Antibodies are a principal determinant of immunity for most RNA viruses and have promise to reduce infection or disease during major epidemics. The novel coronavirus SARS-CoV-2 has caused a global ...pandemic with millions of infections and hundreds of thousands of deaths to date
. In response, we used a rapid antibody discovery platform to isolate hundreds of human monoclonal antibodies (mAbs) against the SARS-CoV-2 spike (S) protein. We stratify these mAbs into five major classes on the basis of their reactivity to subdomains of S protein as well as their cross-reactivity to SARS-CoV. Many of these mAbs inhibit infection of authentic SARS-CoV-2 virus, with most neutralizing mAbs recognizing the receptor-binding domain (RBD) of S. This work defines sites of vulnerability on SARS-CoV-2 S and demonstrates the speed and robustness of advanced antibody discovery platforms.
Most human monoclonal antibodies (mAbs) neutralizing SARS-CoV-2 recognize the spike (S) protein receptor-binding domain and block virus interactions with the cellular receptor angiotensin-converting ...enzyme 2. We describe a panel of human mAbs binding to diverse epitopes on the N-terminal domain (NTD) of S protein from SARS-CoV-2 convalescent donors and found a minority of these possessed neutralizing activity. Two mAbs (COV2-2676 and COV2-2489) inhibited infection of authentic SARS-CoV-2 and recombinant VSV/SARS-CoV-2 viruses. We mapped their binding epitopes by alanine-scanning mutagenesis and selection of functional SARS-CoV-2 S neutralization escape variants. Mechanistic studies showed that these antibodies neutralize in part by inhibiting a post-attachment step in the infection cycle. COV2-2676 and COV2-2489 offered protection either as prophylaxis or therapy, and Fc effector functions were required for optimal protection. Thus, natural infection induces a subset of potent NTD-specific mAbs that leverage neutralizing and Fc-mediated activities to protect against SARS-CoV-2 infection using multiple functional attributes.
Display omitted
•Natural SARS-2 infection induces a subset of potent N-terminal domain-specific mAbs•N-terminal domain reactive human monoclonal antibodies can neutralize live virus•COV2-2676 and COV2-2489 offer protection in a hACE2-transgenic mouse model•COV2-2676 and COV2-2489 Fc-effector functions are essential for optimal protection
Suryadevara et al. find human neutralizing antibodies to the spike protein N-terminal domain that arise from natural infection with SARS-CoV-2. These antibodies inhibit post-attachment steps of the viral cycle and initiate protective immune responses via the antibody Fc domain.
The coronavirus disease 2019 pandemic has made deployment of an effective vaccine a global health priority. We evaluated the protective activity of a chimpanzee adenovirus-vectored vaccine encoding a ...prefusion stabilized spike protein (ChAd-SARS-CoV-2-S) in challenge studies with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and mice expressing the human angiotensin-converting enzyme 2 receptor. Intramuscular dosing of ChAd-SARS-CoV-2-S induces robust systemic humoral and cell-mediated immune responses and protects against lung infection, inflammation, and pathology but does not confer sterilizing immunity, as evidenced by detection of viral RNA and induction of anti-nucleoprotein antibodies after SARS-CoV-2 challenge. In contrast, a single intranasal dose of ChAd-SARS-CoV-2-S induces high levels of neutralizing antibodies, promotes systemic and mucosal immunoglobulin A (IgA) and T cell responses, and almost entirely prevents SARS-CoV-2 infection in both the upper and lower respiratory tracts. Intranasal administration of ChAd-SARS-CoV-2-S is a candidate for preventing SARS-CoV-2 infection and transmission and curtailing pandemic spread.
Display omitted
•Chimpanzee adenoviral vaccines encoding stabilized S induce neutralizing Abs•Chimpanzee adenoviral vaccines protect against SARS-CoV-2 infection and pneumonia•Intranasal vaccine delivery generates robust mucosal B and T cell responses•Intranasal ChAd-SARS-CoV-2 prevents upper and lower respiratory tract infection
Intranasal or intramuscular immunization of ChAd-SARS-CoV-2, a chimpanzee adenoviral vaccine encoding stabilized spike protein, prevents SARS-CoV-2 lung infection and pneumonia in mice. In particular, intranasally delivered ChAd-SARS-CoV-2 uniquely prevents both upper and lower respiratory tract infections, potentially protecting against SARS-CoV-2 infection and transmission.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic with millions of human infections. One limitation to the evaluation of potential therapies and vaccines to inhibit ...SARS-CoV-2 infection and ameliorate disease is the lack of susceptible small animals in large numbers. Commercially available laboratory strains of mice are not readily infected by SARS-CoV-2 because of species-specific differences in their angiotensin-converting enzyme 2 (ACE2) receptors. Here, we transduced replication-defective adenoviruses encoding human ACE2 via intranasal administration into BALB/c mice and established receptor expression in lung tissues. hACE2-transduced mice were productively infected with SARS-CoV-2, and this resulted in high viral titers in the lung, lung pathology, and weight loss. Passive transfer of a neutralizing monoclonal antibody reduced viral burden in the lung and mitigated inflammation and weight loss. The development of an accessible mouse model of SARS-CoV-2 infection and pathogenesis will expedite the testing and deployment of therapeutics and vaccines.
Display omitted
•Adenovirus transduction of human ACE2 enables SARS-CoV-2 infection of BALB/c mice•High levels of viral RNA and infectious SARS-CoV-2 accumulate in lungs•Mice transduced with human ACE2 develop viral pneumonia after SARS-CoV-2 infection•Neutralizing mAbs protect from SARS-CoV-2-induced lung infection and inflammation
Laboratory mice transduced with adenoviruses encoding human ACE2 are permissive for SARS-CoV-2 and develop pneumonia. Passive transfer of a neutralizing monoclonal antibody reduces lung infection, inflammation, and disease.
Rapidly emerging SARS-CoV-2 variants jeopardize antibody-based countermeasures. Although cell culture experiments have demonstrated a loss of potency of several anti-spike neutralizing antibodies ...against variant strains of SARS-CoV-2
, the in vivo importance of these results remains uncertain. Here we report the in vitro and in vivo activity of a panel of monoclonal antibodies (mAbs), which correspond to many in advanced clinical development by Vir Biotechnology, AbbVie, AstraZeneca, Regeneron and Lilly, against SARS-CoV-2 variant viruses. Although some individual mAbs showed reduced or abrogated neutralizing activity in cell culture against B.1.351, B.1.1.28, B.1.617.1 and B.1.526 viruses with mutations at residue E484 of the spike protein, low prophylactic doses of mAb combinations protected against infection by many variants in K18-hACE2 transgenic mice, 129S2 immunocompetent mice and hamsters, without the emergence of resistance. Exceptions were LY-CoV555 monotherapy and LY-CoV555 and LY-CoV016 combination therapy, both of which lost all protective activity, and the combination of AbbVie 2B04 and 47D11, which showed a partial loss of activity. When administered after infection, higher doses of several mAb cocktails protected in vivo against viruses with a B.1.351 spike gene. Therefore, many-but not all-of the antibody products with Emergency Use Authorization should retain substantial efficacy against the prevailing variant strains of SARS-CoV-2.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused millions of human infections, and an effective vaccine is critical to mitigate coronavirus-induced disease 2019 (COVID-19). ...Previously, we developed a replication-competent vesicular stomatitis virus (VSV) expressing a modified form of the SARS-CoV-2 spike gene in place of the native glycoprotein gene (VSV-eGFP-SARS-CoV-2). Here, we show that vaccination with VSV-eGFP-SARS-CoV-2 generates neutralizing immune responses and protects mice from SARS-CoV-2. Immunization of mice with VSV-eGFP-SARS-CoV-2 elicits high antibody titers that neutralize SARS-CoV-2 and target the receptor binding domain that engages human angiotensin-converting enzyme-2 (ACE2). Upon challenge with a human isolate of SARS-CoV-2, mice that expressed human ACE2 and were immunized with VSV-eGFP-SARS-CoV-2 show profoundly reduced viral infection and inflammation in the lung, indicating protection against pneumonia. Passive transfer of sera from VSV-eGFP-SARS-CoV-2-immunized animals also protects naive mice from SARS-CoV-2 challenge. These data support development of VSV-SARS-CoV-2 as an attenuated, replication-competent vaccine against SARS-CoV-2.
Display omitted
•A replicating VSV-SARS-CoV-2 vaccine induces high-titer neutralizing antibodies•Infectious SARS-CoV-2 is undetectable in the lung of vaccinated mice post-challenge•SARS-CoV-2-induced lung inflammation and pathology is decreased in vaccinated mice•Transfer of vaccine-derived immune sera to naive mice protects against SARS-CoV-2
Case, Rothlauf et al. report the efficacy of a replicating VSV-based SARS-CoV-2 vaccine. Immunized hACE2-expressing mice challenged with SARS-CoV-2 are protected against lung infection, inflammation, and pneumonia. Neutralizing antibodies are a correlate of this protection, as passive transfer of vaccine-derived immune sera protects naive mice from subsequent SARS-CoV-2 challenge.
SARS-CoV-2 variants that attenuate antibody neutralization could jeopardize vaccine efficacy. We recently reported the protective activity of an intranasally administered spike protein-based ...chimpanzee adenovirus-vectored vaccine (ChAd-SARS-CoV-2-S) in animals, which has advanced to human trials. Here, we assessed its durability, dose response, and cross-protective activity in mice. A single intranasal dose of ChAd-SARS-CoV-2-S induced durably high neutralizing and Fc effector antibody responses in serum and S-specific IgG and IgA secreting long-lived plasma cells in the bone marrow. Protection against a historical SARS-CoV-2 strain was observed across a 100-fold vaccine dose range and over a 200-day period. At 6 weeks or 9 months after vaccination, serum antibodies neutralized SARS-CoV-2 strains with B.1.351, B.1.1.28, and B.1.617.1 spike proteins and conferred almost complete protection in the upper and lower respiratory tracts after challenge with variant viruses. Thus, in mice, intranasal immunization with ChAd-SARS-CoV-2-S provides durable protection against historical and emerging SARS-CoV-2 strains.
Display omitted
•Immunization with ChAd-SARS-CoV-2 S induces durable immunity•Intranasal ChAd-SARS-CoV-2 S induces inhibitory IgG and IgA Abs•Abs induced by intranasal ChAd-SARS-CoV-2 S have robust Fc effector functions•Intranasal ChAd-SARS-CoV-2 S confers cross-protection against variants of concern
Hassan et al. show that immunization with ChAd-SARS-CoV-2-S is durably immunogenic and protects against SARS-CoV-2 challenge in a dose-dependent manner. Many months after single-dose intranasal immunization, ChAd-SARS-CoV-2 confers protection against variants of concerns of SARS-CoV-2 in both the upper and lower respiratory tracts of mice.
Although mRNA vaccines encoding the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) prevent COVID-19, the emergence of new viral variants jeopardizes their efficacy. ...Here, we assessed the immunogenicity and protective activity of historical (mRNA-1273, designed for Wuhan-1 spike protein) or modified (mRNA-1273.351, designed for B.1.351 spike protein) Moderna mRNA vaccines in 129S2 and K18-hACE2 mice. Mice were immunized with either high-dose or low-dose formulations of the mRNA vaccines, where low-dose vaccination modeled suboptimal immune responses. Immunization with formulations at either dose induced neutralizing antibodies in serum against ancestral SARS-CoV-2 WA1/2020 and several virus variants, although serum titers were lower against the B.1.617.2 (Delta) virus. Protection against weight loss and lung pathology was observed with all high-dose vaccines against all viruses. However, low-dose formulations of the vaccines, which produced lower magnitude antibody and T cell responses, showed breakthrough lung infections with B.1.617.2 and development of pneumonia in K18-hACE2 mice. Thus, in individuals with reduced immunity after mRNA vaccination, breakthrough infection and disease may occur with some SARS-CoV-2 variants.
, a Gram-negative coccobacillus, has become a prevalent nosocomial health threat affecting the majority of hospitals both in the U.S. and around the globe. Microbial cell surface hydrophobicity (CSH) ...has previously been correlated with virulence, uptake by immune cells, and attachment to epithelial cells. A mutant strain of
(ΔtrxA) lacking the redox protein thioredoxin A was found to be more hydrophobic than its wild type (WT) and complemented counterparts, as measured by both Microbial Adhesion to Hydrocarbon (MATH) and salt aggregation. The hydrophobicity of the mutant could be abrogated through treatment with sodium cyanoborohydride (SCBH). This modulation correlated with reduction of disulfide bonds, as SCBH was able to reduce 5,5'-dithio-bis-2-nitrobenzoic acid and treatment with the known disulfide reducer, β-mercaptoethanol, also decreased ΔtrxA CSH. Additionally, the ΔtrxA mutant was more readily taken up than WT by J774 macrophages and this differential uptake could be abrogated though SCBH treatment. When partitioned into aqueous and hydrophobic phases, ΔtrxA recovered from the hydrophobic partition was phagocytosed more readily than from the aqueous phase further supporting the contribution of CSH to
uptake by phagocytes. A second Gram-negative bacterium,
, also showed the association of TrxA deficiency (FnΔtrxA) with increased hydrophobicity and uptake by J774 cells. We previously have demonstrated that modification of the type IV pilus system (T4P) was associated with the
ΔtrxA phenotype, and the
FnΔtrxA mutant also was found to have a marked T4P deficiency. Interestingly, a
mutant lacking pilT also showed increased hydrophobicity over FnWT. Collective evidence presented in this study suggests that Gram-negative bacterial thioredoxin mediates CSH through multiple mechanisms including disulfide-bond reduction and T4P modulation.
Acute Graft versus host disease (GVHD) remains the primary cause of morbidity and nonrelapse mortality after allogeneic hematopoietic cell transplantation (HCT). Despite the current standard ...prophylaxis, approximately 50% of patients will develop acute GVHD. the outcome of patients with steroid-refractory and/or severe GVHD is poor; hence, more effective prophylaxis reducing the risk of these scenarios is greatly needed. In recent years, considerable attention has been directed toward mesenchymal stromal cells (MSCs) as prophylaxis and therapy for acute GVHD. Even though the immunosuppressive and immunomodulatory properties of MSCs are enhanced by proinflammatory cytokines such as interferon γ (γMSCs), relatively less attention has been focused on these cells. Our laboratory has shown human γMSCs are effective in GVHD prophylaxis in a fully mismatched murine HCT/GVHD model. When infused IV on day +1 after HCT, γMSCs traffic to the spleen, liver, and gut-associated lymphoid tissue (GALT) which is essential for effective prophylaxis. This may be a key element of the mechanism of prophylaxis as the gut and GALT are thought to be paramount for the development of GVHD. We now sought to understand the mechanism(s) of γMSC suppression of alloreactive T cells suppressive activity in this specific clinical and biological setting. In vivo, control mice transplanted with a fully MHC mismatched graft developed GVHD in 6-8 days (0% GVHD-free survival) while human γMSCs administration at day +1 completely prevented GVHD in the first 17 days (100% GVHD survival) but the animals developed acute GVHD in 21-28 days. Hence, a single dose of γMSCs is 100% effective early but is insufficient for overall GVHD prevention. The mechanism is highly effective but of relatively short duration, and seemingly reversible. In vitro, the reversible T cell suppressive activity was entirely contact-independent, while cell-to-cell contact could induce T cell death. Moreover, γMSC IDO1 expression, alone, was sufficient to suppress T cells. After 24 hours of T cell activation or γMSC suppression in vitro, we found the T cells were arrested in G0 inconsistent with most cell cycle regulation which would arrest cells in late G1 at the so-called restriction point. In scRNA Seq, we identified 16,478 expressed genes in both groups but only 98 genes were significantly differentially expressed (≥20% FC, Padj ≤ 10 -4). ROS response pathways were significantly enriched in the suppressed cells suggesting oxidative stress. Paradoxically, flow cytometric analysis of CM-H2DCFDA stained T cells revealed less ROS in suppressed cells compared to controls. Additionally, total NAD is elevated and the NAD/NADH ratio was 10-fold less in suppressed T cells compared to activated cells indicating an excess of reducing equivalents in the suppressed cells. Collectively these data define reductive stress. On this note, the upregulation of ROS response genes is also consistent with reductive stress. IDO1 activity seems to have induced reductive stress. The IDO1 catalytic activity converts tryptophan (Trp) to kynurenine (Kyn). We then found the addition of Trp (10 µM) to γMSC CM abolished suppression; supplementing fresh media with 15-fold excess Kyn had no effect. Moreover, RPMI without Trp and with dialyzed FBS also conferred suppression to activated T cells. These data suggest that among the many metabolites consumed or generated in γMSC CM, the lack of Trp induces T cell suppression. Trp supplementation of γMSC CM showed the expected level of ROS in activated T cells suggesting the Trp deficiency caused the reduced ROS. Supplementing γMSC CM with a graded concentration of hydrogen peroxide, we found that 100 nM hydrogen peroxide was not toxic to control cells, had ROS similar to activated T cells, and abolished suppression indicating the reduced ROS directly caused suppression. Kyn, nutritional deficiency, or an integrated stress response did not seem to play a measurable role in this setting. Collectively, our data suggest that IDO1-mediated Trp deficiency perturbs redox homeostasis (reductive stress) which suppresses activated T cells. We postulate that once redox homeostasis is restored, T cell activation proceeds. Future efforts will be directed toward identifying the link between Trp deficiency and redox homeostasis in this setting, and the genomic changes that directly mediate the lack of T cell proliferation.
PDF
