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chuka_lisЛетом 2021 у меня был пост о суперантигенных свойствах коронавируса, как продолжение серии постов об его патологических особенностях.
Известно, что не все вирусы обладают свойствами суперантигена, а редкие, и вроде бы, непосредственно для коронавирусных антигенов, ситуация не была прозрачной (что они напрямую связываются с толл-рецепторами и запускают неспецифическую активацию лимфоцитов).
Однако, такое свойство может быть и опосредованным.
Возможно, так и есть в случае ковида- что поясняет разное течение болезни у разных людей. Т.к. добавляется промежуточный "помощник", или фактор, (а может даже и не один, а несколько), который усложняет картину.
Международная группа ученых выявила, что коронавирус активирует ретровирусные гены, которые давным давно "вшиты" в человеческий геном и являются оприходованными на пользу (или хотя бы обезоруженными) следами былых ретровирусных инфекций, прокатывавшихся через популяцию.
Ретровирусы обладают особенностью встраиваться в геном, а потом их оттуда не выпилить, и сейчас эти следы древних вирусов в нашем геноме представляют собой транспозоны, или прыгающие гены, или повторяющиеся последовательности, функции которых чаще - регуляторные, в основном, и постепенно выясняются наукой. Эти участки не способны, даже при активации, привести к ретровирусной ре-инфекции, тк "ущербны". Однако, когда они активированы, они функциональны, и эта функциональность - не всегда на пользу. Известно, что иногда при активации эти гены могут запускать в иммунных клетках механизмы, приводящие к неспецифической воспалительной активации "изнутри", после чего клетка, например, выдает на свои мембраны много маркеров воспаления, меток смерти, или продуцирует множество медиаторов, запускающих воспалительные реакции вокруг нее, или активирует каскадно другие иммунны клетки, которые с ней обычно взаимодействуют, или начинает усиленно размножаться. Результат работы ретровирусного остатка (эндогенного) может быть такой же, как и суперантигенное воздействие- сильная, системная активация, попутно и реакция воспаления, с возможным разрушением некоторых тканей, запускание аутоиммунных реакций (тк будут активироваться любые В-лимфоциты, широким фронтом, в том числе и производящие антитела (и на свои антигены, которые в норме глубоко супрессированы регуляторной системой). Одна из "задач" ретровируса - простимулировать пролиферацию иммунных клеток, которые он заражает и использует. Ретровирусный остаток - заразить не может, а активировать одни и сделать при это уязвимыми другие клетки - может. При определенных условиях, конечно. Когда что-то "сбойнуло". Под действием внешнего фактора.
В этой работе авторы обнаружили, что экспозиция к коронавирусу моноядерных клеток периферической крови здоровых людей, активирует в них, примерно у трети, репликацию РНК с ретровирусных вставок HERV-K и HERV-W, и даже экспрессию суперкапсидного мембранного белка, который кодирует этот остаток HERV-W .
При этом, сами эти моноядерные клетки коронавирусом -не заражаются, а только меняют свою активность в его присутствии.
То же происходит и со зрелыми и "стареющими" Т лимфоцитами здоровых людей, при контакте с коронавирусом " в пробирке".
За стимулирующий ретровирусные гены эффект был ответственнен шипик коронавируса- потому что "обработка" им суспензий или моноядерных клеток периферической крови некоторых здоровых волонтеров, или Т лимфоцитов, показала результат, аналогичный эффекту воздействия целого коронавируса.
Далее- этот же ретровирусный белок, кодируемый HERV-W, обнаруживался в повышенных количествах в крови, плазме симптоматично больных ковидом. Тяжесть течения ковида в буольных коррелирует и с "растворимым" ретровирусным белком, и с количеством белка HERV-W на мембранах Т лимофицтов, экспрессирующих CD3+ маркер (зрелые).
Что интересно, когда этот маркер померяли у тех, кто не госпитализировался- то он тоже обнаружился у большинства заразившихся ковидом (но его не было в крови у здоровых доноров).
Так же, у тех, кто умер от острого тяжелого ковида, в тканях легких, сердца, мозга, обонятельных луковиц, слизистой носа и других органов, вовлеченных в патологию ковида, обнаруживался экспрессируемый HERV-W белок, в клетках, которые принимали участие в развитии патологии (макрофаги, эндотелиальные клетки селезенки, сердца, сосудов, глия, этс), но не были поражены коронавирусом (не экспрессировали антигены коронавируса, в отличие от соседних эпителиальных клеток, или пневмоцитов, этс).
Авторами не обнаружен коронавирус в тканях сердца или в мозгу, зато обнаружены активированные им клетки, продуцирующие ретровирусный белок (а для контролей с похожими патологиями (ткань возле опухоли), таких белков не обнаружилось, что подтверждает, что толчком была инфекция).
Не то чтобы это прямые, скорее косвенные доказательства, но вполне "логичные".
Авторы не исключают, что активация HERV-W может быть вовлечена в развитие симптоматики не только ковида, но и постковидного симптома.
Может быть, тк повышенные уровни этого белка обнаруживаются у больных рассеянным склерозом, например (кое-где симптоматика с постковидом пересекается) или с нейропсихиатрическими отклонениями. Имумнные клетки, которые сбили свои настройки, могут жить долго, и зачастую оказываются "влиятельными" по своей природе, что может доводить до патологии.
Конечно, они проверили только по вот этим двум ретровирусным остаткам, а ведь в организме среди нашего генома их содержится около 8%. И не все они изучены, как возможные факторы влияния. Эти 2 авторы выбрали потому, что по ним было уже кое- что известно, и оказалось что один вариант из выбранных ими - экспрессируется и может быть маркером и нести ответственность как минимум за часть эффектов (а за продукцию Ил6, например, нет). Вообще же связать одно с другим, особенно если явления отсроченные (фактор-время- эффект), непросто.
Patients with COVID-19 may develop abnormal inflammatory response and lymphopenia, followed in some cases by delayed-onset syndromes, often long-lasting after the initial SARS-CoV-2 infection. As viral infections may activate human endogenous retroviral elements (HERV), we studied the effect of SARS-CoV-2 on HERV-W and HERV-K envelope (ENV) expression, known to be involved in immunological and neurological pathogenesis of human diseases. Our results have showed that the exposure to SARS-CoV-2 virus activates early HERV-W and K transcription but only HERV-W ENV protein expression, in an infection- and ACE2-independent way within peripheral blood mononuclear cell cultures from one-third of healthy donors. Moreover, HERV-W ENV protein was significantly increased in serum and plasma of COVID-19 patients, correlating with its expression in CD3+ lymphocytes and with disease severity. Finally, HERV-W ENV was found expressed in post-mortem tissues of lungs, heart, brain olfactory bulb and nasal mucosa from acute COVID-19 patients in cell-types relevant for COVID-19-associated pathogenesis within affected organs, but different from those expressing of SARS-CoV-2 antigens. Altogether, the present study revealed that SARS-CoV-2 can induce HERV-W ENV expression in cells from individuals with symptomatic and severe COVID-19. Our data suggest that HERV-W ENV is likely to be involved in pathogenic features underlying symptoms of acute and post-acute COVID. It highlights the importance to further understand patients genetic susceptibility to HERV-W activation and the relevance of this pathogenic element as a prognostic marker and a therapeutic target in COVID-19 associated syndromes. 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 [20-22], in addition to adaptive immune defects resulting in lymphopenia and neutrophils/lymphocytes imbalance [23, 24]. A deficient interferon response has also been shown to favor or result from SARS-CoV-2 infection [25, 26]. Such multifaceted immunological dysregulations are thus underlying hyper-immune reactions such as the “cytokine storm” syndrome, the multisystem inflammatory syndrome in children (MIS-C), the dysregulation of coagulation, as well as neurological and various other manifestations [27-29]. The present COVID-19 pandemic has thus raised many questions about the pathophysiological mechanisms explaining the many symptoms or 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 (HERVs) present in the human genome [30, 31]. In particular, the resulting production of endogenous proteins of retroviral origin with pathogenic effects may generate clinical symptoms corresponding to the organ, tissue or cells in which they are expressed, according to the specific tropism of the triggering infectious agent [32-40]. HERVs represent about 8% of human chromosomal sequences and comprise about 22 families independently acquired during evolution from exogenous retroviruses via an infection of germ line cells [41, 42]. Abnormal expression may then become self-sustained, thus creating lifelong chronic expression from host‘ s genome copies in affected tissues [43], e.g., with cytokine-mediated feedback loops [44] or, possibly, mediated by their own envelope proteins [45]. Such a sustained expression has been shown to be involved in brain lesions with lifelong expansion in patients with multiple sclerosis (MS) [43, 46-48]. Different HERV envelope proteins were shown to exert major immunopathogenic [49-55] and/or neuropathogenic [46, 51-58] effects in vitro and in vivo, associated with pathognomonic features of human diseases. We therefore studied whether SARS-CoV-2 could activate HERV copies considered as ‘dormant enemies within’ [59]. We focused on HERV families already shown to be involved in the pathogenesis of human diseases, HERV-W and HERV-K [57], to evaluate their potential association with COVID-19 and associated syndromes. This question became critical after a recent study has revealed the significant expression of HERV-W envelope protein (ENV) in lymphoid cells from COVID-19 patients, correlating with disease outcome and markers of lymphocyte exhaustion or senescence [60].In the present study, we initially addressed the potential role of SARS-CoV-2 in directly triggering the activation of a pathogenic HERV protein expression as reported with other viruses in, e.g., MS and in type 1 diabetes [61-63]. We further analyzed its expression in white blood cells and its possible detection in plasma of patients with COVID-19 presenting various clinical forms at early and late time-points. We finally examined this HERV expression in affected tissues from COVID-19 post-mortem samples. Also, since a major concern beyond the initial COVID-19 infectious phase is foreseen to result from the occurrence of long lasting symptoms with more-or-less delayed onset and often involves neurological impairment [17, 64-66], we examined SARS-CoV-2 and HERV-W expression in brain parenchyma. For comparison with dominantly or frequently affected organs, we also examined these antigens expression in lung and cardiac tissues.Our results showed that (i) SARS-CoV-2 can activate the production of HERV-W ENV in cultured blood mononuclear cells from a sub-group of healthy donors, (ii) HERV-W ENV is expressed on T-lymphocytes from COVID-19 patients, (iii) HERV-W ENV antigen is detected in all tested plasma or sera samples from severe cases in intensive care unit, but only in about 20% of PCR positive cases after early diagnosis, (iv) the level of HERV-W ENV antigenemia increases with disease severity and (iv) HERV-W ENV expression is observed by immunohistochemistry in cell-types relevant for COVID-19 associated pathogenesis within affected organs and particularly in brain microglia. HERV-W ENV protein expression having been observed in CD3+ T-cells of COVID- 19 patients [60], we analyzed whether direct exposure to SARS-CoV-2 could induce HERV-W ENV in T lymphocytes from healthy donors. We analyzed HERV-W ENV expression in PBMC cultures from three healthy donors, with or without exposure to SARS-CoV-2, using cytofluorometry analysis on non-permeabilized cells. As illustrated in Figure 2, CD3+ T lymphocytes were identified with the gating strategy in non-infected (NI) cultures. CD3+ cells comprised CD3high T-cells, physiologically representing naïve/non-activated cells, and CD3+ cells with increased size, normally representing activated T-cells following, e.g., antigenic stimulation, further decreasing CD3 exposure at heir surface and corresponding to the CD3low subpopulation [71, 72]. Furthermore, in order to avoid confusion between CD14+ monocytes and larger CD3low T-cells, we have evaluated T-cell populations by eliminating CD14+ cells and gating specifically CD14- cells (Figure 2A-D). Indeed, when inoculated with SARSCoV- 2, CD3+ cells showed HERV-W ENV cell-surface expression. Minimal fluorescence associated with HERV-W ENV detection (compatible with technical noise) was observed in sham-inoculated CD3+ T-cells (Figure 2A), whereas a significant increase was characterized in SARS-CoV-2-associated cultures mainly observed in CD3low cells (Figure 2B). In the presence of SARS-CoV-2, CD3low cells may also reflect the predicted superantigenic properties of the Spike protein [73]. Double-labeling with anti-CD3 and anti-HERV-W ENV specific antibodies indicated that a significant proportion of CD3low T-cells was positive for HERV-W ENV at the cell-surface both at 24h and at 72h after exposure to the virus (Figure 2A and 2C, CD3+ top panels) compared to CD3high T-cells (Figure 2B and 2D, CD3+ bottom panels). Sham-infected cultures did not express notable levels of HERV-W envelope antigen in CD3+ T-lymphocytes (Figure 2A and 2C, CD3+ top and bottom panels). The percentage of HERV-W ENV positive cells in each condition is represented in 24h and 72h post-inoculation with SARS-CoV-2. Exposure to SARS-CoV-2 recombinant trimeric Spike protein triggers HERV-W ENV protein production in PBMC of some heathy individuals. Of interest, donor #30 was the only one tested positive for anti-SARS CoV-2 serum antibodies (data not shown), which did not prevent HERV transcriptional activation by this recombinant spike trimer but coincided with delayed peak of HERV RNA. IL-6 secretion was significantly increased after exposure to SARS-CoV-2 S antigen in PBMC from all tested donors, including those not responding in terms of HERV activation. Thus, HERV activation occurred independently from IL-6 production and, beyond RNA analysis, HERV-W ENV protein production was confirmed by immunofluorescence analysis at 72h in cultured PBMC of 2 out of four other donors inoculated with spike trimers (0.5 μg/mL) and not in mock-control cultures. HERV-W ENV positive cells were detected, as previously seen with the infectious virus. Moreover, a relative increase in HERV-W ENV RNA was quantified, despite a low proportion of activated cells within the cultures. Of interest, the absence of ACE2 expression in PBMC (Supplementary Figure S3 A) suggests an interaction of the spike protein with another receptor. Cell viability was measured and did not vary significantly within the culture period of experiments. 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 antigen in plasma. a highly significant difference was observed between COVID-19 patients and HBD controls on CD3+ T-cells (p=0.0005 for CD3low T-cells and p=0.0023 for CD3high T-cells) (Figure 4A and 4B). However, while the amount of CD3high T-cells positive for HERV-W ENV was low but significant when compared to HBD (p=0.0005) (Figure 4A), significant but much higher levels were seen in CD3high T-cells of COVID-19 individuals (p=0.0023 Vs HBD; Figure 4B). Interestingly, whereas the levels of CD14+ monocytes stained for HERV-W ENV remained equivalent between HBD and COVID-19 patients (Figure 4C), a significant difference was also observed in CD19+ B-cells between HBD and COVID-19 groups (p=0.031) (Figure 4D). HERV-W ENV soluble hexameric antigen was also detected in plasma of COVID-19 patients and its correlation with HERV-W ENV expression levels previously determined by cytofluorometry on each cell population was assessed (Figure 4 E-H). A moderate but significant correlation was observed between the HERV-W ENV antigen released in plasma and the one expressed on the membrane of both naïve CD3+ and activated CD3+ T-lymphocytes (p<0.02 and p<0.03, respectively)Finally, SARS-CoV-2 IgG serology for multiple antigens was also analyzed, but no significant correlation was observed between anti-SARS-CoV-2 IgG titers and HERVW ENV hexamer antigenemia in plasma samples. To study HERV-W ENV antigenemia in patients representing a wider range of clinical status at early COVID-19 diagnosis (compared to patients with other diseases and healthy controls-HC) its soluble antigen was quantified in sera from a third cohort consisting in two successive sampling series of SARS-CoV-2 PCR positive cases with heterogeneous clinical presentation (described in Supplementary Table S4 ). As shown in Figure 5H, a significant difference was found between all COVID-19 patients and HBD or other diseases (Mann Whitney U-test, respectively p=0.0010 and p=0.0011). 21% of COVID-19 sera were positive for HERV-W ENV among the 144 SARS-CoV-2 PCR positive cases, while none of the 44 PCR negative HBD were positive for HERV-W ENV (Chi2=8.796, p<0.001). Similarly, HERV-W ENV antigen was not detected in 43 sera from non-COVID-19 other diseases either (Chi2=8.604, p<0.001). Finally, the ratio between neutrophils and lymphocytes counts (N/L), previously suggested as a biomarker of COVID-19 severity [23], was significantly increased in COVID-19 cases with HERV-W ENV positive antigenemia.To better understand the consistency and magnitude of HERV-W ENV expression in SARS-CoV-2-infected patients, we next analyzed the expression of this antigen in tissues from COVID-19 patients. Post-Mortem lung, heart, nasal mucosa and brain tissue samples were obtained from patients deceased from severe acute forms of COVID-19. Lung tissue staining with monoclonal antibodies targeting SARS-CoV-2 Nucleocapsid (N-Protein) or HERV-W ENV.SARS-CoV-2 antigen was readily detected in lung epithelial cells but not in alveolar macrophages HERV-W ENV antigen was strongly expressed at the cell membrane of macrophages but not in neighboring SARS-CoV-2 positive pneumocytes. The specificity of both SARS-CoV-2 N and HERV-W ENV proteins was confirmed by the absence of staining within similar sections of lung tissue from non-COVID controls, i.e., normal tissue surrounding lung tumors Endothelial nature of HERV-W ENV positive cells was confirmed with CD31 staining in similar vessel structures from neighboring slides. SARS-CoV-2 antigen was not detected in cardiac tissue from studied samples. To further address the expression of HERV-W ENV in the central nervous system (CNS) of COVID-19 patients, as previously observed in multiple sclerosis lesions [46], we analyzed sections from tissue samples taken across the cribriform plate, comprising areas of the olfactory bulb and of the nasal mucosa (Figure 8A). This anatomical region was chosen since suggested to be a most probable route of coronavirus passage to the brainstem via olfactory nerve roots within nasal mucosa [75] and since frequently reported anosmia in COVID-19 patients indicates pathological involvement of the olfactory bulb parenchyma [76, 77]. Sections from samples with the upper anatomical brain region, i.e. the frontal lobe, were also made available. Immunohistology results for both SARS-CoV-2 and HERV-W ENV antigens in brain parenchyma or nasal mucosa areas are presented Figure 8. In sections at the CNSnasal tissue interface presented in Figure 8B, a strong SARS-CoV-2 N antigen staining indicating viral replication was seen in nasal mucosa but not in neighboring CNS areas of the olfactory bulb. SARS-CoV-2 antigen was not detected in various areas of CNS sections within olfactory bulb or frontal brain parenchyma, as illustrated in Figure 8C. 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 8D and 8E). Conversely to SARS-CoV-2 antigen, HERV-ENV was also detected in the olfactory bulb parenchyma in glial cells (Figure 8F), which revealed to have the morphology of microglia and were stained by microglial maker, Iba-1 (Figure 8G and 8H). Higher magnifications of HERV-W ENV positive microglial cells within brain parenchyma are boxed and presented in Figure 8G and 8H. The absence of staining with the isotype control further confirmed the specificity of previous staining in COVID-19 tissue samples (Figure 8 D’-F’). To further confirm the expression of HERV-W ENV in microglia with COVID-19 brain parenchyma, a double immunostaining was performed with the fluorophore-labeled same monoclonal antibodies against HERV-W ENV and Iba-1. Microglia-like cells positive for HERV-W ENV revealed to be co-stained with the antibody against Iba-1, and could thus be confirmed to be microglial cells (Figure 8 I-K, 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 detection. In parallel, sections from nasal mucosa showed staining of infiltrated lymphoid cells also consistently negative for Iba-1. In the present study we have analyzed the induction of HERV expression during COVID-19 disease. Firstly, in vitro results showed that SARS-CoV-2 can trigger both HERV-W and HERV-K ENV RNA transcription, in short-term PBMC cultures from healthy individuals, after a single exposure to SARS-CoV-2 virus. In contrast to the induced HERV-W and -K transcription, only HERV-W ENV protein was detected during short-term primary cultures of PBMC from about 30% of healthy donors. As wild-type SARS-CoV-2 spike protein trimer induced the production of IL-6 from PBMC of all donors, either responding or non-responding with HERV activation, IL-6 cannot be responsible for the induction of HERV-W ENV expression. The observed HERV-W activation is therefore not likely to be induced by cytokines or by inflammation due to viral infection, but by SARS-CoV-2 spike protein itself, as further shown. Cytofluorometry analysis confirmed that HERV-W ENV protein expression was predominantly and strongly induced in CD3low T-lymphocytes within the CD3+ Tcell population. 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 [72]. Alternatively, in COVID-19 patients, our observations may corroborate previous reports describing superantigen motifs of SARS-CoV-2 spike protein [73], and may involve cellular mechanisms associated to lymphopenia and hyperinflammation as already characterized for other emerging viruses (e.g., Ebola or Lassa) [78, 79]. However, because HERV-W ENV has also been shown to display superantigen-like effect [51], the origin of this short-term effect on T-lymphocytes may be questioned. Results from a recent study potentially provide a first answer, since showing correlated expression between HERV-W ENV and markers of exhaustion in T-lymphocytes from severe COVID-19 cases [60]. Of note, HERV activation occurs without signs of infection of lymphocytes or monocytes by SARS-CoV-2, while a recombinant trimer of its spike protein without stabilizing mutations [74] 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. This type of HERV activation mediated by an interaction between a triggering virus and a specific receptor on certain cells has already been described, e.g., with HHV-6A [33]. Because SARS-CoV-2 induced HERV-W ENV expression in human lymphoid cells that neither express ACE2 nor TMPRSS2 and does not infect them, additional undetermined receptors are expected to mediate HERV activation by SARS-CoV-2, which should now be further studied with recent data on alternative receptors [80].The study further addressed the frequency of HERV expression in COVID-19 patients. In hospitalized individuals with different severity status, HERV-W ENV protein was confirmed to be expressed at the membrane surface of T-lymphocytes but mainly in CD3low T-cells. A lower but significant detection was also found in Blymphocytes, which corresponds to a cell type already known to be permissive for HERV-W expression [81, 82]. However, HERV-W expression was not observed in Tcells before its first observation in COVID-19 [60]. After the recent characterization of a soluble hexameric form of HERV-W ENV in MS brain lesions [70], we here observed its presence as a circulating antigen in COVID- 19 plasma or sera. In sample series from patients in intensive care unit, a significant correlation was found between HERV-W ENV detection on T-lymphocytes and the soluble antigen in plasma. Though moderate, this correlation was seen between blood cells and plasma devoid of cells, which indicates a relationship between expressing lymphocytes and the release of this soluble antigen in blood. Plasma HERV-W ENV antigenemia was confirmed to be significantly detected in hospitalized patients with more-or-less severe COVID-19 but not in tested healthy controls, whereas HERV-K ENV protein was never detected in plasma or serum. Importantly, HERV-W ENV antigenic levels strongly correlated with disease severity and were significantly different from healthy controls. All severe COVID-19 cases were tested positive for HERV-W ENV protein in plasma, which points to a marker of disease severity as already shown with blood lymphocytes [60]. We also report HERV-W ENV levels in a series of COVID-19 cases that were selected based on positive SARSCoV- 2 PCR, independent of disease severity. Within this series, HERV-W ENV antigenemia was detected in about 20% of samples only, a number quite similar to the percentage of PBMC donors that showed HERV-W ENV positivity in reaction to SARS-CoV-2 challenge in vitro. Altogether, these data suggest that a percentage of individuals with an underlying susceptibility to symptomatic and/or severe evolution may be linked to the activation of HERV-W ENV expression. This is also consistent with the significant increase in frequency and in antigenemia with disease severity in the previous series with hospitalized patients (Cf. Figure 5D), similarly to the previously shown increased and predictive expression on T-lymphocytes of such inpatients [60]. In short term PBMC cultures non-responding to SARS-CoV-2 exposure, we had observed HERV RNA levels below the levels of non-exposed controls. Such decreased levels may be explained by the activation of HERVinhibitory 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. In fact, most PCR positive individuals do not develop major symptomatology after SARS-CoV-2 infection, including about 35% of asymptomatic cases [83].Furthermore, analyses of other blood parameters showed that the neutrophils counts, reported to be most often increased in COVID-19 with worsening evolution [84, 85], also paralleled HERV-W ENV antigenemia with values above the upper normal limit in all hospitalized patients with ENV positive plasma (Cf. Supplementary Table S3). Moreover, since patients from different geographic areas and time periods of the pandemic were analyzed and were confirmed to have been infected by different variants of SARS-CoV-2 (Supplementary Table S3), the global results are not expected to be influenced by such variables. To study HERV-W ENV expression beyond blood cells and plasma or serum, we have analyzed slides from tissue samples obtained at autopsy from patients deceased from acute COVID-19. SARS-CoV-2 N antigen corroborating viral replication was readily detected in epithelial cells within lungs and nasal mucosa but not in studied sections from brain parenchyma, even in olfactory bulb sections neighboring nasal mucosa with noticeable ongoing infection. The present results did not show any infection by SARS-CoV-2 of the central nervous system (CNS) nor of the cardiac tissues in the studied cases. HERV-W ENV was strongly expressed in lymphoid infiltrates in tissues surrounding lung alveola and within nasal mucosa. It was clearly and frequently detected in blood vessels endothelium from all tissues including CNS, which revealed quite numerous within cardiac muscle and pericardiac fatty tissue. In the CNS, HERV-W ENV expression was found in scattered cells that were confirmed to be microglia. Finally, strong HERV-W ENV staining was often detected in aggregated cells corresponding to thrombotic structures in blood vessels from the lung sections. Globally, positive endothelial cells from blood vessels were seen in all studied tissues and all HERV-W ENV expressing cells did not correspond to phenotypes seen to be infected with SARS-CoV-2. This discrepancy is well illustrated by infected lung epithelial cells without HERV-W ENV expression and noninfected alveolar macrophages presenting strong HERV-W ENV membrane staining within the same lung section. This is consistent with our observations of HERV-W ENV protein presence on lymphocytes without infection by SARS-CoV-2 in COVID- 19 patients and in PBMC, after in vitro activation with infectious SARS-CoV-2. Most importantly, it shows HERV-W ENV expression in tissue-infiltrated lymphocytes and macrophages within affected organs, like in blood of COVID-19 patients. Therefore, results from immunohistochemistry analyses indicate that HERV-W ENV expression is intimately associated with organs and cells involved in COVID-19 associated or superimposed pathology beyond tissue inflammation mediated by immune cells or cytokines, e.g., in vasculitis or intravascular thrombotic processes [29, 55]. Moreover, given known HERV-W involvement in MS pathogenesis [43, 46, 48] or in certain psychoses associated with inflammatory biomarkers [58, 86], 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 postacute COVID-19 period [14, 17, 64, 66, 77]. In acute primary infection, one must also consider the pathogenic effects on immune cells resulting in an hyperactivation of the innate immunity via HERV-W ENVmediated TLR4 activation [53] with a possible contribution to the frequently observed lymphopenia along with an adaptive immune defect. The induction of autoimmune manifestations [87-90] as previously shown to be induced with HERV-W ENV (previously named MSRV) in a humanized mouse model [47], should also be considered. 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 pathogenic player contributing to the disease severity. To date, various studies have considered many parameters in COVID-19 patients, but none has addressed HERV activation and expression of HERV proteins initiating and perpetuating pathogenic pathways underlying worsening clinical evolution and long-term pathology as seen with the now emerging post-COVID “wave”. The latter represents millions of patients suffering from various and numerous symptoms, who develop long-term disabling active pathology for which no rationalized understanding nor therapeutic perspective can be proposed to date. In face of this challenging situation, data from the present study altogether call for evaluating HERV-W ENV as a marker of severity but also as a potential therapeutic target in COVID-19 associated syndromes.
Известно, что не все вирусы обладают свойствами суперантигена, а редкие, и вроде бы, непосредственно для коронавирусных антигенов, ситуация не была прозрачной (что они напрямую связываются с толл-рецепторами и запускают неспецифическую активацию лимфоцитов).
Однако, такое свойство может быть и опосредованным.
Возможно, так и есть в случае ковида- что поясняет разное течение болезни у разных людей. Т.к. добавляется промежуточный "помощник", или фактор, (а может даже и не один, а несколько), который усложняет картину.
Международная группа ученых выявила, что коронавирус активирует ретровирусные гены, которые давным давно "вшиты" в человеческий геном и являются оприходованными на пользу (или хотя бы обезоруженными) следами былых ретровирусных инфекций, прокатывавшихся через популяцию.
Ретровирусы обладают особенностью встраиваться в геном, а потом их оттуда не выпилить, и сейчас эти следы древних вирусов в нашем геноме представляют собой транспозоны, или прыгающие гены, или повторяющиеся последовательности, функции которых чаще - регуляторные, в основном, и постепенно выясняются наукой. Эти участки не способны, даже при активации, привести к ретровирусной ре-инфекции, тк "ущербны". Однако, когда они активированы, они функциональны, и эта функциональность - не всегда на пользу. Известно, что иногда при активации эти гены могут запускать в иммунных клетках механизмы, приводящие к неспецифической воспалительной активации "изнутри", после чего клетка, например, выдает на свои мембраны много маркеров воспаления, меток смерти, или продуцирует множество медиаторов, запускающих воспалительные реакции вокруг нее, или активирует каскадно другие иммунны клетки, которые с ней обычно взаимодействуют, или начинает усиленно размножаться. Результат работы ретровирусного остатка (эндогенного) может быть такой же, как и суперантигенное воздействие- сильная, системная активация, попутно и реакция воспаления, с возможным разрушением некоторых тканей, запускание аутоиммунных реакций (тк будут активироваться любые В-лимфоциты, широким фронтом, в том числе и производящие антитела (и на свои антигены, которые в норме глубоко супрессированы регуляторной системой). Одна из "задач" ретровируса - простимулировать пролиферацию иммунных клеток, которые он заражает и использует. Ретровирусный остаток - заразить не может, а активировать одни и сделать при это уязвимыми другие клетки - может. При определенных условиях, конечно. Когда что-то "сбойнуло". Под действием внешнего фактора.
В этой работе авторы обнаружили, что экспозиция к коронавирусу моноядерных клеток периферической крови здоровых людей, активирует в них, примерно у трети, репликацию РНК с ретровирусных вставок HERV-K и HERV-W, и даже экспрессию суперкапсидного мембранного белка, который кодирует этот остаток HERV-W .
При этом, сами эти моноядерные клетки коронавирусом -не заражаются, а только меняют свою активность в его присутствии.
То же происходит и со зрелыми и "стареющими" Т лимфоцитами здоровых людей, при контакте с коронавирусом " в пробирке".
За стимулирующий ретровирусные гены эффект был ответственнен шипик коронавируса- потому что "обработка" им суспензий или моноядерных клеток периферической крови некоторых здоровых волонтеров, или Т лимфоцитов, показала результат, аналогичный эффекту воздействия целого коронавируса.
Далее- этот же ретровирусный белок, кодируемый HERV-W, обнаруживался в повышенных количествах в крови, плазме симптоматично больных ковидом. Тяжесть течения ковида в буольных коррелирует и с "растворимым" ретровирусным белком, и с количеством белка HERV-W на мембранах Т лимофицтов, экспрессирующих CD3+ маркер (зрелые).
Что интересно, когда этот маркер померяли у тех, кто не госпитализировался- то он тоже обнаружился у большинства заразившихся ковидом (но его не было в крови у здоровых доноров).
Так же, у тех, кто умер от острого тяжелого ковида, в тканях легких, сердца, мозга, обонятельных луковиц, слизистой носа и других органов, вовлеченных в патологию ковида, обнаруживался экспрессируемый HERV-W белок, в клетках, которые принимали участие в развитии патологии (макрофаги, эндотелиальные клетки селезенки, сердца, сосудов, глия, этс), но не были поражены коронавирусом (не экспрессировали антигены коронавируса, в отличие от соседних эпителиальных клеток, или пневмоцитов, этс).
Авторами не обнаружен коронавирус в тканях сердца или в мозгу, зато обнаружены активированные им клетки, продуцирующие ретровирусный белок (а для контролей с похожими патологиями (ткань возле опухоли), таких белков не обнаружилось, что подтверждает, что толчком была инфекция).
Не то чтобы это прямые, скорее косвенные доказательства, но вполне "логичные".
Авторы не исключают, что активация HERV-W может быть вовлечена в развитие симптоматики не только ковида, но и постковидного симптома.
Может быть, тк повышенные уровни этого белка обнаруживаются у больных рассеянным склерозом, например (кое-где симптоматика с постковидом пересекается) или с нейропсихиатрическими отклонениями. Имумнные клетки, которые сбили свои настройки, могут жить долго, и зачастую оказываются "влиятельными" по своей природе, что может доводить до патологии.
Конечно, они проверили только по вот этим двум ретровирусным остаткам, а ведь в организме среди нашего генома их содержится около 8%. И не все они изучены, как возможные факторы влияния. Эти 2 авторы выбрали потому, что по ним было уже кое- что известно, и оказалось что один вариант из выбранных ими - экспрессируется и может быть маркером и нести ответственность как минимум за часть эффектов (а за продукцию Ил6, например, нет). Вообще же связать одно с другим, особенно если явления отсроченные (фактор-время- эффект), непросто.
Patients with COVID-19 may develop abnormal inflammatory response and lymphopenia, followed in some cases by delayed-onset syndromes, often long-lasting after the initial SARS-CoV-2 infection. As viral infections may activate human endogenous retroviral elements (HERV), we studied the effect of SARS-CoV-2 on HERV-W and HERV-K envelope (ENV) expression, known to be involved in immunological and neurological pathogenesis of human diseases. Our results have showed that the exposure to SARS-CoV-2 virus activates early HERV-W and K transcription but only HERV-W ENV protein expression, in an infection- and ACE2-independent way within peripheral blood mononuclear cell cultures from one-third of healthy donors. Moreover, HERV-W ENV protein was significantly increased in serum and plasma of COVID-19 patients, correlating with its expression in CD3+ lymphocytes and with disease severity. Finally, HERV-W ENV was found expressed in post-mortem tissues of lungs, heart, brain olfactory bulb and nasal mucosa from acute COVID-19 patients in cell-types relevant for COVID-19-associated pathogenesis within affected organs, but different from those expressing of SARS-CoV-2 antigens. Altogether, the present study revealed that SARS-CoV-2 can induce HERV-W ENV expression in cells from individuals with symptomatic and severe COVID-19. Our data suggest that HERV-W ENV is likely to be involved in pathogenic features underlying symptoms of acute and post-acute COVID. It highlights the importance to further understand patients genetic susceptibility to HERV-W activation and the relevance of this pathogenic element as a prognostic marker and a therapeutic target in COVID-19 associated syndromes. 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 [20-22], in addition to adaptive immune defects resulting in lymphopenia and neutrophils/lymphocytes imbalance [23, 24]. A deficient interferon response has also been shown to favor or result from SARS-CoV-2 infection [25, 26]. Such multifaceted immunological dysregulations are thus underlying hyper-immune reactions such as the “cytokine storm” syndrome, the multisystem inflammatory syndrome in children (MIS-C), the dysregulation of coagulation, as well as neurological and various other manifestations [27-29]. The present COVID-19 pandemic has thus raised many questions about the pathophysiological mechanisms explaining the many symptoms or 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 (HERVs) present in the human genome [30, 31]. In particular, the resulting production of endogenous proteins of retroviral origin with pathogenic effects may generate clinical symptoms corresponding to the organ, tissue or cells in which they are expressed, according to the specific tropism of the triggering infectious agent [32-40]. HERVs represent about 8% of human chromosomal sequences and comprise about 22 families independently acquired during evolution from exogenous retroviruses via an infection of germ line cells [41, 42]. Abnormal expression may then become self-sustained, thus creating lifelong chronic expression from host‘ s genome copies in affected tissues [43], e.g., with cytokine-mediated feedback loops [44] or, possibly, mediated by their own envelope proteins [45]. Such a sustained expression has been shown to be involved in brain lesions with lifelong expansion in patients with multiple sclerosis (MS) [43, 46-48]. Different HERV envelope proteins were shown to exert major immunopathogenic [49-55] and/or neuropathogenic [46, 51-58] effects in vitro and in vivo, associated with pathognomonic features of human diseases. We therefore studied whether SARS-CoV-2 could activate HERV copies considered as ‘dormant enemies within’ [59]. We focused on HERV families already shown to be involved in the pathogenesis of human diseases, HERV-W and HERV-K [57], to evaluate their potential association with COVID-19 and associated syndromes. This question became critical after a recent study has revealed the significant expression of HERV-W envelope protein (ENV) in lymphoid cells from COVID-19 patients, correlating with disease outcome and markers of lymphocyte exhaustion or senescence [60].In the present study, we initially addressed the potential role of SARS-CoV-2 in directly triggering the activation of a pathogenic HERV protein expression as reported with other viruses in, e.g., MS and in type 1 diabetes [61-63]. We further analyzed its expression in white blood cells and its possible detection in plasma of patients with COVID-19 presenting various clinical forms at early and late time-points. We finally examined this HERV expression in affected tissues from COVID-19 post-mortem samples. Also, since a major concern beyond the initial COVID-19 infectious phase is foreseen to result from the occurrence of long lasting symptoms with more-or-less delayed onset and often involves neurological impairment [17, 64-66], we examined SARS-CoV-2 and HERV-W expression in brain parenchyma. For comparison with dominantly or frequently affected organs, we also examined these antigens expression in lung and cardiac tissues.Our results showed that (i) SARS-CoV-2 can activate the production of HERV-W ENV in cultured blood mononuclear cells from a sub-group of healthy donors, (ii) HERV-W ENV is expressed on T-lymphocytes from COVID-19 patients, (iii) HERV-W ENV antigen is detected in all tested plasma or sera samples from severe cases in intensive care unit, but only in about 20% of PCR positive cases after early diagnosis, (iv) the level of HERV-W ENV antigenemia increases with disease severity and (iv) HERV-W ENV expression is observed by immunohistochemistry in cell-types relevant for COVID-19 associated pathogenesis within affected organs and particularly in brain microglia. HERV-W ENV protein expression having been observed in CD3+ T-cells of COVID- 19 patients [60], we analyzed whether direct exposure to SARS-CoV-2 could induce HERV-W ENV in T lymphocytes from healthy donors. We analyzed HERV-W ENV expression in PBMC cultures from three healthy donors, with or without exposure to SARS-CoV-2, using cytofluorometry analysis on non-permeabilized cells. As illustrated in Figure 2, CD3+ T lymphocytes were identified with the gating strategy in non-infected (NI) cultures. CD3+ cells comprised CD3high T-cells, physiologically representing naïve/non-activated cells, and CD3+ cells with increased size, normally representing activated T-cells following, e.g., antigenic stimulation, further decreasing CD3 exposure at heir surface and corresponding to the CD3low subpopulation [71, 72]. Furthermore, in order to avoid confusion between CD14+ monocytes and larger CD3low T-cells, we have evaluated T-cell populations by eliminating CD14+ cells and gating specifically CD14- cells (Figure 2A-D). Indeed, when inoculated with SARSCoV- 2, CD3+ cells showed HERV-W ENV cell-surface expression. Minimal fluorescence associated with HERV-W ENV detection (compatible with technical noise) was observed in sham-inoculated CD3+ T-cells (Figure 2A), whereas a significant increase was characterized in SARS-CoV-2-associated cultures mainly observed in CD3low cells (Figure 2B). In the presence of SARS-CoV-2, CD3low cells may also reflect the predicted superantigenic properties of the Spike protein [73]. Double-labeling with anti-CD3 and anti-HERV-W ENV specific antibodies indicated that a significant proportion of CD3low T-cells was positive for HERV-W ENV at the cell-surface both at 24h and at 72h after exposure to the virus (Figure 2A and 2C, CD3+ top panels) compared to CD3high T-cells (Figure 2B and 2D, CD3+ bottom panels). Sham-infected cultures did not express notable levels of HERV-W envelope antigen in CD3+ T-lymphocytes (Figure 2A and 2C, CD3+ top and bottom panels). The percentage of HERV-W ENV positive cells in each condition is represented in 24h and 72h post-inoculation with SARS-CoV-2. Exposure to SARS-CoV-2 recombinant trimeric Spike protein triggers HERV-W ENV protein production in PBMC of some heathy individuals. Of interest, donor #30 was the only one tested positive for anti-SARS CoV-2 serum antibodies (data not shown), which did not prevent HERV transcriptional activation by this recombinant spike trimer but coincided with delayed peak of HERV RNA. IL-6 secretion was significantly increased after exposure to SARS-CoV-2 S antigen in PBMC from all tested donors, including those not responding in terms of HERV activation. Thus, HERV activation occurred independently from IL-6 production and, beyond RNA analysis, HERV-W ENV protein production was confirmed by immunofluorescence analysis at 72h in cultured PBMC of 2 out of four other donors inoculated with spike trimers (0.5 μg/mL) and not in mock-control cultures. HERV-W ENV positive cells were detected, as previously seen with the infectious virus. Moreover, a relative increase in HERV-W ENV RNA was quantified, despite a low proportion of activated cells within the cultures. Of interest, the absence of ACE2 expression in PBMC (Supplementary Figure S3 A) suggests an interaction of the spike protein with another receptor. Cell viability was measured and did not vary significantly within the culture period of experiments. 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 antigen in plasma. a highly significant difference was observed between COVID-19 patients and HBD controls on CD3+ T-cells (p=0.0005 for CD3low T-cells and p=0.0023 for CD3high T-cells) (Figure 4A and 4B). However, while the amount of CD3high T-cells positive for HERV-W ENV was low but significant when compared to HBD (p=0.0005) (Figure 4A), significant but much higher levels were seen in CD3high T-cells of COVID-19 individuals (p=0.0023 Vs HBD; Figure 4B). Interestingly, whereas the levels of CD14+ monocytes stained for HERV-W ENV remained equivalent between HBD and COVID-19 patients (Figure 4C), a significant difference was also observed in CD19+ B-cells between HBD and COVID-19 groups (p=0.031) (Figure 4D). HERV-W ENV soluble hexameric antigen was also detected in plasma of COVID-19 patients and its correlation with HERV-W ENV expression levels previously determined by cytofluorometry on each cell population was assessed (Figure 4 E-H). A moderate but significant correlation was observed between the HERV-W ENV antigen released in plasma and the one expressed on the membrane of both naïve CD3+ and activated CD3+ T-lymphocytes (p<0.02 and p<0.03, respectively)Finally, SARS-CoV-2 IgG serology for multiple antigens was also analyzed, but no significant correlation was observed between anti-SARS-CoV-2 IgG titers and HERVW ENV hexamer antigenemia in plasma samples. To study HERV-W ENV antigenemia in patients representing a wider range of clinical status at early COVID-19 diagnosis (compared to patients with other diseases and healthy controls-HC) its soluble antigen was quantified in sera from a third cohort consisting in two successive sampling series of SARS-CoV-2 PCR positive cases with heterogeneous clinical presentation (described in Supplementary Table S4 ). As shown in Figure 5H, a significant difference was found between all COVID-19 patients and HBD or other diseases (Mann Whitney U-test, respectively p=0.0010 and p=0.0011). 21% of COVID-19 sera were positive for HERV-W ENV among the 144 SARS-CoV-2 PCR positive cases, while none of the 44 PCR negative HBD were positive for HERV-W ENV (Chi2=8.796, p<0.001). Similarly, HERV-W ENV antigen was not detected in 43 sera from non-COVID-19 other diseases either (Chi2=8.604, p<0.001). Finally, the ratio between neutrophils and lymphocytes counts (N/L), previously suggested as a biomarker of COVID-19 severity [23], was significantly increased in COVID-19 cases with HERV-W ENV positive antigenemia.To better understand the consistency and magnitude of HERV-W ENV expression in SARS-CoV-2-infected patients, we next analyzed the expression of this antigen in tissues from COVID-19 patients. Post-Mortem lung, heart, nasal mucosa and brain tissue samples were obtained from patients deceased from severe acute forms of COVID-19. Lung tissue staining with monoclonal antibodies targeting SARS-CoV-2 Nucleocapsid (N-Protein) or HERV-W ENV.SARS-CoV-2 antigen was readily detected in lung epithelial cells but not in alveolar macrophages HERV-W ENV antigen was strongly expressed at the cell membrane of macrophages but not in neighboring SARS-CoV-2 positive pneumocytes. The specificity of both SARS-CoV-2 N and HERV-W ENV proteins was confirmed by the absence of staining within similar sections of lung tissue from non-COVID controls, i.e., normal tissue surrounding lung tumors Endothelial nature of HERV-W ENV positive cells was confirmed with CD31 staining in similar vessel structures from neighboring slides. SARS-CoV-2 antigen was not detected in cardiac tissue from studied samples. To further address the expression of HERV-W ENV in the central nervous system (CNS) of COVID-19 patients, as previously observed in multiple sclerosis lesions [46], we analyzed sections from tissue samples taken across the cribriform plate, comprising areas of the olfactory bulb and of the nasal mucosa (Figure 8A). This anatomical region was chosen since suggested to be a most probable route of coronavirus passage to the brainstem via olfactory nerve roots within nasal mucosa [75] and since frequently reported anosmia in COVID-19 patients indicates pathological involvement of the olfactory bulb parenchyma [76, 77]. Sections from samples with the upper anatomical brain region, i.e. the frontal lobe, were also made available. Immunohistology results for both SARS-CoV-2 and HERV-W ENV antigens in brain parenchyma or nasal mucosa areas are presented Figure 8. In sections at the CNSnasal tissue interface presented in Figure 8B, a strong SARS-CoV-2 N antigen staining indicating viral replication was seen in nasal mucosa but not in neighboring CNS areas of the olfactory bulb. SARS-CoV-2 antigen was not detected in various areas of CNS sections within olfactory bulb or frontal brain parenchyma, as illustrated in Figure 8C. 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 8D and 8E). Conversely to SARS-CoV-2 antigen, HERV-ENV was also detected in the olfactory bulb parenchyma in glial cells (Figure 8F), which revealed to have the morphology of microglia and were stained by microglial maker, Iba-1 (Figure 8G and 8H). Higher magnifications of HERV-W ENV positive microglial cells within brain parenchyma are boxed and presented in Figure 8G and 8H. The absence of staining with the isotype control further confirmed the specificity of previous staining in COVID-19 tissue samples (Figure 8 D’-F’). To further confirm the expression of HERV-W ENV in microglia with COVID-19 brain parenchyma, a double immunostaining was performed with the fluorophore-labeled same monoclonal antibodies against HERV-W ENV and Iba-1. Microglia-like cells positive for HERV-W ENV revealed to be co-stained with the antibody against Iba-1, and could thus be confirmed to be microglial cells (Figure 8 I-K, 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 detection. In parallel, sections from nasal mucosa showed staining of infiltrated lymphoid cells also consistently negative for Iba-1. In the present study we have analyzed the induction of HERV expression during COVID-19 disease. Firstly, in vitro results showed that SARS-CoV-2 can trigger both HERV-W and HERV-K ENV RNA transcription, in short-term PBMC cultures from healthy individuals, after a single exposure to SARS-CoV-2 virus. In contrast to the induced HERV-W and -K transcription, only HERV-W ENV protein was detected during short-term primary cultures of PBMC from about 30% of healthy donors. As wild-type SARS-CoV-2 spike protein trimer induced the production of IL-6 from PBMC of all donors, either responding or non-responding with HERV activation, IL-6 cannot be responsible for the induction of HERV-W ENV expression. The observed HERV-W activation is therefore not likely to be induced by cytokines or by inflammation due to viral infection, but by SARS-CoV-2 spike protein itself, as further shown. Cytofluorometry analysis confirmed that HERV-W ENV protein expression was predominantly and strongly induced in CD3low T-lymphocytes within the CD3+ Tcell population. 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 [72]. Alternatively, in COVID-19 patients, our observations may corroborate previous reports describing superantigen motifs of SARS-CoV-2 spike protein [73], and may involve cellular mechanisms associated to lymphopenia and hyperinflammation as already characterized for other emerging viruses (e.g., Ebola or Lassa) [78, 79]. However, because HERV-W ENV has also been shown to display superantigen-like effect [51], the origin of this short-term effect on T-lymphocytes may be questioned. Results from a recent study potentially provide a first answer, since showing correlated expression between HERV-W ENV and markers of exhaustion in T-lymphocytes from severe COVID-19 cases [60]. Of note, HERV activation occurs without signs of infection of lymphocytes or monocytes by SARS-CoV-2, while a recombinant trimer of its spike protein without stabilizing mutations [74] 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. This type of HERV activation mediated by an interaction between a triggering virus and a specific receptor on certain cells has already been described, e.g., with HHV-6A [33]. Because SARS-CoV-2 induced HERV-W ENV expression in human lymphoid cells that neither express ACE2 nor TMPRSS2 and does not infect them, additional undetermined receptors are expected to mediate HERV activation by SARS-CoV-2, which should now be further studied with recent data on alternative receptors [80].The study further addressed the frequency of HERV expression in COVID-19 patients. In hospitalized individuals with different severity status, HERV-W ENV protein was confirmed to be expressed at the membrane surface of T-lymphocytes but mainly in CD3low T-cells. A lower but significant detection was also found in Blymphocytes, which corresponds to a cell type already known to be permissive for HERV-W expression [81, 82]. However, HERV-W expression was not observed in Tcells before its first observation in COVID-19 [60]. After the recent characterization of a soluble hexameric form of HERV-W ENV in MS brain lesions [70], we here observed its presence as a circulating antigen in COVID- 19 plasma or sera. In sample series from patients in intensive care unit, a significant correlation was found between HERV-W ENV detection on T-lymphocytes and the soluble antigen in plasma. Though moderate, this correlation was seen between blood cells and plasma devoid of cells, which indicates a relationship between expressing lymphocytes and the release of this soluble antigen in blood. Plasma HERV-W ENV antigenemia was confirmed to be significantly detected in hospitalized patients with more-or-less severe COVID-19 but not in tested healthy controls, whereas HERV-K ENV protein was never detected in plasma or serum. Importantly, HERV-W ENV antigenic levels strongly correlated with disease severity and were significantly different from healthy controls. All severe COVID-19 cases were tested positive for HERV-W ENV protein in plasma, which points to a marker of disease severity as already shown with blood lymphocytes [60]. We also report HERV-W ENV levels in a series of COVID-19 cases that were selected based on positive SARSCoV- 2 PCR, independent of disease severity. Within this series, HERV-W ENV antigenemia was detected in about 20% of samples only, a number quite similar to the percentage of PBMC donors that showed HERV-W ENV positivity in reaction to SARS-CoV-2 challenge in vitro. Altogether, these data suggest that a percentage of individuals with an underlying susceptibility to symptomatic and/or severe evolution may be linked to the activation of HERV-W ENV expression. This is also consistent with the significant increase in frequency and in antigenemia with disease severity in the previous series with hospitalized patients (Cf. Figure 5D), similarly to the previously shown increased and predictive expression on T-lymphocytes of such inpatients [60]. In short term PBMC cultures non-responding to SARS-CoV-2 exposure, we had observed HERV RNA levels below the levels of non-exposed controls. Such decreased levels may be explained by the activation of HERVinhibitory 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. In fact, most PCR positive individuals do not develop major symptomatology after SARS-CoV-2 infection, including about 35% of asymptomatic cases [83].Furthermore, analyses of other blood parameters showed that the neutrophils counts, reported to be most often increased in COVID-19 with worsening evolution [84, 85], also paralleled HERV-W ENV antigenemia with values above the upper normal limit in all hospitalized patients with ENV positive plasma (Cf. Supplementary Table S3). Moreover, since patients from different geographic areas and time periods of the pandemic were analyzed and were confirmed to have been infected by different variants of SARS-CoV-2 (Supplementary Table S3), the global results are not expected to be influenced by such variables. To study HERV-W ENV expression beyond blood cells and plasma or serum, we have analyzed slides from tissue samples obtained at autopsy from patients deceased from acute COVID-19. SARS-CoV-2 N antigen corroborating viral replication was readily detected in epithelial cells within lungs and nasal mucosa but not in studied sections from brain parenchyma, even in olfactory bulb sections neighboring nasal mucosa with noticeable ongoing infection. The present results did not show any infection by SARS-CoV-2 of the central nervous system (CNS) nor of the cardiac tissues in the studied cases. HERV-W ENV was strongly expressed in lymphoid infiltrates in tissues surrounding lung alveola and within nasal mucosa. It was clearly and frequently detected in blood vessels endothelium from all tissues including CNS, which revealed quite numerous within cardiac muscle and pericardiac fatty tissue. In the CNS, HERV-W ENV expression was found in scattered cells that were confirmed to be microglia. Finally, strong HERV-W ENV staining was often detected in aggregated cells corresponding to thrombotic structures in blood vessels from the lung sections. Globally, positive endothelial cells from blood vessels were seen in all studied tissues and all HERV-W ENV expressing cells did not correspond to phenotypes seen to be infected with SARS-CoV-2. This discrepancy is well illustrated by infected lung epithelial cells without HERV-W ENV expression and noninfected alveolar macrophages presenting strong HERV-W ENV membrane staining within the same lung section. This is consistent with our observations of HERV-W ENV protein presence on lymphocytes without infection by SARS-CoV-2 in COVID- 19 patients and in PBMC, after in vitro activation with infectious SARS-CoV-2. Most importantly, it shows HERV-W ENV expression in tissue-infiltrated lymphocytes and macrophages within affected organs, like in blood of COVID-19 patients. Therefore, results from immunohistochemistry analyses indicate that HERV-W ENV expression is intimately associated with organs and cells involved in COVID-19 associated or superimposed pathology beyond tissue inflammation mediated by immune cells or cytokines, e.g., in vasculitis or intravascular thrombotic processes [29, 55]. Moreover, given known HERV-W involvement in MS pathogenesis [43, 46, 48] or in certain psychoses associated with inflammatory biomarkers [58, 86], 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 postacute COVID-19 period [14, 17, 64, 66, 77]. In acute primary infection, one must also consider the pathogenic effects on immune cells resulting in an hyperactivation of the innate immunity via HERV-W ENVmediated TLR4 activation [53] with a possible contribution to the frequently observed lymphopenia along with an adaptive immune defect. The induction of autoimmune manifestations [87-90] as previously shown to be induced with HERV-W ENV (previously named MSRV) in a humanized mouse model [47], should also be considered. 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 pathogenic player contributing to the disease severity. To date, various studies have considered many parameters in COVID-19 patients, but none has addressed HERV activation and expression of HERV proteins initiating and perpetuating pathogenic pathways underlying worsening clinical evolution and long-term pathology as seen with the now emerging post-COVID “wave”. The latter represents millions of patients suffering from various and numerous symptoms, who develop long-term disabling active pathology for which no rationalized understanding nor therapeutic perspective can be proposed to date. In face of this challenging situation, data from the present study altogether call for evaluating HERV-W ENV as a marker of severity but also as a potential therapeutic target in COVID-19 associated syndromes.
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