Bispecific antibodies combine breadth, potency, and avidity of parental antibodies to neutralize sarbecoviruses

SARS-CoV-2 mutational variants evade humoral immune responses elicited by vaccines and current monoclonal antibody (mAb) therapies. Novel antibody-based treatments will thus need to exhibit broad neutralization against different variants. Bispecific antibodies (bsAbs) combine the specificities of two distinct antibodies into one antibody taking advantage of the avidity, synergy and cooperativity provided by targeting two different epitopes. Here we used controlled Fab-arm exchange (cFAE), a versatile and straightforward method, to produce bsAbs that neutralize SARS-CoV and SARS-CoV-2 variants, including Omicron and its subvariants, by combining potent SARS-CoV-2-specific neutralizing antibodies with broader but less potent antibodies that also neutralize SARS-CoV. We demonstrate that the parental IgGs rely on avidity for their neutralizing activity by comparing their potency to bsAbs containing one irrelevant "dead" Fab arm. We used single particle mass photometry to measure formation of antibody:spike complexes, and determined that bsAbs increase binding stoichiometry compared to corresponding cocktails, without a loss of binding affinity. The heterogeneous binding pattern of bsAbs to spike (S), observed by negative-stain electron microscopy and mass photometry provided evidence for both intra- and inter-spike crosslinking. This study highlights the utility of cross-neutralizing antibodies for designing bivalent or multivalent agents to provide a robust activity against circulating variants, as well as future SARS-like coronaviruses.

IgG1 responses following SARS-CoV-2 infection are polyclonal and highly personalized, whereby each donor and each clone displays a distinct pattern of cross-reactivity against SARS-CoV-2 variants

Using a recently introduced efficient mass spectrometry-based approach we monitored individual donors IgG1 clonal responses in molecular detail, examining SARS-CoV-2 spike-protein-specific IgG1 repertoires. We monitored the plasma clonal IgG1 profiles of 8 donors (4 male and 4 female) who had recently experienced an infection by either the wild type Wuhan Hu-1 virus or one of 3 VOCs (Alpha, Beta and Gamma). In these donors we charted the full plasma IgG1 repertoires as well as the IgG1 repertoires targeting the SARS-CoV-2 spike protein trimer as antigen. We observed that shortly after infection in between <0.1% to almost 10% of all IgG1 antibody molecules present in plasma did bind to the spike protein. Each donor displayed a unique plasma IgG1 repertoire, but also each donor displayed a unique and polyclonal antibody response against the SARS-CoV-2 spike-protein variants. Our analyses revealed that certain clones exhibit (alike) binding affinity towards all four tested spike-protein variants, whereas other clones displayed strong unique mutant-specific affinity. We conclude that each infected person generates a unique polyclonal response following infection, whereby some of these clones can bind multiple viral variants, whereas other clones do not display such cross-reactivity. In general, by assessing IgG1 repertoires following infection it becomes possible to identify and select fully matured human plasma antibodies that target specific antigens, and display either high specificity or cross-reactivity versus mutated versions of the antigen, which will aid in selecting antibodies that may be developed into biotherapeutics.

Probing Affinity, Avidity, Anti-Cooperativity, and Competition in Antibody and Receptor Binding to the SARS-CoV-2 Spike by Single Particle Mass Analyses

Determining how antibodies interact with the spike (S) protein of the SARS-CoV-2 virus is critical for combating COVID-19. Structural studies typically employ simplified, truncated constructs that may not fully recapitulate the behaviour of the original complexes. Here, we combine two single particle mass analysis techniques (mass photometry and charge-detection mass spectrometry) to enable measurement of full IgG binding to the trimeric SARS-CoV-2 S ectodomain. Our experiments reveal that antibodies targeting the S-trimer typically prefer stoichiometries lower than the symmetry-predicted 3:1 binding. We determine that this behaviour arises from the interplay of steric clashes and avidity effects that are not reflected in common antibody constructs (i.e. Fabs). Surprisingly, these sub-stoichiometric complexes are fully effective at blocking ACE2 binding despite containing free receptor binding sites. Our results highlight the importance of studying antibody/antigen interactions using complete, multimeric constructs and showcase the utility of single particle mass analyses in unraveling these complex interactions.

Is there a serum proteome signature to predict mortality in severe COVID-19 patients?

Here we recorded serum proteome profiles of 33 COVID-19 patients admitted to respiratory and intensive care units because of respiratory failure. We received, for most patients, blood samples just after admission and at two more later timepoints. We focused on serum proteins different in abundance between the group of survivors and non-survivors and observed that a rather small panel of about a dozen proteins were significantly different in abundance between these two groups. The four structurally and functionally related type-3 cystatins AHSG, FETUB, HRG and KNG1 were all more abundant in the survivors. The family of inter--trypsin inhibitors, ITIH1, ITIH2, ITIH3 and ITIH4, were all found to be differentially abundant in between survivors and non-survivors, whereby ITIH1 and ITIH2 were more abundant in the survivor group and ITIH3 and ITIH4 more abundant in the non-survivors. ITIH1/ITIH2 and ITIH3/ITIH4 also did show opposite trends in protein abundance during disease progression. This panel of eight proteins, complemented with a few more, may represent a panel for mortality risk assessment and eventually even for treatment, by administration of exogenous proteins possibly aiding survival. Such administration is not unprecedented, as administration of exogenous inter--trypsin inhibitors is already used in the treatment of patients with severe sepsis and Kawasaki disease. The mortality risk panel defined here is in excellent agreement with findings in two recent COVID-19 serum proteomics studies on independent cohorts, supporting our findings. This panel may not be unique for COVID-19, as some of the proteins here annotated as mortality risk factors have previously been annotated as mortality markers in aging and in other diseases caused by different pathogens, including bacteria.

Breastmilk; a source of SARS-CoV-2 specific IgA antibodies

BackgroundSince the outbreak of COVID-19, many put their hopes in the rapid development of effective immunizations. For now patient isolation, physical distancing and good hygiene are the sole measures for prevention. Processed breast milk with antibodies against SaRS-CoV-2 may serve as additional protection. We aimed to determine the presence and neutralization capacity of antibodies against SaRS-CoV-2 in breastmilk of mothers who have recovered from COVID-19. MethodsThis prospective case control study included lactating mothers, recovered from (suspected) COVID-19 and healthy controls. Serum and breastmilk was collected. To assess the presence of antibodies in breastmilk and serum, we used multiple complementary assays, namely ELISA with the SARS-CoV-2 spike protein, SARS-CoV-2 receptor binding domain (RBD) and with the SARS-CoV-2 nucleocapsid (N) protein for IgG and bridging ELISA with the SARS-CoV-2 RBD and N protein for total Ig. To assess the effect of pasteurization breastmilk was exposed to Holder Pasteurization and High Pressure Pasteurization. ResultsBreastmilk contained antibodies against SARS-CoV-2 using any of the assays in 24 out of 29 (83%) proven cases, in six out of nine (67%) suspected cases and in none of the 13 controls. In vitro neutralization of SARS-CoV-2 clinical isolate virus strain was successful in a subset of serum (13%) and milk samples (26%). Although after pasteurization of the milk SARS-CoV-2 antibodies were detected with both methods of pasteurization, virus neutralizing capacity of those antibodies was only retained with the HPP approach. ConclusionBreastmilk of mothers who recovered from COVID-19 contains significant amounts of IgA against SARS-CoV-2, both before and after pasteurization. Key PointsO_ST_ABSQuestionC_ST_ABSDoes breastmilk of mothers who have recovered from coronavirus disease 2019 (COVID-19) contain antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)? FindingsWe provide multiple lines of evidence on the presence of a variety of antibodies against SARS-CoV-2, with no such antibodies present in the controls. These antibodies are capable of neutralizing a clinical isolate of SARS-CoV-2 in vitro. We furthermore show that high pressure pasteurization hardly affects antibody levels and efficacy. MeaningBreastmilk, obtained from mothers who have recovered from COVID-19, may serve as a safe and widely applicable preventive strategy for vulnerable high risk populations