19 + B cells but decreased from mild to severe cases. Comparison of CD-19 + /HERV-W ENV + B cell percentage increased with series of patients from Wuhan to Delta and Omicron variants ( Figure 3B) and with decreasing severity of COVID-19, suggesting that HERV-W ENV + B cells may be depleted with increasing disease severity. Finally, despite several cases with positive CD14 + cells, no statistical difference with asymptomatic cases or healthy controls was observed. The global evolution of the percentage of patients with HERV-W ENV + PBMC sub-populations according to COVID-19 severity scale compared to controls is summarized in Figure 4E.

Expression of SARS-CoV-2 and HERV-W ENV in tissues from autopsies of acute COVID-19 patients
To better understand the extent of HERV-W ENV expression in SARS-CoV-2-infected patients, we next analyzed the expression of this protein in tissues from COVID-19 patients (Figures 5, 6, and S4). Postmortem tissue samples from lung, heart, gastrointestinal tract, nasal mucosa, and brain were obtained from patients deceased from severe acute forms of COVID-19 (clinical data are provided in https://doi.org/ 10.17632/hmz5sm67rb.1).
Following the absence of HERV-K ENV detection in plasma of COVID-19 patients ( Figure S3), despite positivity for HERV-W ENV ( Figure 4C), we performed immunohistochemistry (IHC) staining with an already validated anti-HERV-K ENV monoclonal antibody targeting a highly conserved epitope. 15,35 In accordance with results obtained with plasma, all tissue samples from all autopsied cases were negative for HERV-K ENV ( Figure S3B).
Unlike the absence of the detection of HERV-K ENV, the lung tissue was readily stained for HERV-W ENV as well as SARS-CoV-2 N, although in different cell types ( Figure 5). SARS-CoV-2 antigen was detected in lung epithelial cells but not in alveolar macrophages ( Figure 5A). Conversely, HERV-W ENV antigen was strongly expressed at the cell membrane of CD68 + macrophages but not in neighboring SARS-CoV-2-positive epithelial cells (Figures 5B-5A). Furthermore, neither HERV-W ENV nor SARS-CoV-2 N staining was found within similar sections of lung tissue from non-tumoral regions of non-COVID patients with lung cancer, confirming the absence of non-specific staining ( Figure 5A). In wider areas of COVID-19 lung tissue, HERV-W ENV expression was observed in scattered infiltrating lymphoid cells ( Figure 5B) and with a sub-epithelial strong staining of cellular aggregates ( Figure 5B), in parallel with the absence of staining with an isotype control in the same tissue samples ( Figure 5B). HERV-W ENV-positive cells are presented with higher magnification in Figure 5C, which shows massive infiltrates of lymphoid cells, some of them diffusing within alveola and endothelial cells with aggregated cells or clots within blood vessels. In addition, an isolated blood clot representing a circulating micro-thrombus with strong HERV-W ENV staining was observed ( Figure 5C).
In COVID-19 heart tissue samples ( Figures S4A-S4C), HERV-W ENV was mainly found in endothelial cells from numerous small blood vessels, within cardiac muscle ( Figure S4A) and in the pericardial fatty tissue ( Figure S4B). The endothelial nature of HERV-W ENV-positive cells was confirmed with CD31 staining in similar vessel structures from neighboring slides ( Figures S4A and S4B), and no detectable staining was  iScience Article seen with an isotype control ( Figure S4C), confirming the absence of non-specific labeling. Surprisingly, no SARS-CoV-2 antigen was detected in cardiac tissues from studied COVID-19 samples.
Tissues from the gastrointestinal tract presented areas of strong labeling with SARS-CoV-2 anti-nucleocapsid antigen, mostly in epithelial cells but also in the gastric antral mucosa, in and around intestine sub-mucosal glands as well as goblet ting lymphoid cells ( Figure S4E) and immune cells from mucosa-associated lymphoid tissue (MALT; Figure S4E). Here again, HERV-W ENV-expressing cells were different from those infected with SARS-CoV-2. However, a faint HERV-W ENV staining was often seen at the apical top of intestine epithelial cells ( Figure S4E).
To further address the expression of HERV-W ENV in the central nervous system (CNS) of COVID-19 patients, as previously observed in MS lesions, 19 we analyzed sections from tissue samples taken across the cribriform plate, comprising areas of the olfactory bulb and of the nasal mucosa ( Figure 6). This anatomical region was chosen since it is suggested to be a most probable route of coronavirus passage to the brainstem via olfactory nerve roots within nasal mucosa 40 and since frequently reported anosmia in COVID-19 patients indicates pathological involvement of the olfactory bulb parenchyma. 41,42 Sections from samples with the upper anatomical brain region, i.e., the frontal lobe, were also analyzed.
Immunohistology analysis revealed both SARS-CoV-2 and HERV-W ENV protein expression in nasal mucosa areas. In sections at the CNS-nasal tissue interface ( Figure 6B), a strong SARS-CoV-2 N staining was observed, indicating viral replication in nasal mucosa but not in neighboring CNS areas of the olfactory bulb. SARS-CoV-2 antigen was detected neither in various areas of CNS sections within the olfactory bulb nor in the frontal brain parenchyma ( Figure 6B). A strong HERV-W ENV staining was seen in nasal mucosa, involving infiltrated lymphoid cells and, as already seen in pulmonary and cardiac tissues, the endothelium of blood vessels ( Figure 6C). Conversely to SARS-CoV-2 antigen, HERV-ENV was detected both in the olfactory bulb and in the brain parenchyma within cells presenting the morphology of microglia ( Figure 6E). Cells with the same morphology were stained by a microglial maker, Iba-1 ( Figure 6E). Higher magnifications of HERV-W ENV-positive microglial cells within brain parenchyma are boxed and presented in Figure 6E, and the specificity of this staining was confirmed with isotype controls ( Figure 6D). To further confirm the expression of HERV-W ENV in microglia within COVID-19 brain parenchyma, a double immunostaining was performed with HERV-W ENV and Iba-1-specific antibodies. Microglia-like cells positive for HERV-W ENV were co-stained with the antibody against Iba-1, thereby confirmed to be microglial cells ( Figure 6F, boxed in yellow with higher magnification on the right side). On the same pictures, endothelial cells from an HERV-W ENV-positive blood vessel wall were consistently negative for Iba-1 ( Figure 6F). In parallel, sections from nasal mucosa showed HERV-W ENV staining of infiltrated lymphoid cells, also negative for Iba-1 ( Figure 6F). (B) SARS-CoV-2 virus was searched for in brain parenchyma (Bi and Bii) and nasal mucosa (Bi) using anti-N SARS-CoV-2 immunodetection (brown-red staining). The black dotted line on Bi represents the boundary between brain parenchyma and nasal mucosa (position of the cribriform plate). SARS-CoV-2 nucleocapsid immunostaining was only observed in nasal mucosa and not in neighboring brain parenchyma (Bi). Bars = 100 mm.
(C) HERV-W ENV immunodetection using the murine IgG1 antibody GN_mAb_Env01 (brown-red staining) revealed that HERV-W ENV IHC staining was detected in nasal epithelium (Ci), nasal mucosa (Cii), and brain parenchyma (Ciii). Bars = 100 mm. Immunohistochemical results for HERV-W ENV and SARS-CoV-2 N protein staining from studied tissues of COVID-19 autopsies are summarized in Figure 7. Altogether, these results revealed that HERV-W ENV is expressed in postmortem tissues of lungs, gut, heart, brain parenchyma, and nasal mucosa from acute COVID-19 patients in cell types relevant for COVID-19-associated pathogenesis within affected organs.

DISCUSSION
Despite rapid advances in basic and translational science, COVID-19 continues to pose an important global health threat. This new disease revealed heterogeneous clinical profiles in the evolution of acute and postacute COVID-19, with symptoms not directly related to the viral infection. In the present study we have analyzed the induction of HERV expression during COVID-19 disease. Our initial in vitro results showed that SARS-CoV-2 can trigger both HERV-W and HERV-K ENV RNA transcription after a single exposure to SARS-CoV-2 virus, but only HERV-W ENV protein expression in short-term PBMC cultures from about 30% of healthy donors. These divergent outcomes suggest heterogeneity in the healthy population for SARS-CoV-2-induced HERV activation.
Recombinant wild-type SARS-CoV-2 spike protein trimer induced the production of IL-6 as previously reported 43 in PBMC from all donors either responding or not with HERV activation, at time points posterior to the activation of HERVs. Thus, a cytokine expression such as IL-6 secretion is not likely to be responsible for the induction of HERV-W ENV expression. Cytofluorometry analysis confirmed that HERV-W ENV protein in vitro early expression was predominantly and strongly induced in CD3 low T lymphocytes within the CD3 + T cell population. Consistently, T lymphocytes that underwent a very recent contact with antigenic components of SARS-CoV-2 may dynamically become activated CD3 + T cells as previously described. 37 Alternatively, in COVID-19 patients, our observations may corroborate previous reports describing superantigen motifs of SARS-CoV-2 spike protein, 44 which may involve cellular mechanisms associated with lymphopenia and hyperinflammation as already characterized for other emerging viruses (e.g., Ebola or Lassa). 45,46 However, because HERV-W ENV has also been shown to display superantigen-like effect, 22 the origin of this short-term effect on T lymphocytes may be questioned. Results from a recent study potentially provide an answer, since showing correlated expression between HERV-W ENV and markers of exhaustion in T lymphocytes from severe COVID-19 cases. 27 Altogether, results showed (i) lymphopenia in all HERV-W ENV-positive COVID-19 hospitalized cases from the same cohort along with (ii) a significant difference for the lymphocyte/neutrophils ratio between cohorts of early-diagnosed patients, asymptomatic or with mild disease, versus hospitalized patients in later stages of COVID-19. These observations suggest a negative impact of this endogenous protein on the fate of expressing lymphocytes, as previously suggested by the co-labeling of HERV-W ENV with exhaustion markers on T cells from COVID-19 patients. 27 HERV activation occurs without signs of infection of lymphocytes by SARS-CoV-2. Consistently, a recombinant trimer of its wild-type spike protein without stabilizing mutations 39 appeared sufficient to reproduce similar HERV-W and -K ENV RNA stimulation as well as HERV-W ENV protein production in lymphoid cells, albeit in a subset of donors like with the infectious virus. This type of HERV-W activation mediated by an interaction between a triggering virus and a specific receptor on certain cells has already been described, e.g., CD46 receptor with HHV-6A. 38 As SARS-CoV-2 induces HERV-W ENV expression in human lymphoid cells that do not express ACE2, yet undetermined receptor(s) are expected to mediate HERV activation by SARS-CoV-2, which should now be further studied with recent data on alternative receptors. 47 In addition to the SARS-CoV-2-induced activation of HERV-W expression in cultured lymphocytes, this study has demonstrated their activation in COVID-19 patients. In hospitalized individuals with different severity status, HERV-W ENV protein was confirmed to be expressed at the membrane of significant proportion of both CD3 low and CD3 high T cells. A significant expression was also found in B lymphocytes, which corresponds to a cell type already shown to be permissive for HERV-W expression. 48,49 Interestingly, HERV-W ENV + /CD19 + B lymphocytes were most prevalent and represented the most significant percentage in PBMCs from patients infected with more recent SARS-CoV-2 variants, Delta and Omicron, which might be related to particular properties of this variants. This would suggest a more rapid detrimental effect on HERV-W ENV-expressing B versus T cells, thereby possibly depleting positive B lymphocytes in later and more severe disease evolution. Of note, HERV-W expression was not previously observed in T cells in any pathological conditions, before its first observation in COVID-19, 27 suggesting the specific capacity of SARS-CoV-2 spike to trigger its expression in T lymphocytes, possibly followed by a longer survival in the ll OPEN ACCESS iScience 26, 106604, May 19, 2023 iScience Article bloodstream than B lymphocytes or monocytes, which were seen as strongly expressing macrophages after in vitro induction in the present study. Furthermore, analyses of other blood parameters from studied patients showed that their neutrophil counts, reported to be most often increased in COVID-19 with worsening evolution, 50,51 also paralleled HERV-W ENV presence in the serum, with values above the upper normal limit in all hospitalized patients with ENV-positive plasma.
After the recent characterization of a soluble hexameric form of HERV-W ENV in MS brain lesions, 15 the present study has shown its presence in the form of a circulating protein in COVID-19 plasma or sera. In a series from patients in intensive care unit, a significant correlation was found between HERV-W ENV detection on CD3 low and CD3 high T lymphocytes and the soluble protein in plasma. Though moderate, this nonetheless statistically significant correlation was seen between blood cells and the corresponding plasma devoid of cells, which should indicate a relationship between HERV-W-expressing lymphocytes and the release of this soluble protein in blood. The presence of HERV-W ENV in plasma was confirmed to be significantly increased in hospitalized patients with severe COVID-19 but was not detected in healthy controls, whereas HERV-K ENV protein was never detected in plasma or serum. Since all severe COVID-19 cases were tested positive for HERV-W ENV protein in plasma, this points to a potential marker of disease severity as already shown with blood T lymphocytes. 27 We also found HERV-W ENV in plasma of about 20% of early COVID-19 cases who were included after positive SARS-CoV-2 PCR result, independently of disease severity. This percentage is similar to that of healthy donors who showed HERV-W ENV positivity in PBMC exposed to SARS-CoV-2 challenge in vitro. Altogether, these data suggest that a percentage of individuals with an underlying susceptibility to more symptomatic and/or severe evolution may be linked to the activation of HERV-W ENV expression. In PBMC cultures non-responding to SARS-CoV-2 exposure, we had observed HERV RNA levels below the levels of non-exposed controls, which may be explained by the activation of HERV-inhibitory pathways and effectors. Thus, an inter-individual variability in the potency of HERV-inhibitory mechanisms, possibly with an (epi)genetic origin, may provide an explanation for non-universal activation of HERV-W ENV upon SARS-CoV-2 challenge, similarly to individuals with COVID-19. Indeed, most SARS-CoV-2 RT-PCRpositive individuals do not develop major symptomatology after infection, including asymptomatic cases who may represent about 35% of PCR-positive cases. 52 Moreover, since patients from different geographic areas and time periods of the pandemic, infected by different variants of SARS-CoV-2 were analyzed, the global results of this study are not expected to be influenced by such variables.
The analysis of tissues obtained at autopsy from patients deceased from acute COVID-19 revealed that HERV-W ENV was strongly expressed in numerous tissues, including lymphoid infiltrates surrounding lung alveola, within nasal or intestinal mucosa, and in endothelial cells from blood vessels of all tissues including the CNS parenchyma. In addition, HERV-W ENV expression in the CNS was found within scattered cells that were confirmed to be microglia. Strong HERV-W ENV staining was also often detected in aggregated cells corresponding to thrombotic structures in blood vessels of lung samples. SARS-CoV-2 nucleocapsid, corroborating viral replication, was readily detected in epithelial cells within lungs or intestines, as well as nasal mucosa, but not in analyzed sections from brain parenchyma, nor in the cardiac tissues of the studied cases. It was not even detected in the CNS parenchyma from olfactory bulb sections neighboring nasal mucosa with noticeable ongoing SARS-CoV-2 infection. Finally, a very strong expression of SARS-CoV-2 was observed in intestinal epithelium coinciding with numerous HERV-W ENV-positive infiltrated lymphoid cells in the mucosa. HERV-W ENV expression was also found in intestine, in lymphoid tissue (MALT) next to SARS-CoV-2-positive areas. Beyond this detection in the gastrointestinal tract of acute COVID-19 cases, it should be relevant to mention that persisting SARS-CoV-2 infection in the digestive system has been reported. 53 Of note, the specificity of anti-HERV-W ENV antibody used in this study and the absence of significant cross-reactivity on postmortem healthy human tissues from all organs as well as the absence of any pharmacokinetic or toxic effect related to elevated doses in clinical trials with healthy volunteers have been documented and reported previously. 54,55 This was more specifically further studied for brain, blood vessels, and pancreas tissues with same results. 18,19,24,28,56,57 In addition, HERV-K ENV has not been detected in the human sera and tissues from healthy donors when using the same antibody as used in this study; nevertheless, the same antibody was previously shown to efficiently detect HERV-K ENV in the cerebrospinal fluid of patients with amyotrophic lateral sclerosis. 35 Globally, cells expressing HERV-W ENV did not correspond to cell phenotypes seen to be infected with SARS-CoV-2, consistently with our observations of HERV-W ENV protein expression in lymphocytes without infection by SARS- iScience Article tissue-infiltrated lymphoid cells within affected organs, similarly to what is observed in blood of COVID-19 patients. Therefore, results from IHC analyses indicate that HERV-W ENV expression is intimately associated with organs and cells involved in COVID-19-associated or superimposed pathology, e.g., in vasculitis or intravascular thrombotic processes. 8,57 Moreover, given the known HERV-W involvement in the microglia-driven pathogenesis of MS 18,19,24 or of certain psychoses associated with inflammatory biomarkers, 58,59 the presently observed HERV-W ENV expression in microglia strongly suggests a role in neurological symptoms and cognitive impairment mostly occurring or persisting during the post-acute COVID-19 period. 41,42,[60][61][62] In acute primary infection, the pathogenic effects of HERV-W ENV on immune cells need to be further considered to better understand COVID-19 immunopathogenesis. HERV-W ENV production may result in a hyperactivation of the innate immunity via HERV-W ENV-mediated Toll-like receptor 4 (TLR4) activation 63 and in a possible contribution to the frequently observed lymphopenia along with an adaptive immune defect. The induction of autoimmune manifestations [64][65][66][67] as previously shown to be provoked with HERV-W ENV, initially called multiple sclerosis associated retrovirus (MSRV) envelope protein, 21 as well as the capacity of HERVs to modulate innate immunity should also be considered. 68 Altogether, these data indicate that HERV-W ENV does not simply represent a biomarker of COVID-19 severity or evolution but is also likely to be a superimposed pathogenic player contributing to the disease severity and may help to explain the inter-individual variability in COVID-19 manifestations. In addition, it may play a role in the clinical evolution with possible long-term pathology as seen with the now-emerging post-COVID secondary pandemic 60,69 representing millions of patients suffering from various symptoms and long-term disabling pathology for which no rationalized understanding or therapeutic perspective can be proposed to date. In face of this challenging situation, data from the present study strongly suggest HERV-W ENV as a marker of severity and as a potential therapeutic target for personalized medical approaches in COVID-19-associated syndromes.

Limitations of the study
Further understanding the regulation of the expression of HERV-W and the signaling pathway used by SARS-CoV-2 to induce it will be important in the future work, to better apprehend the implicated mechanisms. In addition, the exploration of the effect of HERV-W ENV on the hyperactivation of innate immunity seen in COVID-19 patients and further in-depth studies of its role in the fate of T lymphocytes should help in explaining the role of HERV-W in the immunopathogenesis of certain COVID-19-associated syndromes.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

DECLARATION OF INTERESTS
HP, BC, JB, JP, and NQ receive compensation from GeNeuro-Innovation for their work. PK received consulting fees from GeNeuro SA. Other co-authors do not declare conflict of interest.

Lead contact
Further information and requests for reagents and resources should be directed to and will be fulfilled by the lead contact.

Materials availability
Requests for new materials generated in this paper are to be directed to and will be fulfilled (pending MTA and associated restrictions) by the lead contact.
Data and code availability d Clinical data of patients cohort have been deposited at Mendeley data website.
d This paper does not report original code.

Study design
The objective of this study was to analyze the implication of endogenous retroviruses (HERVs), and in particular the envelope protein of the HERV-W family (HERV-W ENV), in the pathophysiology of COVID-19. Certain exogenous viruses are known to be capable of awakening HERV sequences which may have synergistic pathogenic effect with the agent initially responsible for the infection. In order to analyze if that may be the case with SARS-CoV-2 infection, we opted for a two-step strategy: 1. Study in culture in vitro whether SARS-CoV-2 and more particularly the Spike protein is capable of inducing the expression of HERV-W ENV in PBMCs of healthy blood donors; 2. Analysis of samples from COVID-19 patients, including PBMCs, plasma, serum and post-mortem tissues of lungs, hear, gastrointestinal tract and nervous tissue. All experiments were reproduced at least three times when possible depending on the availability of the biological material studied. All graphical plots show the mean +standard deviation. All other imaging or flow cytometry data show a representative example of the indicated total number of experiments. For all the immunodetection, all the specificity controls were carried out (isotype control, positive control, negative control, evaluation of the background noise in the absence of primary antibody).

Ethical approval
All healthy blood donors signed a written Informed Consent Form, documented at the French Blood Center (EFS), allowing the use of their blood and blood components for medical research after anonymization.