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1.
Eur J Immunol ; 2021 Aug 06.
Article in English | MEDLINE | ID: covidwho-1589126

ABSTRACT

TRIANNI mice carry an entire set of human immunoglobulin V region gene segments and are a powerful tool to rapidly isolate human monoclonal antibodies. After immunizing these mice with DNA encoding the spike protein of SARS-CoV-2 and boosting with spike protein, we identified 29 hybridoma antibodies that reacted with the SARS-CoV-2 spike protein. Nine antibodies neutralize SARS-CoV-2 infection at IC50 values in the subnanomolar range. ELISA-binding studies and DNA sequence analyses revealed one cluster of three clonally related neutralizing antibodies that target the receptor-binding domain and compete with the cellular receptor hACE2. A second cluster of six clonally related neutralizing antibodies bind to the N-terminal domain of the spike protein without competing with the binding of hACE2 or cluster 1 antibodies. SARS-CoV-2 mutants selected for resistance to an antibody from one cluster are still neutralized by an antibody from the other cluster. Antibodies from both clusters markedly reduced viral spread in mice transgenic for human ACE2 and protected the animals from SARS-CoV-2-induced weight loss. The two clusters of potent noncompeting SARS-CoV-2 neutralizing antibodies provide potential candidates for therapy and prophylaxis of COVID-19. The study further supports transgenic animals with a human immunoglobulin gene repertoire as a powerful platform in pandemic preparedness initiatives.

2.
Immunity ; 54(12): 2908-2921.e6, 2021 12 14.
Article in English | MEDLINE | ID: covidwho-1521063

ABSTRACT

Viral mutations are an emerging concern in reducing SARS-CoV-2 vaccination efficacy. Second-generation vaccines will need to elicit neutralizing antibodies against sites that are evolutionarily conserved across the sarbecovirus subgenus. Here, we immunized mice containing a human antibody repertoire with diverse sarbecovirus receptor-binding domains (RBDs) to identify antibodies targeting conserved sites of vulnerability. Antibodies with broad reactivity against diverse clade B RBDs targeting the conserved class 4 epitope, with recurring IGHV/IGKV pairs, were readily elicited but were non-neutralizing. However, rare class 4 antibodies binding this conserved RBD supersite showed potent neutralization of SARS-CoV-2 and all variants of concern. Structural analysis revealed that the neutralizing ability of cross-reactive antibodies was reserved only for those with an elongated CDRH3 that extends the antiparallel beta-sheet RBD core and orients the antibody light chain to obstruct ACE2-RBD interactions. These results identify a structurally defined pathway for vaccine strategies eliciting escape-resistant SARS-CoV-2 neutralizing antibodies.


Subject(s)
Betacoronavirus/physiology , COVID-19 Vaccines/immunology , Coronavirus Infections/immunology , SARS Virus/physiology , Spike Glycoprotein, Coronavirus/metabolism , Animals , Antibodies, Neutralizing/metabolism , Antibodies, Viral/metabolism , Conserved Sequence/genetics , Evolution, Molecular , Humans , Immunization , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Protein Binding , Protein Domains/genetics , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology
3.
Immunity ; 2021.
Article in English | EuropePMC | ID: covidwho-1489418

ABSTRACT

Viral mutations are an emerging concern in reducing SARS-CoV-2 vaccination efficacy. Burnett et al. immunized humanized mice with different diverse sarbecovirus RBDs to elicit antibodies targeting conserved sites. Non-neutralizing cross-reactive antibodies targeting the conserved class 4 epitope were readily elicited. Neutralizing ability was reserved only for antibodies binding this conserved supersite through an elongated CDRH3 that obstructed ACE2-RBD interactions.

4.
Nat Commun ; 12(1): 1961, 2021 03 30.
Article in English | MEDLINE | ID: covidwho-1169399

ABSTRACT

The pathogenesis of severe COVID-19 reflects an inefficient immune reaction to SARS-CoV-2. Here we analyze, at the single cell level, plasmablasts egressed into the blood to study the dynamics of adaptive immune response in COVID-19 patients requiring intensive care. Before seroconversion in response to SARS-CoV-2 spike protein, peripheral plasmablasts display a type 1 interferon-induced gene expression signature; however, following seroconversion, plasmablasts lose this signature, express instead gene signatures induced by IL-21 and TGF-ß, and produce mostly IgG1 and IgA1. In the sustained immune reaction from COVID-19 patients, plasmablasts shift to the expression of IgA2, thereby reflecting an instruction by TGF-ß. Despite their continued presence in the blood, plasmablasts are not found in the lungs of deceased COVID-19 patients, nor does patient IgA2 binds to the dominant antigens of SARS-CoV-2. Our results thus suggest that, in severe COVID-19, SARS-CoV-2 triggers a chronic immune reaction that is instructed by TGF-ß, and is distracted from itself.


Subject(s)
Antibodies, Viral/immunology , COVID-19/immunology , SARS-CoV-2/immunology , Transforming Growth Factor beta/immunology , Adult , Aged , Aged, 80 and over , COVID-19/virology , Female , Humans , Immunoglobulin A/immunology , Immunoglobulin G/immunology , Interleukins/immunology , Male , Middle Aged , Plasma Cells/immunology , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology
5.
Eur J Immunol ; 51(6): 1436-1448, 2021 06.
Article in English | MEDLINE | ID: covidwho-1156872

ABSTRACT

COVID-19 is a life-threatening disease leading to bilateral pneumonia and respiratory failure. The underlying reasons why a smaller percentage of patients present with severe pulmonary symptoms whereas the majority is only mildly affected are to date not well understood. Comparing the immunological phenotype in healthy donors and patients with mild versus severe COVID-19 shows that in COVID-19 patients, NK-/B-cell activation and proliferation are enhanced independent of severity. As an important precondition for effective antibody responses, T-follicular helper cells and antibody secreting cells are increased both in patients with mild and severe SARS-CoV-2 infection. Beyond this, T cells in COVID-19 patients exhibit a stronger activation profile with differentiation toward effector cell phenotypes. Importantly, when looking at the rates of pulmonary complications in COVID-19 patients, the chemokine receptor CCR4 is higher expressed by both CD4 and CD8 T cells of patients with severe COVID-19. This raises the hypothesis that CCR4 upregulation on T cells in the pathogenesis of COVID-19 promotes stronger T-cell attraction to the lungs leading to increased immune activation with presumably higher pulmonary toxicity. Our study contributes significantly to the understanding of the immunological changes during COVID-19, as new therapeutic agents, preferentially targeting the immune system, are highly warranted.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , COVID-19/immunology , Lung/immunology , Lymphocyte Activation , Receptors, CCR4/immunology , SARS-CoV-2/immunology , Up-Regulation/immunology , Adult , CD8-Positive T-Lymphocytes/pathology , COVID-19/pathology , Female , Humans , Lung/pathology , Lung/virology , Male , Middle Aged , Severity of Illness Index
6.
Eur J Clin Microbiol Infect Dis ; 40(4): 751-759, 2021 Apr.
Article in English | MEDLINE | ID: covidwho-880323

ABSTRACT

SARS-CoV-2 has emerged as a previously unknown zoonotic coronavirus that spread worldwide causing a serious pandemic. While reliable nucleic acid-based diagnostic assays were rapidly available, only a limited number of validated serological assays were available in the early phase of the pandemic. Here, we evaluated a novel flow cytometric approach to assess spike-specific antibody responses.HEK 293T cells expressing SARS-CoV-2 spike protein in its natural confirmation on the surface were used to detect specific IgG and IgM antibody responses in patient sera by flow cytometry. A soluble angiotensin-converting-enzyme 2 (ACE-2) variant was developed as external standard to quantify spike-specific antibody responses on different assay platforms. Analyses of 201 pre-COVID-19 sera proved a high assay specificity in comparison to commercially available CLIA and ELISA systems, while also revealing the highest sensitivity in specimens from PCR-confirmed SARS-CoV-2-infected patients. The external standard allowed robust quantification of antibody responses among different assay platforms. In conclusion, our newly established flow cytometric assay allows sensitive and quantitative detection of SARS-CoV-2-specific antibodies, which can be easily adopted in different laboratories and does not rely on external supply of assay kits. The flow cytometric assay also provides a blueprint for rapid development of serological tests to other emerging viral infections.


Subject(s)
Antibodies, Viral/immunology , COVID-19 Serological Testing/methods , COVID-19/diagnosis , Immunoglobulin G/immunology , Immunoglobulin M/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Angiotensin-Converting Enzyme 2 , Enzyme-Linked Immunosorbent Assay , Flow Cytometry/methods , HEK293 Cells , Humans , Reference Standards , Reproducibility of Results , Sensitivity and Specificity
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