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Human Angiotensin-Converting Enzyme 2 (hACE2) is the major receptor enabling host cell invasion by SARS-CoV-2 via interaction with Spike glycoprotein. The murine ACE2 ortholog does not interact efficiently with SARS-CoV-2 Spike and therefore the conventional laboratory mouse strains are not permissive to SARS-CoV-2 replication. Here, we generated new hACE2 transgenic mice, which harbor the hACE2 gene under the human keratin 18 promoter, in C57BL/6 "HHD-DR1" background. HHD-DR1 mice are fully devoid of murine Major Histocompatibility Complex (MHC) molecules of class-I and -II and express only MHC molecules from Human Leukocyte Antigen (HLA) HLA 02.01, DRA01.01, DRB1.01.01 alleles, widely expressed in human populations. We selected three transgenic strains, with various hACE2 mRNA expression levels and distinctive profiles of lung and/or brain permissiveness to SARS-CoV-2 replication. Compared to the previously available B6.K18-ACE22Prlmn/JAX mice, which have limited permissiveness to SARS-CoV-2 Omicron replication, these three new hACE2 transgenic strains display higher levels of hACE2 mRNA expression, associated with high permissiveness to the replication of SARS-CoV-2 Omicron sub-variants. As a first application, one of these MHC- and ACE2-humanized strains was successfully used to show the efficacy of a lentiviral vector-based COVID-19 vaccine candidate.
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As the COVID-19 pandemic continues and new SARS-CoV-2 variants of concern emerge, the adaptive immunity initially induced by the first-generation COVID-19 vaccines wains and needs to be strengthened and broadened in specificity. Vaccination by the nasal route induces mucosal humoral and cellular immunity at the entry point of SARS-CoV-2 into the host organism and has been shown to be the most effective for reducing viral transmission. The lentiviral vaccination vector (LV) is particularly suitable for this route of immunization because it is non-cytopathic, non-replicative and scarcely inflammatory. Here, to set up an optimized cross-protective intranasal booster against COVID-19, we generated an LV encoding stabilized Spike of SARS-CoV-2 Beta variant (LV::SBeta-2P). mRNA vaccine-primed and -boosted mice, with waning primary humoral immunity at 4 months post-vaccination, were boosted intranasally with LV::SBeta-2P. Strong boost effect was detected on cross-sero-neutralizing activity and systemic T-cell immunity. In addition, mucosal anti-Spike IgG and IgA, lung resident B cells, and effector memory and resident T cells were efficiently induced, correlating with complete pulmonary protection against the SARS-CoV-2 Delta variant, demonstrating the suitability of the LV::SBeta-2P vaccine candidate as an intranasal booster against COVID-19.
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ObjectivesOur aims were to evaluate Systemic Lupus Erythematosus (SLE) disease activity and SARS-CoV-2 specific immune responses after BNT162b2 vaccination. MethodsIn this prospective study, disease activity and clinical assessments were recorded from the first dose of vaccine, until day 15 after the second dose in 126 SLE patients. SARS-CoV-2 antibody responses were measured against wild-type spike antigen while serum-neutralizing activity was assessed against the SARS-CoV-2 historical strain and variants of concerns (VOCs). Vaccine-specific T-cell responses were quantified by Interferon (IFN)-{gamma} release assay after the second dose. ResultsBNT162b2 was well tolerated and no statistically significant variations of BILAG and SLEDAI scores were observed throughout the study in SLE patients with active and inactive disease at baseline. Mycophenolate Mofetil (MMF) and Methotrexate (MTX) treatments were associated with drastically reduced BNT162b2 antibody-response ({beta}=-78; p=0.007, {beta}=-122; p<0.001, respectively). Anti-spike antibody response was positively associated with baseline total IgG serum levels, naive B cell frequencies ({beta}=2; p=0.018, {beta}=2.5; p=0.003) and SARS-CoV-2-specific T cell response (r=0.462; p=0.003). In responders, serum neutralization activity decreased against VOCs bearing the E484K mutation but remained detectable in a majority of patients. ConclusionMMF, MTX and poor baseline humoral immune status, particularly: low naive B cell frequencies, are independently associated with impaired BNT162b2 mRNA antibody response, delineating SLE patients who might need adapted vaccine regimens and follow-up. KEY MESSAGESO_ST_ABSWhat is already known about this subject?C_ST_ABSO_LIBNT162b2 efficacy and safety has been described in studies mixing different RMDs C_LI What does this study add?O_LINo serious adverse effects, nor SLE flares have been documented after BNT162b2 in SLE patients. C_LIO_LINot only MMF and MTX, but also a poor humoral immune status at baseline impair vaccine antibody response C_LIO_LIAlbeit decreased, serum neutralizing activity against VOCs is conferred to vaccine-responders. C_LI How might this impact on clinical practice or future developments?O_LIThese parameters could be helpful for physicians to delineate which patients should have antibody measurement after full BNT162b2 vaccination and should be proposed a third injection of BNT162b2 vaccine. C_LI
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Non-integrative, non-cytopathic and non-inflammatory lentiviral vectors are particularly suitable for mucosal vaccination and recently emerge as a promising strategy to elicit sterilizing prophylaxis against SARS-CoV-2 in preclinical animal models. Here, we demonstrate that a single intranasal administration of a lentiviral vector encoding a prefusion form of SARS-CoV-2 spike glycoprotein induces full protection of respiratory tracts and totally avoids pulmonary inflammation in the susceptible hamster model. More importantly, we generated a new transgenic mouse strain, expressing the human Angiotensin Converting Enzyme 2, with unprecedent brain permissibility to SARS-CoV-2 replication and developing a lethal disease in <4 days post infection. Even though the neurotropism of SARS-CoV-2 is now well established, so far other vaccine strategies under development have not taken into the account the protection of central nervous system. Using our highly stringent transgenic model, we demonstrated that an intranasal booster immunization with the developed lentiviral vaccine candidate achieves full protection of both respiratory tracts and brain against SARS-CoV-2.
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A large proportion of SARS-CoV-2 infected individuals remains asymptomatic. Little is known about the extent and quality of their antiviral humoral response. Here, we analyzed antibody functions in 52 asymptomatic infected individuals, 119 mild and 21 hospitalized COVID-19 patients. We measured anti-Spike antibody levels with the S-Flow assay and mapped SARS-CoV-2 Spike- and N-targeted regions by Luminex. Neutralization, complement deposition and Antibody-Dependent Cellular Cytotoxicity (ADCC) were evaluated using replication-competent SARS-CoV-2 or reporter cell systems. We show that COVID-19 sera mediate complement deposition and kill infected cells by ADCC. Sera from asymptomatic individuals neutralize the virus, activate ADCC and trigger complement deposition. Antibody levels and activities are slightly lower in asymptomatic individuals. The different functions of the antibodies are correlated, independently of disease severity. Longitudinal samplings show that antibody functions follow similar kinetics of induction and contraction, with minor variations. Overall, asymptomatic SARS-CoV-2 infection elicits polyfunctional antibodies neutralizing the virus and targeting infected cells. - Sera from convalescent COVID-19 patients activate the complement and kill infected cells by ADCC. - Asymptomatic and symptomatic SARS-CoV-2-infected individuals harbor polyfunctional antibodies. - Antibody levels and functions are slightly lower in asymptomatic individuals - The different antiviral activities of anti-Spike antibodies are correlated regardless of disease severity. - Functions of anti-Spike antibodies have similar kinetics of induction and contraction.
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Although the COVID-19 pandemic peaked in March/April 2020 in France, the prevalence of infection is barely known. Herein, we assessed using high-throughput methods the serological response against the SARS-CoV-2 virus of 1847 participants working in one institution in Paris. 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 proteins 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. The population studied is representative of active workers in Paris. The short lifespan of the serological systemic responses suggests an underestimation the true prevalence of infection.
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BackgroundAssessment of cumulative incidence of SARS-CoV-2 infections is critical for monitoring the course and the extent of the epidemic. As asymptomatic or mild cases were typically not captured by surveillance data in France, we implemented nationwide serological surveillance. We present estimates for prevalence of anti-SARS-CoV-2 antibodies in the French population and the proportion of infected individuals who developed potentially protective neutralizing antibodies throughout the first epidemic wave. MethodsWe performed serial cross-sectional sampling of residual sera over three periods: prior to (9-15 March), during (6-12 April) and following (11-17 May) a nationwide lockdown. Each sample was tested for anti-SARS-CoV-2 IgG antibodies targeting the Nucleoprotein and Spike using two Luciferase-Linked ImmunoSorbent Assays, and for neutralising antibodies using a pseudo-neutralisation assay. We fitted a general linear mixed model of seropositivity in a Bayesian framework to derive prevalence estimates stratified by age, sex and region. FindingsIn total, sera from 11 021 individuals were analysed. Nationwide seroprevalence of SARS-CoV-2 antibodies was estimated at 0.41% [0.05-0.88] mid-March, 4.14% [3.31-4.99] mid-April and 4.93% [4.02-5.89] mid-May. Approximately 70% of seropositive individuals had detectable neutralising antibodies. Seroprevalence was higher in regions where circulation occurred earlier and was more intense. Seroprevalence was lowest in children under 10 years of age (2.72% [1.10-4.87]). InterpretationSeroprevalence estimates confirm that the nationwide lockdown substantially curbed transmission and that the vast majority of the French population remains susceptible to SARS-CoV-2. Low seroprevalence in school age children suggests limited susceptibility and/or transmissibility in this age group. Our results show a clear picture of the progression of the first epidemic wave and provide a framework to inform the ongoing public health response as viral transmission is picking up again in France and globally. FundingSante publique France.
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BackgroundA nationwide lockdown was implemented in France on 17 March 2020 to control the COVID-19 pandemic. People living in precarious conditions were relocated by the authorities to emergency shelters, hotels and large venues. Medecins sans Frontieres (MSF) then intervened to provide medical care in several of these locations in Paris and in Seine-Saint-Denis, one of its suburbs, between March and June 2020. A seroprevalence survey was conducted to assess the level of exposure to COVID-19 among the population living in the sites. To our knowledge, this is the first assessment of the impact of the pandemic on populations living in insecure conditions in Europe. MethodsWe conducted a cross-sectional seroprevalence study in the food distribution sites, emergency shelters and workers residences supported by MSF in Paris and Seine-Saint-Denis, to determine the extent of COVID-19 exposure as determined by SARS-CoV2 antibody seropositivity. The detection of SARS-COV2 antibodies in serum was performed at the Institut Pasteur of Paris using two LuLISA (Luciferase-Linked Immunosorbent Assay) assays and a Pseudo Neutralization Test. A questionnaire covering sociodemographic characteristics, living conditions, adherence to sanitary recommendations and symptom manifestations was also completed. We describe here the seroprevalence site by site and identify the risk factors for seropositivity using a multivariable logistic regression model with site random effects. We also investigated associations between seropositivity and symptoms eventually reported. FindingsOverall, 426/818 individuals tested positive in the 14 sites investigated. Seroprevalence varied significantly with the type of site (chi2 p<0.001). It was highest at 88.7% (95%CI 81.8-93.2) among individuals living in workers residences, followed by 50.5% (95%CI 46.3-54.7) in emergency shelters and 27.8 % (95%CI 20.8-35.7) among individuals recruited from the food distribution sites. Seroprevalence also varied significantly between sites of the same type. Among other risk factors, the odds for seropositivity were higher among individuals living in crowded sites (medium: adj. OR 2.7, 95%CI 1.5-5.1, p=0.001; high: adj. OR 3.4, 95%CI 1.7-6.9, p<0.001) compared with individuals from low crowding sites and among those who reported transit accommodation in a gymnasium before the lockdown (adj. OR 3.1, 95%CI 1.2-8.1, p=0.023). More than two-thirds of the seropositive individuals (68.3%; 95%CI 64.2-72.2) did not report any symptoms during the recall period. InterpretationThe results demonstrate rather high exposure to SARS-COV-2 with important variations between study sites. Living in crowded conditions was identified as the most important explanatory factor for differences in levels of exposure. This study describes the key factors which determine the risk of exposure and illustrates the importance of identifying populations at high risk of exposure in order to orient and adapt prevention and control strategies to their specific needs.
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It is currently unknown whether acquired immunity to common alpha- and beta-coronaviruses provides cross-protection against SARS-CoV-2. In this study, we found that certain patient sera and intravenous immunoglobulins (IVIG) collected prior to the COVID-19 outbreak were cross-reactive to SARS-CoV-2 full-length Spike, S2 domain, and Nucleocapsid. However, their presence did not translate into neutralizing activity against SARS-CoV-2 in vitro. Importantly, we detected serum IgG reactivity to common coronaviruses in the early sera of patients with severe COVID-19 before the appearance of anti-SARS-CoV-2 antibodies. Collectively, the results of our study indicate that pre-existing immunity to common coronaviruses does not confer cross-protection against SARS-CoV-2 in vivo.
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To develop a vaccine candidate against COVID-19, we generated a Lentiviral Vector (LV), eliciting neutralizing antibodies against the Spike glycoprotein of SARS-CoV-2. Systemic vaccination by this vector in mice, in which the expression of the SARS-CoV-2 receptor hACE2 has been induced by transduction of respiratory tract cells by an adenoviral vector, conferred only partial protection, despite an intense serum neutralizing activity. However, targeting the immune response to the respiratory tract through an intranasal boost with this LV resulted in > 3 log10 decrease in the lung viral loads and avoided local inflammation. Moreover, both integrative and non-integrative LV platforms displayed a strong vaccine efficacy and inhibited lung deleterious injury in golden hamsters, which are naturally permissive to SARS-CoV-2 replication and restitute the human COVID-19 physiopathology. Our results provide evidence of marked prophylactic effects of the LV-based vaccination against SARS-CoV-2 and designate the intranasal immunization as a powerful approach against COVID-19. HighlightsA lentiviral vector encoding for Spike predicts a promising COVID-19 vaccine Targeting the immune response to the upper respiratory tract is key to protection Intranasal vaccination induces protective mucosal immunity against SARS-CoV-2 Lung anti-Spike IgA responses correlate with protection and reduced inflammation
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BackgroundThe extent of SARS-CoV-2 transmission among pupils in primary schools and their families is unknown. MethodsBetween 28-30 April 2020, a retrospective cohort study was conducted among pupils, their parents and relatives, and staff of primary schools exposed to SARS-CoV-2 in February and March 2020 in a city north of Paris, France. Participants completed a questionnaire that covered sociodemographic information and history of recent symptoms. A blood sample was tested for the presence of anti-SARS-CoV-2 antibodies using a flow-cytometry-based assay. ResultsThe infection attack rate (IAR) was 45/510 (8.8%), 3/42 (7.1%), 1/28 (3.6%), 76/641 (11.9%) and 14/119 (11.8%) among primary school pupils, teachers, non-teaching staff, parents, and relatives, respectively (P = 0.29). Prior to school closure on February 14, three SARS-CoV-2 infected pupils attended three separate schools with no secondary cases in the following 14 days among pupils, teachers and non-teaching staff of the same schools. Familial clustering of cases was documented by the high proportion of antibodies among parents and relatives of infected pupils (36/59 = 61.0% and 4/9 = 44.4%, respectively). In children, disease manifestations were mild, and 24/58 (41.4%) of infected children were asymptomatic. InterpretationIn young children, SARS-CoV-2 infection was largely mild or asymptomatic and there was no evidence of onwards transmission from children in the school setting.
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A major dogma in immunology has it that the IgM antibody response precedes secondary memory responses built on the production of IgG, IgA and, occasionaly, IgE. Here, we measured acute humoral responses to SARS-CoV-2, including the frequency of antibody-secreting cells and the presence of specific, neutralizing, antibodies in serum and broncho-alveolar fluid of 145 patients with COVID-19. Surprisingly, early SARS-CoV-2-specific humoral responses were found to be typically dominated by antibodies of the IgA isotype. Peripheral expansion of IgA-plasmablasts with mucosal-homing potential was detected shortly after the onset of symptoms and peaked during the third week of the disease. While the specific antibody response included IgG, IgM and IgA, the latter contributed to a much larger extent to virus neutralization, as compared to IgG. However, specific IgA serum levels notably decrease after one month of evolution. These results represent a challenging observation given the present uncertainty as to which kind of humoral response would optimally protect against re-infection, and whether vaccine regimens should consider boosting a potent, although, at least in blood, fading IgA response. One sentence SummaryWhile early specific antibody response included IgG, IgM and IgA, the latter contributed to a much larger extent to virus neutralization.
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BackgroundThe serologic response of individuals with mild forms of SARS-CoV-2 infection is poorly characterized. MethodsHospital staff who had recovered from mild forms of PCR-confirmed SARS-CoV-2 infection were tested for anti-SARS-CoV-2 antibodies using two assays: a rapid immunodiagnostic test (99.4% specificity) and the S-Flow assay ([~]99% specificity).The neutralizing activity of the sera was tested with a pseudovirus-based assay. ResultsOf 162 hospital staff who participated in the investigation, 160 reported SARS-CoV-2 infection that had not required hospital admission and were included in these analyses. The median time from symptom onset to blood sample collection was 24 days (IQR: 21-28, range 13-39). The rapid immunodiagnostic test detected antibodies in 153 (95.6%) of the samples and the S-Flow assay in 159 (99.4%), failing to detect antibodies in one sample collected 18 days after symptom onset (the rapid test did not detect antibodies in that patient). Neutralizing antibodies (NAbs) were detected in 79%, 92% and 98% of samples collected 13-20, 21-27 and 28-41 days after symptom onset, respectively (P=0.02). ConclusionAntibodies against SARS-CoV-2 were detected in virtually all hospital staff sampled from 13 days after the onset of COVID-19 symptoms. This finding supports the use of serologic testing for the diagnosis of individuals who have recovered from SARS-CoV-2 infection. The neutralizing activity of the antibodies increased overtime. Future studies will help assess the persistence of the humoral response and its associated neutralization capacity in recovered patients.
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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.
