SARS-CoV2 E and 3a proteins are inducers of pannexin currents (preprint)

Controversial reports have suggested that SARS-CoV E and 3a proteins may be viroporins that conduct currents through the plasma membrane of the infected cells. If true, these proteins would represent accessible targets for the development of new antiviral drugs by using high-throughput patch-clamp techniques. Here we aimed at better characterizing the cell responses induced by E or 3a protein with a particular focus on the ion conductances measured at the cell surface. First, we show that expression of SARS-CoV-2 E or 3a protein in CHO cells gives rise to cells with newly-acquired round shape, tending to detach from the Petri dish. This suggests that cell death is induced upon expression of E or 3a protein. We confirmed this hypothesis by using flow cytometry, in agreement with earlier reports on other cell types. In adhering cells expressing E or 3a protein, whole-cell currents were in fact not different from the control condition indicating that E and 3a proteins are not plasma membrane viroporins. In contrast, recording currents on detached cells uncovered outwardly-rectifying currents, much larger than those observed in control. The current characteristics are reminiscent of what was previously observed in cells expressing SARS-CoV-1 E or 3a proteins. Herein, we illustrate for the first time that carbenoxolone blocks these outward currents suggesting that they are conducted by pannexin channels, mostly likely activated by cell morphology change and/or cell death. Alongside we also demonstrate that truncation of the C-terminal PDZ binding motifs reduces the proportion of dying cells but does not prevent pannexin currents suggesting distinct pathways for cell death and pannexin currents induced by E and 3a proteins. We conclude that SARS-CoV-2 E and 3a proteins are not acting as viroporins expressed at the plasma membrane.

COVID-19 in Tunisia (North Africa): IgG and IgG subclass antibody responses to SARS-CoV-2 according to disease severity (preprint)

Coronavirus disease 2019 (COVID 19) expresses a wide spectrum of disease severity. We investigated the profile of IgG and IgG subclass antibody responses to SARS CoV 2 in Tunisian patients with COVID 19 according to disease severity (86 patients with severe disease and 63 with mild to moderate disease). Two in house developed ELISA with excellent performance were used to test for antibodies to the nucleocapsid (N) protein and the receptor-binding domain of the spike antigen (S-RBD) of SARS CoV 2. IgG, IgG1 and IgG3 antibodies were significantly higher in patients with severe disease compared to non-severe disease. Antibodies to S-RBD or the N protein were dominated by IgG1 and IgG3 or IgG1/IgG3 and IgG2 subclasses respectively. In patients with severe disease, IgG antibodies' appearance to S RBD was delayed compared to the N protein. IgG subclass imbalance may reflect the pathophysiology of COVID 19 and may herald disease aggravation. This study brings information on the immune responses to SARS CoV 2 in North African patients and completes the picture drawn on COVID 19 in different African populations and worldwide.

Low Seroprevalence of COVID-19 in Lao PDR, Late 2020 (preprint)

Background: Lao PDR has low reported COVID-19 cases but it is unclear whether this masks silent transmission. A nationwide seroprevalence study was done between August and September 2020 to determine SARS-CoV-2 exposure.Methods: Participants were from the general community/healthcare workers in five provinces. Bat/wildlife contacts were from Vientiane province. ELISAs detected anti-Nucleoprotein (N; n=3173 tested) and anti-Spike (S; n=1417 tested) SARS-CoV-2 antibodies. Double-positive samples were checked by IgM/IgG rapid tests. Positive controls were confirmed COVID-19 cases and negative controls were pre-COVID-19 samples.Findings: In pre-COVID-19 samples, 14/265 (5·3%, [95% CI=3·1-8·7%]) were anti-N antibody single-positive and 3/265 (1·1% [0·3-3·5%]) were anti-S antibody single positive. None were anti-N and anti-S antibody double positive. Anti-N and anti-S antibodies were detected in 131/2417 (5·4% [4·5-6·3%]) and 19/1061 (1·8% [1·1-2·8%]) of the general population, 13/666 (2·0% [1·1-3·3%]) and 4/282 (1·4% [0·5-3·7%]) of healthcare workers and 15/74 (20·3% [12·6-31·0%]) and 5/74 (6·8% [2·8-15·3%]) of bat/wildlife contacts. Only 2/3173 (0·1% [0·02-0·3%]) were double positive for anti-N and anti-S antibodies (rapid test negative).Interpretation: We find no evidence for significant circulation of SARS-CoV-2 in Lao PDR during or before September 2020. This likely results from decisive measures taken by the government early in the pandemic, social behavior, and low population density. High anti-N antibodies/low anti-S antibodies in bat/wildlife contacts may indicate exposure to cross-reactive animal coronaviruses with threat of emerging novel viruses.Funding: Core; Agence Française de Développement. Additional support; Institut Pasteur du Laos, Institute Pasteur, Paris and Luxembourg Ministry of Foreign and European Affairs (“PaReCIDS II”). Declaration of Interests: The authors have no conflict of interest to declare.Ethics Approval Statement: The study protocol was approved by all participating institutes and by the Lao National Ethics Committee for Health Research (NECHR) (Ref #052/2020).

Nasopharyngeal and serological anti SARS-CoV-2 IgG/IgA responses in COVID-19 patients. (preprint)

BackgroundThe systemic antibody responses to SARS-CoV-2 in COVID-19 patients has been extensively studied. However, much less is known about the mucosal responses in the upper airways at the site of initial SARS-CoV-2 replication. Local antibody responses in the nasopharyngeal epithelium, that are likely to determine the course of infection, have not been analysed so far nor their correlation with antibody responses in serum. MethodsThe IgG and IgA antibody responses were analysed in the plasma as well as in nasopharyngeal swabs (NPS) from the first four COVID-19 patients confirmed by RT-qPCR in France. Two were pauci-symptomatic while two developed severe disease. Taking advantage of a comprehensive series of plasma and nasopharyngeal samples, we characterized their antibody profiles from the second week post symptoms onset, by using an in-house ELISA to detect anti-SARS-CoV-2 Nucleoprotein (N) IgG and IgA. ResultsAnti-N IgG and IgA antibodies were detected in the NPS of severe patients. Overall, the levels of IgA and IgG antibodies in plasma and NPS appeared specific to each patient. ConclusionsAnti-N IgG and IgA antibodies are detected in NPS, and their levels are related to antibody levels in plasma. The two patients with severe disease exhibited different antibody profiles that may reflect different disease outcome. For the pauci-symptomatic patients, one showed a low anti-N IgG and IgA response in the plasma only, while the other one did not exhibit overt serological response.

High seroprevalence but short-lived immune response to SARS-CoV-2 infection in Paris (preprint)

Background Although the COVID-19 pandemic peaked in March/April 2020 in France, the prevalence of infection is barely known. Herein, we assessed the serological response against the SARS-CoV-2 virus in a large population working in one institution of the Paris conurbation. We set up two high-throughput and sensitive methods to assess SARS CoV-2 Nucleoprotein and Spike protein-specific IgG response along with a pseudo-neutralization assay in sera. We studied 1847 participants who also answered a web-based survey on clinical symptoms. Methods and Results In May-July 2020, 11% (95% CI: 9.7-12.6) of serums were positive for IgG against the SARS-CoV-2 N and S protein and 9.5% (CI:8.2-11.0) were pseudo-neutralizer. The prevalence of immunization was 11.6% (CI:10.2-13.2) considering positivity in at least one assays. In 5% (CI:3.9-7.1) of RT-qPCR positive individuals, no systemic IgGs were detected. Among immune individuals, 21% had been asymptomatic. Anosmia and ageusia occurred in 52% of the IgG-positive individuals and in 3% of the negative ones. In contrast, 30% of the anosmia-ageusia cases were seronegative suggesting that the true prevalence of infection may reach 16.6%. In sera obtained 4-8 weeks after the first sampling anti-N and anti-S IgG titers and pseudo-neutralization activity declined by 31%, 17% and 53%, respectively with half-life of 35, 87 and 28 days, respectively. Conclusions The population studied being not particularly exposed to SARS-CoV-2 infection is representative of active workers in the Paris conurbation, suggesting that the current epidemiological models may underestimate the true prevalence of infection. The short lifespan of the serological systemic responses hinders retrospective assessment of the epidemic extent.

Intranasal Immunization with a Lentiviral Vector Coding for SARS-CoV-2 Spike Protein Confers Vigorous Protection in Pre-Clinical Animal Models (preprint)

We developed a potent vaccination strategy, based on lentiviral vector (LV), capable of inducing neutralizing antibodies specific to the Spike glycoprotein (S) of SARS-CoV-2, the etiologic agent of CoronaVirus Disease 2019 (COVID-19). Among several LV encoding distinct variants of S, a single one encoding the full-length, membrane anchored S (LV::SFL) triggered high antibody titers in mice, with neutralization activities comparable to patients recovered from COVID-19. LV::SFL systemic vaccination in mice, in which the expression of the CoV2 receptor hACE2 was induced by transduction of the respiratory tract cells by an adenoviral type 5 (Ad5) vector, despite an intense serum neutralizing activity, only {approx}1 log10 reduction of lung viral loads was observed after SARS-CoV2 challenge. We thus explored the strategy of targeting the immune response to the upper respiratory tract through an intranasal boost administration. Even though, after a prime and target regimen, the systemic neutralizing activity did not increase substantially, {approx}5 log10 decrease in lung viral loads was achieved, with the loads in some animals under the limit of detection of a highly sensitive RT-PCR assay. The conferred protection also avoided largely pulmonary inflammation. We confirmed the vaccine efficacy and inhibition of lung inflammation using both integrative and non-integrative LV platforms in golden hamsters, naturally permissive to SARS-CoV2 replication and restituting human COVID-19 physiopathology. Our results provide the proof-of-principle evidence of marked prophylactic effects of an LV-based vaccination strategy against SARS-CoV-2 in two pre-clinical animal models and designate the intranasal LV::SFL-based immunization as a vigorous and promising vaccine approach against COVID-19.

SARS-CoV-2 serological analysis of COVID-19 hospitalized patients, pauci-symptomatic individuals and blood donors. (preprint)

It is of paramount importance to evaluate the prevalence of both asymptomatic and symptomatic cases of SARS-CoV-2 infection and their antibody response profile. Here, we performed a pilot study to assess the levels of anti-SARS-CoV-2 antibodies in samples taken from 491 pre- epidemic individuals, 51 patients from Hopital Bichat (Paris), 209 pauci-symptomatic individuals in the French Oise region and 200 contemporary Oise blood donors. Two in-house ELISA assays, that recognize the full-length nucleoprotein (N) or trimeric Spike (S) ectodomain were implemented. We also developed two novel assays: the S-Flow assay, which is based on the recognition of S at the cell surface by flow-cytometry, and the LIPS assay that recognizes diverse antigens (including S1 or N C- terminal domain) by immunoprecipitation. Overall, the results obtained with the four assays were similar, with differences in sensitivity that can be attributed to the technique and the antigen in use. High antibody titers were associated with neutralisation activity, assessed using infectious SARS-CoV- 2 or lentiviral-S pseudotypes. In hospitalized patients, seroconversion and neutralisation occurred on 5-14 days post symptom onset, confirming previous studies. Seropositivity was detected in 29% of pauci-symptomatic individuals within 15 days post-symptoms and 3 % of blood of healthy donors collected in the area of a cluster of COVID cases. Altogether, our assays allow for a broad evaluation of SARS-CoV2 seroprevalence and antibody profiling in different population subsets.