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1.
Preprint in English | bioRxiv | ID: ppbiorxiv-517073

ABSTRACT

Humans display vast clinical variability upon SARS-CoV-2 infection1-3, partly due to genetic and immunological factors4. However, the magnitude of population differences in immune responses to SARS-CoV-2 and the mechanisms underlying such variation remain unknown. Here we report single-cell RNA-sequencing data for peripheral blood mononuclear cells from 222 healthy donors of various ancestries stimulated with SARS-CoV-2 or influenza A virus. We show that SARS-CoV-2 induces a weaker, but more heterogeneous interferon-stimulated gene activity than influenza A virus, and a unique pro-inflammatory signature in myeloid cells. We observe marked population differences in transcriptional responses to viral exposure that reflect environmentally induced cellular heterogeneity, as illustrated by higher rates of cytomegalovirus infection, affecting lymphoid cells, in African-descent individuals. Expression quantitative trait loci and mediation analyses reveal a broad effect of cell proportions on population differences in immune responses, with genetic variants having a narrower but stronger effect on specific loci. Additionally, natural selection has increased immune response differentiation across populations, particularly for variants associated with SARS-CoV-2 responses in East Asians. We document the cellular and molecular mechanisms through which Neanderthal introgression has altered immune functions, such as its impact on the myeloid response in Europeans. Finally, colocalization analyses reveal an overlap between the genetic architecture of immune responses to SARS-CoV-2 and COVID-19 severity. Collectively, these findings suggest that adaptive evolution targeting immunity has also contributed to current disparities in COVID-19 risk.

2.
Preprint in English | bioRxiv | ID: ppbiorxiv-516888

ABSTRACT

Convergent evolution of SARS-CoV-2 Omicron BA.2, BA.4 and BA.5 lineages has led to the emergence of several new subvariants, including BA.2.75.2, BA.4.6. and BQ.1.1. The subvariants BA.2.75.2 and BQ.1.1 are expected to become predominant in many countries in November 2022. They carry an additional and often redundant set of mutations in the spike, likely responsible for increased transmissibility and immune evasion. Here, we established a viral amplification procedure to easily isolate Omicron strains. We examined their sensitivity to 6 therapeutic monoclonal antibodies (mAbs) and to 72 sera from Pfizer BNT162b2-vaccinated individuals, with or without BA.1/BA.2 or BA.5 breakthrough infection. Ronapreve (Casirivimab and Imdevimab) and Evusheld (Cilgavimab and Tixagevimab) lost any antiviral efficacy against BA.2.75.2 and BQ.1.1, whereas Xevudy (Sotrovimab) remained weakly active. BQ.1.1 was also resistant to Bebtelovimab. Neutralizing titers in triply vaccinated individuals were low to undetectable against BQ.1.1 and BA.2.75.2, 4 months after boosting. A BA.1/BA.2 breakthrough infection increased these titers, which remained about 18-fold lower against BA.2.75.2 and BQ.1.1, than against BA.1. Reciprocally, a BA.5 breakthrough infection increased more efficiently neutralization against BA.5 and BQ.1.1 than against BA.2.75.2. Thus, the evolution trajectory of novel Omicron subvariants facilitated their spread in immunized populations and raises concerns about the efficacy of most currently available mAbs.

3.
Preprint in English | medRxiv | ID: ppmedrxiv-22278699

ABSTRACT

The emergence of novel Omicron lineages, such as BA.5, may impact the therapeutic efficacy of anti-SARS-CoV-2 neutralizing monoclonal antibodies (mAbs). Here, we evaluated the neutralization and ADCC activity of 6 therapeutic mAbs against Delta, BA.2, BA.4 and BA.5 isolates. The Omicron sub-variants escaped most of the antibodies but remained sensitive to Bebtelovimab and Cilgavimab. Consistent with their shared spike sequence, BA.4 and BA.5 displayed identical neutralization profiles. Sotrovimab was the most efficient at eliciting ADCC. We also analyzed 121 sera from 40 immunocompromised individuals up to 6 months after infusion of 1200 mg of Ronapreve (Imdevimab + Casirivimab), and 300 or 600 mg of Evusheld (Cilgavimab + Tixagevimab). Sera from Ronapreve-treated individuals did not neutralize Omicron subvariants. Evusheld-treated individuals neutralized BA.2 and BA.5, but titers were reduced by 41- and 130-fold, respectively, compared to Delta. A longitudinal evaluation of sera from Evusheld-treated patients revealed a slow decay of mAb levels and neutralization. The decline was more rapid against BA.5. Our data shed light on the antiviral activities of therapeutic mAbs and the duration of effectiveness of Evusheld pre-exposure prophylaxis.

4.
Preprint in English | bioRxiv | ID: ppbiorxiv-501370

ABSTRACT

COVID-19 is a respiratory disease affecting multiple organs including the central nervous system (CNS), with a characteristic loss of smell and taste. Although frequently reported, the neurological symptoms remain enigmatic. There is no consensus on the extent of CNS infection. Here, we derived human induced pluripotent stem cells (hiPSC) into neural progenitor cells (NPCs) and cortical excitatory neurons to study their permissiveness to SARS-CoV-2 infection. Flow cytometry and western blot analysis indicated that NPCs and neurons do not express detectable levels of the SARS-CoV-2 receptor ACE2. We thus generated cells expressing ACE2 by lentiviral transduction to analyze in a controlled manner the properties of SARS-CoV-2 infection relative to ACE2 expression. Sensitivity of parental and ACE2 expressing cells was assessed with GFP- or luciferase-carrying pseudoviruses and with authentic SARS-CoV-2 Wuhan, D614G, Alpha or Delta variants. SARS-CoV-2 replication was assessed by microscopy, RT-qPCR and infectivity assays. Pseudoviruses infected only cells overexpressing ACE2. Neurons and NPCs were unable to efficiently replicate SARS-CoV-2, whereas ACE2 overexpressing neurons were highly sensitive to productive infection. Altogether, our results indicate that primary NPCs and cortical neurons remain poorly permissive to SARS-CoV-2 across the variants spectrum, in the absence of ACE2 expression.

5.
Preprint in English | medRxiv | ID: ppmedrxiv-22277885

ABSTRACT

Since early 2022, Omicron BA.1 has been eclipsed by BA.2, which was in turn outcompeted by BA.5, that displays enhanced antibody escape properties. Here, we evaluated the duration of the neutralizing antibody (Nab) response, up to 16 months after Pfizer BNT162b2 vaccination, in individuals with or without BA.1/BA.2 breakthrough infection. We measured neutralization of the ancestral D614G lineage, Delta and Omicron BA.1, BA.2, BA.5 variants in 291 sera and 35 nasal swabs from 27 individuals. Upon vaccination, serum Nab titers were reduced by 10-, 15-and 25-fold for BA.1, BA.2 and BA.5, respectively, compared with D614G. The duration of neutralization was markedly shortened, from an estimated period of 11.5 months post-boost with D614G to 5.5 months with BA.5. After breakthrough, we observed a sharp increase of Nabs against Omicron subvariants, followed by a plateau and a slow decline after 4-5 months. In nasal swabs, infection, but not vaccination, triggered a strong IgA response and a detectable Omicron neutralizing activity. Thus, BA.5 spread is partly due to abbreviated vaccine efficacy, particularly in individuals who were not infected with previous Omicron variants.

6.
Preprint in English | bioRxiv | ID: ppbiorxiv-492832

ABSTRACT

SARS-CoV-2 remained genetically stable during the first three months of the pandemic, before acquiring a D614G spike mutation that rapidly spread worldwide, and then generating successive waves of viral variants with increasingly high transmissibility. We set out to evaluate possible epistatic interactions between the early occurring D614G mutation and the more recently emerged cleavage site mutations present in spike of the Alpha, Delta, and Omicron variants of concern. The P681H/R mutations at the S1/S2 cleavage site increased spike processing and fusogenicity but limited its incorporation into pseudoviruses. In addition, the higher cleavage rate led to higher shedding of the spike S1 subunit, resulting in a lower infectivity of the P681H/R-carrying pseudoviruses compared to those expressing the Wuhan wild-type spike. The D614G mutation increased spike expression at the cell surface and limited S1 shedding from pseudovirions. As a consequence, the D614G mutation preferentially increased the infectivity of P681H/R-carrying pseudoviruses. This enhancement was more marked in cells where the endosomal route predominated, suggesting that more stable spikes could better withstand the endosomal environment. Taken together, these findings suggest that the D614G mutation stabilized S1/S2 association and enabled the selection of mutations that increased S1/S2 cleavage, leading to the emergence of SARS-CoV-2 variants expressing highly fusogenic spikes. AUTHOR SUMMARYThe successive emergence of SARS-CoV-2 variants is fueling the COVID pandemic, thus causing a major and persistent public health issue. The parameters involved in the emergence of variants with higher pathogenic potential remain incompletely understood. The first SARS-CoV-2 variant that spread worldwide in early 2020 carried a D614G mutation in the viral spike, making this protein more stable in its cleaved form at the surface of virions, and resulting in viral particles with higher infectious capacity. The Alpha and the Delta variants that spread in late 2020 and early 2021, respectively, proved increasingly transmissible and pathogenic when compared to the original SARS-CoV-2 strain. Interestingly, Alpha and Delta both carried mutations in a spike cleavage site that needs to be processed by cellular proteases prior to viral entry. The cleavage site mutations P681H/R made the Alpha and Delta spikes more efficient at viral fusion, by generating a higher fraction of cleaved spikes subunits S1 and S2. We show here that the early D614G mutation and the late P681H/R mutations act synergistically to increase the fusion capacity of SARS-CoV-2 variants. Specifically, viruses with increased spike cleavage due to P681H/R were even more dependent on the stabilizing effect of D614G mutation, which limited the shedding of cleaved S1 subunits from viral particles. These findings suggest that the worldwide spread of the D614G mutation was a prerequisite to the emergence of more pathogenic SARS-CoV-2 variants with highly fusogenic spikes.

7.
Preprint in English | bioRxiv | ID: ppbiorxiv-486719

ABSTRACT

Memory B-cell and antibody responses to the SARS-CoV-2 spike protein contribute to long-term immune protection against severe COVID-19, which can also be prevented by antibody-based interventions. Here, wide SARS-CoV-2 immunoprofiling in COVID-19 convalescents combining serological, cellular and monoclonal antibody explorations, revealed humoral immunity coordination. Detailed characterization of a hundred SARS-CoV-2 spike memory B-cell monoclonal antibodies uncovered diversity in their repertoire and antiviral functions. The latter were influenced by the targeted spike region with strong Fc-dependent effectors to the S2 subunit and potent neutralizers to the receptor binding domain. Amongst those, Cv2.1169 and Cv2.3194 antibodies cross-neutralized SARS-CoV-2 variants of concern including Omicron BA.1 and BA.2. Cv2.1169, isolated from a mucosa-derived IgA memory B cell, demonstrated potency boost as IgA dimers and therapeutic efficacy as IgG antibodies in animal models. Structural data provided mechanistic clues to Cv2.1169 potency and breadth. Thus, potent broadly neutralizing IgA antibodies elicited in mucosal tissues can stem SARS-CoV-2 infection, and Cv2.1169 and Cv2.3194 are prime candidates for COVID-19 prevention and treatment.

8.
Preprint in English | medRxiv | ID: ppmedrxiv-22273175

ABSTRACT

BackgroundThe protective immunity against Omicron following a BNT162b2 Pfizer booster dose among elderly is not well characterized. MethodsThirty-eight residents from three nursing homes were recruited for the study. Antibodies targeting the Spike protein of SARS-CoV-2 were measured with the S-Flow assay. Neutralizing activities in sera were measured as effective dilution 50% (ED50) with the S-Fuse assay using authentic isolates of Delta and Omicron. ResultsAmong the 38 elderly included in the study, with median (inter-quartile range, IQR) age of 88 (81-92) years, 30 (78.9%) had been previously infected. The ED50 of neutralization were lower against Omicron than Delta, and higher among convalescent compared to naive residents. During an Omicron epidemic affecting two of the three nursing homes in December 2021-January 2022, 75% (6/8) of naive residents got infected, compared to 25% (6/24) of convalescents (P=0.03). Antibody levels to Spike and ED50 of neutralization against Omicron after the BNT162b2 booster dose were lower in those with breakthrough infection (n=12) compared to those without (n=20): median of 1256 vs 2523 BAU/mL (P=0.02) and median ED50 of 234 vs 1298 (P=0.0004), respectively. ConclusionThis study confirmed the importance of receiving at least three antigenic exposures to the SARS-CoV-2 Spike protein for achieving satisfactory neutralizing antibody levels. In this population, protection against Omicron infection was increased in individuals who had been previously infected in addition to the three vaccine doses. Thus, a fourth antigenic exposure may be useful in the elderly population to prevent infection with Omicron, a variant known for its high escape immunity properties.

9.
Preprint in English | medRxiv | ID: ppmedrxiv-22272066

ABSTRACT

The SARS-CoV-2 Omicron BA.1 variant has been supplanted in many countries by the BA.2 sub-lineage. BA.2 differs from BA.1 by about 21 mutations in its spike. Human anti-spike monoclonal antibodies (mAbs) are used for prevention or treatment of COVID-19. However, the capacity of therapeutic mAbs to neutralize BA.1 and BA.2 remains poorly characterized. Here, we first compared the sensitivity of BA.1 and BA.2 to neutralization by 9 therapeutic mAbs. In contrast to BA.1, BA.2 was sensitive to Cilgavimab, partly inhibited by Imdevimab and resistant to Adintrevimab and Sotrovimab. Two combinations of mAbs, Ronapreve (Casirivimab + Imdevimab) and Evusheld (Cilgavimab + Tixagevimab), are indicated as a pre-exposure prophylaxis in immunocompromised persons at risk of severe disease. We analyzed sera from 29 such individuals, up to one month after administration of Ronapreve and/or Evusheld. After treatment, all individuals displayed elevated antibody levels in their sera and neutralized Delta with high titers. Ronapreve recipients did not neutralize BA.1 and weakly impaired BA.2. With Evusheld, neutralization of BA.1 and BA.2 was detected in 19 and 29 out of 29 patients, respectively. As compared to Delta, titers were more severely decreased against BA.1 (344-fold) than BA.2 (9-fold). We further report 4 breakthrough Omicron infections among the 29 participants. Therefore, BA.1 and BA.2 exhibit noticeable differences in their sensitivity to therapeutic mAbs. Anti-Omicron activity of Ronapreve, and to a lesser extent that of Evusheld, is reduced in patients sera, a phenomenon associated with decreased clinical efficacy.

10.
Preprint in English | bioRxiv | ID: ppbiorxiv-475248

ABSTRACT

SARS-CoV-2 lineages are continuously evolving. As of December 2021, the AY.4.2 Delta sub-lineage represented 20 % of sequenced strains in UK and has been detected in dozens of countries. It has since then been supplanted by the Omicron variant. AY.4.2 displays three additional mutations (T95I, Y145H and A222V) in the N-terminal domain (NTD) of the spike when compared to the original Delta variant (B.1.617.2) and remains poorly characterized. Here, we analyzed the fusogenicity of the AY.4.2 spike and the sensitivity of an authentic AY.4.2 isolate to neutralizing antibodies. The AY.4.2 spike exhibited similar fusogenicity and binding to ACE2 than Delta. The sensitivity of infectious AY.4.2 to a panel of monoclonal neutralizing antibodies was similar to Delta, except for the anti-RBD Imdevimab, which showed incomplete neutralization. Sensitivity of AY.4.2 to sera from individuals having received two or three doses of Pfizer or two doses of AstraZeneca vaccines was reduced by 1.7 to 2.1 fold, when compared to Delta. Our results suggest that mutations in the NTD remotely impair the efficacy of anti-RBD antibodies. The temporary spread of AY.4.2 was not associated with major changes in spike function but rather to a partially reduced neutralization sensitivity.

11.
Preprint in English | bioRxiv | ID: ppbiorxiv-472630

ABSTRACT

The SARS-CoV-2 Omicron variant was first identified in November 2021 in Botswana and South Africa1,2. It has in the meantime spread to many countries and is expected to rapidly become dominant worldwide. The lineage is characterized by the presence of about 32 mutations in the Spike, located mostly in the N-terminal domain (NTD) and the receptor binding domain (RBD), which may enhance viral fitness and allow antibody evasion. Here, we isolated an infectious Omicron virus in Belgium, from a traveller returning from Egypt. We examined its sensitivity to 9 monoclonal antibodies (mAbs) clinically approved or in development3, and to antibodies present in 90 sera from COVID-19 vaccine recipients or convalescent individuals. Omicron was totally or partially resistant to neutralization by all mAbs tested. Sera from Pfizer or AstraZeneca vaccine recipients, sampled 5 months after complete vaccination, barely inhibited Omicron. Sera from COVID-19 convalescent patients collected 6 or 12 months post symptoms displayed low or no neutralizing activity against Omicron. Administration of a booster Pfizer dose as well as vaccination of previously infected individuals generated an anti-Omicron neutralizing response, with titers 5 to 31 fold lower against Omicron than against Delta. Thus, Omicron escapes most therapeutic monoclonal antibodies and to a large extent vaccine-elicited antibodies.

12.
Preprint in English | medRxiv | ID: ppmedrxiv-21266704

ABSTRACT

In immunocompetent subjects, the effectiveness of SARS-CoV-2 vaccines against the delta variant appears three- to five-fold lower than that observed against the alpha variant. Additionally, three doses of SARS-CoV-2 mRNA-based vaccines might be unable to elicit a sufficient immune response against any variant in immunocompromised kidney transplant recipients. This study describes the kinetics of the neutralizing antibody (NAbs) response against the delta strain before and after a fourth dose of a mRNA vaccine in 67 kidney transplant recipients who had experienced a weak antibody response after three doses. While only 16% of patients harbored NAbs against the delta strain prior to the fourth injection - this percentage raised to 66% afterwards. We also found that, after the fourth dose, the NAbs titer increased significantly (p=0.0001) from <7.5 (IQR : <7.5-15.1) to 47.1 (IQR <7.5-284.2). Collectively, our data indicate that a fourth dose of the mRNA-1273 vaccine in kidney transplant recipients with a weak antibody response after three previous doses improves serum neutralization against the delta variant.

13.
Preprint in English | bioRxiv | ID: ppbiorxiv-466418

ABSTRACT

The definition of correlates of protection is critical for the development of next generation SARS-CoV-2 vaccine platforms. Here, we propose a new framework for identifying mechanistic correlates of protection based on mathematical modelling of viral dynamics and data mining of immunological markers. The application to three different studies in non-human primates evaluating SARS-CoV-2 vaccines based on CD40-targeting, two-component spike nanoparticle and mRNA 1273 identifies and quantifies two main mechanisms that are a decrease of rate of cell infection and an increase in clearance of infected cells. Inhibition of RBD binding to ACE2 appears to be a robust mechanistic correlate of protection across the three vaccine platforms although not capturing the whole biological vaccine effect. The model shows that RBD/ACE2 binding inhibition represents a strong mechanism of protection which required significant reduction in blocking potency to effectively compromise the control of viral replication. One Sentence SummaryA framework for modelling the immune control of viral dynamics is applied to quantify the effect of several SARS-CoV-2 vaccine platforms and to define mechanistic correlates of protection.

14.
Preprint in English | bioRxiv | ID: ppbiorxiv-462234

ABSTRACT

As the coronavirus disease 2019 (COVID-19) pandemic continues, there is a strong need for highly potent monoclonal antibodies (mAbs) that are resistant against severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) variants of concern (VoCs). Here, we evaluate the potency of a previously described mAb J08 against these variants using cell-based assays and delve into the molecular details of the binding interaction using cryo-EM. We show that mAb J08 has low nanomolar affinity against VoCs, binds high on the receptor binding domain (RBD) ridge and is therefore unaffected by most mutations, and can bind in the RBD-up and -down conformations. These findings further validate the phase II/III human clinical trial underway using mAb J08 as a monoclonal therapy. One Sentence SummaryPotent neutralizing monoclonal antibody J08 binds SARS-CoV-2 spike independent of known escape mutations.

15.
Preprint in English | medRxiv | ID: ppmedrxiv-21262027

ABSTRACT

Serological tests are important for understanding the physiopathology and following the evolution of the Covid-19 pandemic. Assays based on flow cytometry (FACS) of tissue culture cells expressing the spike (S) protein of SARS-CoV-2 have repeatedly proven to perform slightly better than the plate-based assays ELISA and CLIA (chemiluminescent immuno-assay), and markedly better than lateral flow immuno-assays (LFIA). Here, we describe an optimized and very simple FACS assay based on staining a mix of two Jurkat cell lines, expressing either high levels of the S protein (Jurkat-S) or a fluorescent protein (Jurkat-R expressing m-Cherry, or Jurkat-G, expressing GFP, which serve as an internal negative control). We show that the Jurkat-S&R-flow test has a much broader dynamic range than a commercial ELISA test and performs at least as well in terms of sensitivity and specificity. Also, it is more sensitive and quantitative than the hemagglutination-based test HAT, which we described recently. The Jurkat-flow test requires only a few microliters of blood; thus, it can be used to quantify various Ig isotypes in capillary blood collected from a finger prick. It can be used also to evaluate serological responses in mice, hamsters, cats and dogs. Whilst the Jurkat-flow test is ill-suited and not intended for clinical use, it offers a very attractive solution for laboratories with access to tissue culture and flow cytometry who want to monitor serological responses in humans or in animals, and how these relate to susceptibility to infection, or re-infection, by the virus, and to protection against Covid-19. NoteThis manuscript has been refereed by Review Commons, and modified thanks to the comments and suggestions from two referees. Those comments, and our replies, are provided at the end of the manuscripts pdf, and can also be accessed by clicking on the box with a little green number found just above the "Abstract " tab in the medRXiv window.

16.
Preprint in English | bioRxiv | ID: ppbiorxiv-458516

ABSTRACT

Mast cells are key actors of innate immunity and Th2 adaptive immune response which counterbalance Th1 response, critical for anti-viral immunity. Clonal Mast Cells Activation Disorders (cMCADs) such as mastocytosis and clonal mast cells activation syndrome are characterized by an abnormal mast cells accumulation and/or activation. No data have been published on the anti-viral immune response of patients with cMCADs. The aims of the study were to collected, in a comprehensive way, outcomes of cMCADs patients who experienced a biologically-proven COVID-19 and to characterize both anti-endemic coronaviruses and specific anti-SARS-CoV-2 immune responses in these patients. Clinical follow-up and outcome data were collected prospectively for one year within the French rare disease network CEREMAST encompassing patients from all over the country. Anti-SARS-CoV-2 and anti-endemic coronaviruses specific T-cells were assessed with an enzyme-linked immunospot assay (EliSpot) and anti-SARS-CoV-2 humoral response with dosage of circulating levels of specific IgG, IgA and neutralizing antibodies. Overall, 32 cMCADs patients were identified. None of them required non-invasive or mechanical ventilation; two patients were hospitalized to receive oxygen and steroid therapy. In 21 patients, a characterization of the SARS-CoV-2-specific immune response has been performed. A majority of patients showed a high proportion of circulating SARS-CoV-2-specific interferon (IFN)-{gamma} producing T-cells and high levels of anti-Spike IgG antibodies with neutralizing activity. In addition, no defects in anti-endemic coronaviruses responses were found in patients with cMCADs compared to non-cMCADs controls. Patients with cMCADs frequently showed a spontaneous IFN-{gamma} T-cell production in absence of any stimulation that correlated with circulating basal tryptase levels, a marker of mast cells burden. These findings underscore that patients with cMCADs might be not at risk of severe COVID-19 and the spontaneous IFN-{gamma} production might explain this observation. Author SummaryMast cells are immune cells involved in many biological processes including the anti-microbial response. However, previous studies suggest that mast cells may have a detrimental role in the response against viruses such as SARS-CoV-2, responsible for COVID-19. When a mutation occurs in mast cells, it can lead to a group of diseases called clonal mast cells activation disorders (cMCADs), characterized by deregulated activation of these cells. Hence, patients with cMCADs might be more susceptible to severe COVID-19 than general population. We therefore conducted a 1-year study in France to collect data from all cMCADs patients included in the CEREMAST rare disease French network and who experienced COVID-19. Interestingly, we did not find any severe COVID-19 (i.e. requiring non-invasive or mechanical ventilation) in spite of well-known risk factors for severe COVID-19 in a part of cMCADs patients. We then have studied the immune response against SARS-CoV-2 and other endemic coronaviruses in these patients. We did not observe any abnormalities in the immune response either at the level of T and B lymphocytes. These findings underscore that these patients might not be at risk of severe COVID-19 as one might have feared.

17.
Preprint in English | bioRxiv | ID: ppbiorxiv-454991

ABSTRACT

A challenge for the development of host-targeted anti-infectives against a large spectrum of AB-like toxin-producing bacteria encompasses the identification of chemical compounds corrupting toxin transport through both endolysosomal and retrograde pathways. Here, we performed a high-throughput screening of small chemical compounds blocking active Rac1 proteasomal degradation triggered by the Cytotoxic Necrotizing Factor-1 (CNF1) toxin, followed by orthogonal screens against two AB toxins hijacking defined endolysosomal (Diphtheria toxin) or retrograde (Shiga-like toxin 1) pathways to intoxicate cells. This led to the identification of the molecule N-(3,3-diphenylpropyl)-1-propyl-4-piperidinamine, referred to as C910. This compound induces the swelling of EEA1-positive early endosomes, in absence of PIKfyve kinase inhibition, and disturbs the trafficking of CNF1 and the B-subunit of Shiga toxin along the endolysosomal or retrograde pathways, respectively. Together, we show that C910 protects cells against 8 bacterial AB toxins including large clostridial glucosylating toxins from Clostridium difficile. Of interest, C910 also reduced viral infection in vitro including influenza A virus subtype H1N1 and SARS-CoV-2. Moreover, parenteral administration of C910 to the mice resulted in its accumulation in lung tissues and reduced lethal influenza infection.

18.
Preprint in English | bioRxiv | ID: ppbiorxiv-455901

ABSTRACT

Assessing the duration of humoral and cellular immunity remains key to overcome the current SARS-CoV-2 pandemic, especially in understudied populations in least developed countries. Sixty-four Cambodian individuals with laboratory-confirmed infection with asymptomatic or mild/moderate clinical presentation were evaluated for humoral immune response to the viral spike protein and antibody effector functions during acute phase of infection and at 6-9 months follow-up. Antigen-specific B cells, CD4+ and CD8+ T cells were characterized, and T cells were interrogated for functionality at late convalescence. Anti-spike (S) antibody titers decreased over time, but effector functions mediated by S-specific antibodies remained stable. S- and nucleocapsid (N)-specific B cells could be detected in late convalescence in the activated memory B cell compartment and are mostly IgG+. CD4+ and CD8+ T cell immunity was maintained to S and membrane (M) protein. Asymptomatic infection resulted in decreased ADCC and frequency of SARS-CoV-2-specific CD4+ T cells at late convalescence. Whereas anti-S antibodies correlated with S-specific B cells, there was no correlation between T cell response and humoral immunity. Hence, all aspects of a protective immune response are maintained up to nine months after SARS-CoV-2 infection in the absence of re-infection. One sentence summaryFunctional immune memory to SARS-CoV-2, consisting of polyfunctional antibodies, memory B cells and memory T cells are maintained up to nine months in a South-East Asian cohort in the absence of re-infection.

19.
Preprint in English | medRxiv | ID: ppmedrxiv-21261766

ABSTRACT

BackgroundThe emergence of strains of SARS-CoV-2 exhibiting increase viral fitness and immune escape potential, such as the Delta variant (B.1.617.2), raises concerns in immunocompromised patients. To what extent Delta evades vaccine-induced immunity in immunocompromised individuals with systemic inflammatory diseases remains unclear. MethodsWe conducted a prospective study in patients with systemic inflammatory diseases (cases) and controls receiving two doses of BNT162b2. Primary end points were anti-spike antibodies levels and cross-neutralization of Alpha and Delta variants after BNT162b2 vaccine. Secondary end points were T-cell responses, breakthrough infections and safety. ResultsSixty-four cases and 21 controls not previously infected with SARS-CoV-2 were analyzed. Kinetics of anti-spike IgG and IgA after BNT162b2 vaccine showed lower and delayed induction in cases, more pronounced with rituximab. Administration of two doses of BNT162b2 generated a neutralizing response against Alpha and Delta in 100% of controls, while sera from only one of rituximab-treated patients neutralized Alpha (5%) and none Delta. Other therapeutic regimens induced a partial neutralizing activity against Alpha, even lower against Delta. All controls and cases except those treated with methotrexate mounted a SARS-CoV-2 specific T-cell response. Methotrexate abrogated T-cell responses after one dose and dramatically impaired T-cell responses after 2 doses of BNT162b2. ConclusionsRituximab and methotrexate differentially impact the immunogenicity of BNT162b2, by impairing B-cell and T-cell responses, respectively. Delta fully escapes the humoral response of individuals treated with rituximab. These findings support efforts to improve BNT162b2 immunogenicity in immunocompromised individuals (Funded by the Fonds IMMUNOV; ClinicalTrials.gov number, NCT04870411).

20.
Preprint in English | medRxiv | ID: ppmedrxiv-21261769

ABSTRACT

Multiple myeloma (MM) patients are at risk of fatal outcome after SARS-CoV-2 infection. Preliminary data suggest that MM patients have an impaired response to vaccination. This prospective study analyzed the humoral and cellular immune responses to two doses of BNT162b2 in 72 MM patients, including 48 receiving anti-CD38 immunotherapy. Results evidenced that MM patients display lower levels of SARS-CoV-2 specific IgG and IgA antibodies and decreased neutralization of alpha and delta variants when compared to healthy controls. They also showed decreased numbers of circulating IFN{gamma}-producing Spike SARS-CoV-2 specific T lymphocytes. This defective immune response was particularly marked in patients receiving anti-CD38 immunotherapy. Furthermore, a retrospective investigation of MM patients among COVID-19-related death in the Paris area suggested a limited efficacy of BNT162b2 in patients treated with anti-CD38. Overall, these results show a decreased immunogenicity of BNT162b2 in MM patients and stress the need for novel strategies to improve SARS-CoV-2 prophylaxis in immunocompromised individuals.

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