Spike Binding Research Articles

On the Nature of the Interactions That Govern COV-2 Mutants Escape from Neutralizing Antibodies

The most fruitful prevention and treatment tools for the COVID-19 pandemic have proven to be vaccines and therapeutic antibodies, which have reduced the spread of the disease to manageable proportions. The search for the most effective antibodies against the widest set of COV-2 variants has required a long time and substantial resources. It would be desirable to have a tool that will enable us to understand the structural basis on which mutants escape at least some of the epitope-bound antibodies, a tool that may substantially reduce the time and resources invested in this effort. In this work, we applied a computational-based tool (employed previously by us to understand COV-2 spike binding to its cognate cell receptor) to the study of the effect of Delta and Omicron mutations on the escape tendencies. Our binding energy predictions agree extremely well with the experimentally observed escape tendencies. They have also allowed us to set forth a structural explanation for the results that could be used for the screening of antibodies. Lastly, our results explain the differences in molecular interactions that govern interaction of the spike variants with the receptor as opposed to those with antibodies.

Evolution at Spike protein position 519 in SARS-CoV-2 facilitated adaptation to humans

As the COVID-19 pandemic enters its fourth year, the pursuit of identifying a progenitor virus to SARS-CoV-2 and understanding the mechanism of its emergence persists, albeit against the backdrop of intensified efforts to monitor the ongoing evolution of the virus and the influx of new mutations. Surprisingly, few residues hypothesized to be essential for SARS-CoV-2 emergence and adaptation to humans have been validated experimentally, despite the importance that these mutations could contribute to the development of effective antivirals. To remedy this, we searched for genomic regions in the SARS-CoV-2 genome that show evidence of past selection around residues unique to SARS-CoV-2 compared with closely related coronaviruses. In doing so, we identified a residue at position 519 in Spike within the receptor binding domain that holds a static histidine in human-derived SARS-CoV-2 sequences but an asparagine in SARS-related coronaviruses from bats and pangolins. In experimental validation, the SARS-CoV-2 Spike protein mutant carrying the putatively ancestral H519N substitution showed reduced replication in human lung cells, suggesting that the histidine residue contributes to viral fitness in the human host. Structural analyses revealed a potential role of Spike residue 519 in mediating conformational transitions necessary for Spike prior to binding with ACE2. Pseudotyped viruses bearing the putatively ancestral N519 also demonstrated significantly reduced infectivity in cells expressing the human ACE2 receptor compared to H519. ELISA data corroborated that H519 enhances Spike binding affinity to the human ACE2 receptor compared to the putatively ancestral N519. Collectively, these findings suggest that the evolutionary transition at position 519 of the Spike protein played a critical role in SARS-CoV-2 emergence and adaptation to the human host. Additionally, this residue presents as a potential drug target for designing small molecule inhibitors tailored to this site.

Emerging variants develop total escape from potent monoclonal antibodies induced by BA.4/5 infection

The rapid evolution of SARS-CoV-2 is driven in part by a need to evade the antibody response in the face of high levels of immunity. Here, we isolate spike (S) binding monoclonal antibodies (mAbs) from vaccinees who suffered vaccine break-through infections with Omicron sub lineages BA.4 or BA.5. Twenty eight potent antibodies are isolated and characterised functionally, and in some cases structurally. Since the emergence of BA.4/5, SARS-CoV-2 has continued to accrue mutations in the S protein, to understand this we characterize neutralization of a large panel of variants and demonstrate a steady attrition of neutralization by the panel of BA.4/5 mAbs culminating in total loss of function with recent XBB.1.5.70 variants containing the so-called ‘FLip’ mutations at positions 455 and 456. Interestingly, activity of some mAbs is regained on the recently reported variant BA.2.86.

The Diverse Nature of the Molecular Interactions That Govern the COV-2 Variants' Cell Receptor Affinity Ranking and Its Experimental Variability.

A critical determinant of infectivity and virulence of the most infectious and or lethal variants of concern (VOCs): Wild Type, Delta and Omicron is related to the binding interactions between the receptor-binding domain of the spike and its host receptor, the initial step in cell infection. It is of the utmost importance to understand how mutations of a viral strain, especially those that are in the viral spike, affect the resulting infectivity of the emerging VOC, knowledge that could help us understand the variant virulence and inform the therapies applied or the vaccines developed. For this sake, we have applied a battery of computational protocols of increasing complexity to the calculation of the spike binding affinity for three variants of concern to the ACE2 cell receptor. The results clearly illustrate that the attachment of the spikes of the Delta and Omicron variants to the receptor originates through different molecular interaction mechanisms. All our protocols unanimously predict that the Delta variant has the highest receptor-binding affinity, while the Omicron variant displays a substantial variability in the binding affinity of the spike that relates to the structural plasticity of the Omicron spike-receptor complex. We suggest that the latter result could explain (at least in part) the variability of the in vitro binding results for this VOC and has led us to suggest a reason for the lower virulence of the Omicron variant as compared to earlier strains. Several hypotheses have been developed around this subject.

Impact of ageing on homologous and human-coronavirus-reactive antibodies after SARS-CoV-2 vaccination or infection

The endemic human coronaviruses (HCoVs) circulate worldwide yet remain understudied and unmitigated. The observation of elevated levels of HCoV reactive antibodies in COVID-19 patients highlights the urgent necessity of better understanding of HCoV specific immunity. Here, we characterized in-depth the de novo SARS-CoV-2 specific antibody responses and the boosting of HCoV-reactive antibodies after SARS-CoV-2 vaccination or infection in individuals up to 98 years old. All the vaccinees were home-dwelling with no documented SARS-CoV-2 infection before receiving the COVID-19 mRNA vaccine (BNT162b2). The first two vaccine doses elicited potent SARS-CoV-2 spike binding antibodies in individuals up to 80 years. The third dose largely boosted the previously low S2 domain binding and neutralizing antibodies in elderly 80–90 years old, but less so in those above 90 years. The endemic betacoronavirus (HKU1 and OC43) reactive antibodies were boosted in all vaccinees, although to a lesser extent in those above 80 years old. COVID-19 patients had potent elevation of alpha- and betacoronavirus (229E, NL63, HKU1 and OC43) reactive antibodies. In both patients and vaccinees, S2 domain specific antibody increases correlated with SARS-CoV-2 neutralizing and HCoV-reactive antibody responses in all ages, indicating S2 domain as a candidate for future universal coronavirus vaccine design.

In vivo antiviral efficacy of LCTG-002, a pooled, purified human milk secretory IgA product, against SARS-CoV-2 in a murine model of COVID-19.

Immunoglobulin A (IgA) is the most abundant antibody (Ab) in human mucosae, with secretory form (sIgA) being dominant and uniquely stable. sIgA is challenging to produce recombinantly but is naturally found in human milk, which could be considered a global resource for this biologic, justifying its development as a mucosal therapeutic. Presently, SARS-CoV-2 was utilized as a model mucosal pathogen, and methods were developed to efficiently extract human milk sIgA from donors who were naïve to SARS-CoV-2 or had recovered from infection that elicited high-titer anti-SARS-CoV-2 Spike sIgA in their milk (pooled to make LCTG-002). Mass spectrometry determined that proteins with a relative abundance of 1% or greater were all associated with sIgA. Western blot demonstrated that all batches consisted predominantly of sIgA. Compared to control IgA, LCTG-002 demonstrated significantly higher Spike binding (mean endpoint of 0.87 versus 5.87). LCTG-002 was capable of blocking the Spike receptor-binding domain - angiotensin-converting enzyme 2 (ACE2) interaction with significantly greater potency compared to control (mean LCTG-002 IC50 154ug/mL versus 50% inhibition not achieved for control), and exhibited significant neutralization activity against Spike-pseudotyped virus infection (mean LCTG-002 IC50 49.8ug/mL versus 114.5ug/mL for control). LCTG-002 was tested for its capacity to reduce viral lung burden in K18+hACE2 transgenic mice inoculated with SARS-CoV-2. LCTG-002 significantly reduced SARS-CoV-2 titers compared to control when administered at 0.25 mg/day or 1 mg/day, with a maximum TCID50 reduction of 4.9 logs. This innovative study demonstrates that LCTG-002 is highly pure and efficacious in vivo, supporting further development of milk-derived, polyclonal sIgA therapeutics.

Two Receptor Binding Strategy of SARS-CoV-2 Is Mediated by Both the N-Terminal and Receptor-Binding Spike Domain.

It is not well understood why severe acute respiratory syndrome (SARS)-CoV-2 spreads much faster than other β-coronaviruses such as SARS-CoV and Middle East respiratory syndrome (MERS)-CoV. In a previous publication, we predicted the binding of the N-terminal domain (NTD) of SARS-CoV-2 spike to sialic acids (SAs). Here, we experimentally validate this interaction and present simulations that reveal a second possible interaction between SAs and the spike protein via a binding site located in the receptor-binding domain (RBD). The predictions from molecular-dynamics simulations and the previously-published 2D-Zernike binding-site recognition approach were validated through flow-induced dispersion analysis (FIDA)─which reveals the capability of the SARS-CoV-2 spike to bind to SA-containing (glyco)lipid vesicles, and flow-cytometry measurements─which show that spike binding is strongly decreased upon inhibition of SA expression on the membranes of angiotensin converting enzyme-2 (ACE2)-expressing HEK cells. Our analyses reveal that the SA binding of the NTD and RBD strongly enhances the infection-inducing ACE2 binding. Altogether, our work provides in silico, in vitro, and cellular evidence that the SARS-CoV-2 virus utilizes a two-receptor (SA and ACE2) strategy. This allows the SARS-CoV-2 spike to use SA moieties on the cell membrane as a binding anchor, which increases the residence time of the virus on the cell surface and aids in the binding of the main receptor, ACE2, via 2D diffusion.

Recombination Events Among SARS-CoV-2 Omicron Subvariants: Impact on Spike Interaction With ACE2 Receptor and Neutralizing Antibodies.

The recombination plays a key role in promoting evolution of RNA viruses and emergence of potentially epidemic variants. Some studies investigated the recombination occurrence among SARS-CoV-2, without exploring its impact on virus-host interaction. In the aim to investigate the burden of recombination in terms of frequency and distribution, the occurrence of recombination was first explored in 44 230 Omicron sequences among BQ subvariants and the under investigation "ML" (Multiple Lineages) denoted sequences, using 3seq software. Second, the recombination impact on interaction between the Spike protein and ACE2 receptor as well as neutralizing antibodies (nAbs), was analyzed using docking tools. Recombination was detected in 56.91% and 82.20% of BQ and ML strains, respectively. It took place mainly in spike and ORF1a genes. For BQ recombinant strains, the docking analysis showed that the spike interacted strongly with ACE2 and weakly with nAbs. The mutations S373P, S375F and T376A constitute a residue network that enhances the RBD interaction with ACE2. Thirteen mutations in RBD (S373P, S375F, T376A, D405N, R408S, K417N, N440K, S477N, P494S, Q498R, N501Y, and Y505H) and NTD (Y240H) seem to be implicated in immune evasion of recombinants by altering spike interaction with nAbs. In conclusion, this "in silico" study demonstrated that the recombination mechanism is frequent among Omicron BQ and ML variants. It highlights new key mutations, that potentially implicated in enhancement of spike binding to ACE2 (F376A) and escape from nAbs (RBD: F376A, D405N, R408S, N440K, S477N, P494S, and Y505H; NTD: Y240H). Our findings present considerable insights for the elaboration of effective prophylaxis and therapeutic strategies against future SARS-CoV-2 waves.

Discovery of High Affinity Cyclic Peptide Ligands for Human ACE2 with SARS-CoV-2 Entry Inhibitory Activity.

The development of effective antiviral compounds is essential for mitigating the effects of the COVID-19 pandemic. Entry of SARS-CoV-2 virions into host cells is mediated by the interaction between the viral spike (S) protein and membrane-bound angiotensin-converting enzyme 2 (ACE2) on the surface of epithelial cells. Inhibition of this viral protein-host protein interaction is an attractive avenue for the development of antiviral molecules with numerous spike-binding molecules generated to date. Herein, we describe an alternative approach to inhibit the spike-ACE2 interaction by targeting the spike-binding interface of human ACE2 via mRNA display. Two consecutive display selections were performed to direct cyclic peptide ligand binding toward the spike binding interface of ACE2. Through this process, potent cyclic peptide binders of human ACE2 (with affinities in the picomolar to nanomolar range) were identified, two of which neutralized SARS-CoV-2 entry. This work demonstrates the potential of targeting ACE2 for the generation of anti-SARS-CoV-2 therapeutics as well as broad spectrum antivirals for the treatment of SARS-like betacoronavirus infection.

Surrogate Virus Neutralisation Test Based on Nanoluciferase-Tagged Antigens to Quantify Inhibitory Antibodies against SARS-CoV-2 and Characterise Omicron-Specific Reactivity in a Vaccination Cohort.

Virus-specific antibodies are crucial for protective immunity against SARS-CoV-2. Assessing functional antibodies through conventional or pseudotyped virus neutralisation tests (pVNT) requires high biosafety levels. Alternatively, the virus-free surrogate virus neutralisation test (sVNT) quantifies antibodies interfering with spike binding to angiotensin-converting enzyme 2. We evaluated secreted nanoluciferase-tagged spike protein fragments as diagnostic antigens in the sVNT in a vaccination cohort. Initially, spike fragments were tested in a capture enzyme immunoassay (EIA), identifying the receptor binding domain (RBD) as the optimal diagnostic antigen. The sensitivity of the in-house sVNT applying the nanoluciferase-labelled RBD equalled or surpassed that of a commercial sVNT (cPass, GenScript Diagnostics) and an in-house pVNT four weeks after the first vaccination (98% vs. 94% and 72%, respectively), reaching 100% in all assays four weeks after the second and third vaccinations. When testing serum reactivity with Omicron BA.1 spike, the sVNT and pVNT displayed superior discrimination between wild-type- and variant-specific serum reactivity compared to a capture EIA. This was most pronounced after the first and second vaccinations, with the third vaccination resulting in robust, cross-reactive BA.1 construct detection. In conclusion, utilising nanoluciferase-labelled antigens permits the quantification of SARS-CoV-2-specific inhibitory antibodies. Designed as flexible modular systems, the assays can be readily adjusted for monitoring vaccine efficacy.

Airway and Systemic Immune Responses Following the Third COVID-19 Vaccination in COPD Patients.

Booster vaccinations are required to maintain protection against COVID-19. COPD patients are at higher risk of developing severe illness following SARS-CoV-2 infection. Previous cross-sectional analysis after the second COVID-19 booster showed similar immune responses in COPD patients and controls, but pre-vaccination samples were not available. This longitudinal study evaluated systemic and airway immune responses in COPD patients using samples obtained pre- and post-third COVID-19 vaccination. Twelve COPD patients were recruited, with plasma, nasal and sputum (n = 10) samples collected pre-vaccination and 4- and 14-weeks post vaccination. Samples were analyzed for anti-spike IgA and IgG and cellular immunity. The ability of plasma and nasal samples to block ACE2-spike protein interaction was assessed for Wild type, Delta, and Omicron spike variants. Vaccinations increased anti-spike IgG in plasma (p < 0.001), nasal (IgG p < 0.001) and sputum (p = 0.002) samples, IgA in plasma (p < 0.001) and blood cellular immunity (p = 0.001). Plasma and nasal anti-spike IgA levels correlated (rho: 0.6, p = 0.02), with similar results for IgG (rho: 0.79, p = 0.003). Post-vaccination nasal (p = 0.002) and plasma (p < 0.001) samples were less effective at blocking Omicron spike binding to ACE2 compared to the Wild type spike variant. Airway and systemic immune responses against SARS-CoV-2 increased in COPD patients following a third COVID-19 vaccination. Nasal and systemic responses in COPD patients were less effective against Omicron variant compared to previous variants.

Pyrogenic and inflammatory mediators are produced by polarized M1 and M2 macrophages activated with D-dimer and SARS-CoV-2 spike immune complexes

Lung macrophages are the first line of defense against invading respiratory pathogens including SARS-CoV-2, yet activation of macrophage in the lungs can lead to hyperinflammatory immune response seen in severe COVID-19. Here we used human M1 and M2 polarized macrophages as a surrogate model of inflammatory and regulatory macrophages and explored whether immune complexes (IC) containing spike-specific IgG can trigger aberrant cytokine responses in macrophages in the lungs and associated lymph nodes. We show that IC of SARS-CoV-2 recombinant S protein coated with spike-specific monoclonal antibody induced production of Prostaglandin E2 (PGE2) in non-polarized (M0) and in M1 and M2-type polarized human macrophages only in the presence of D-dimer (DD), a fibrinogen degradation product, associated with coagulopathy in COVID-19. Importantly, an increase in PGE2 was also observed in macrophages activated with DD and IC of SARS-CoV-2 pseudovirions coated with plasma from hospitalized COVID-19 patients but not from healthy subjects. Overall, the levels of PGE2 in macrophages activated with DD and IC were as follows: M1≫M2>M0 and correlated with the levels of spike binding antibodies and not with neutralizing antibody titers. All three macrophage subsets produced similar levels of IL-6 following activation with DD+IC, however TNFα, IL-1β, and IL-10 cytokines were produced by M2 macrophages only. Our study suggests that high titers of spike or virion containing IC in the presence of coagulation byproducts (DD) can promote inflammatory response in macrophages in the lungs and associated lymph nodes and contribute to severe COVID-19.

Analysis of SARS-CoV-2 Variant-Specific Serum Antibody Post-Vaccination Utilizing Immortalized Human Hepatocyte-Like Cells (HLC) to Assess Development of Immunity.

Our previous studies demonstrated that SARS-CoV-2 spike protein could bind to primary hepatocytes and immortalized Hepatocyte-like cells (HLC) via the asialoglycoprotein receptor-1 (ASGR-1). The binding of biotinylated spike protein could be inhibited by Spike-neutralizing monoclonal antibodies, anti-ASGR-1 antibodies and unlabeled spike protein. The cells were unable to bind Spike S1 and Spike S1 was incapable of blocking labeled Spike protein, suggesting that the Receptor Binding Domain (RBD) was not involved in the binding event. This study was done to investigate the utility of these cells and immortalized alveolar type 2-like (AT-2) cells in studying the development of variant-specific antibodies post-vaccination. Serum was collected from 10 individuals pre- and post-vaccination with the J&J, Moderna or Pfizer vaccines. The serum samples were quantified for variant-specific antibodies in a flow cytometry-based immunofluorescent assay utilizing beads coated with biotinylated variant spike proteins. Inhibition of spike protein binding to HLC and AT-2 cells by donor serum was analyzed by immunofluorescent confocal analysis. All variant spike proteins bound to HLC and AT-2 cells. Post-vaccination serum samples demonstrated increases of SARS-CoV-2 antibody levels from 2 weeks to 2.5 months post-vaccination with associated increased spike-blocking capacity. It was also demonstrated that vaccination with all the available vaccines stimulated antibodies that inhibited binding of all the available variant spike proteins to both HLC and AT-2 cells. HLC, along with AT-2 cells, provides a useful platform to study the development of neutralizing antibodies post-vaccination. Vaccination with the 3 available vaccines all elicited neutralizing serum antibodies that inhibited binding of each of the variant spike proteins to both AT-2 and HLC cells. This study suggests that inhibition of spike binding to target cells may be a more useful technique to assess immunity than gross quantitation of antibody.

Cryptic-site-specific antibodies to the SARS-CoV-2 receptor binding domain can retain functional binding affinity to spike variants.

Multiple SARS-CoV-2 variants of concern have emerged and caused a significant number of infections and deaths worldwide. These variants of concern contain mutations that might significantly affect antigen-targeting by antibodies. It is therefore important to further understand how antibody binding and neutralization are affected by the mutations in SARS-CoV-2 variants. We highlighted how antibody epitope specificity can influence antibody binding to SARS-CoV-2 spike protein variants and neutralization of SARS-CoV-2 variants. We showed that weakened spike binding and neutralization of Beta (B.1.351) and Omicron (BA.1) variants compared to wildtype are not universal among the panel of antibodies and identified antibodies of a specific binding footprint exhibiting consistent enhancement of spike binding and retained neutralization to Beta variant. These data and analysis can inform how antigen-targeting by antibodies might evolve during a pandemic and prepare for potential future sarbecovirus outbreaks.

Cellular Mechanisms Associated with Suboptimal Immune Responses to Sars-Cov-2 Bivalent Booster Vaccination in Patients with Multiple Myeloma

Background: In response to the ever-evolving challenges posed by the antigenically diverse Omicron variants, the deployment of bivalent mRNA booster vaccines encompassing both the ancestral (WA1) and Omicron BA.5 spike components was initiated in fall of 2022. The real-world impact of these bivalent vaccines is largely unknown in patients with Multiple Myeloma (MM). We characterize the humoral and cellular immune responses in patients with MM before and after receiving the bivalent booster implementing cutting edge methodologies to identify patterns associated with continuing vulnerability in the current post-pandemic era. Methods: We studied the humoral and cellular immune responses before and after bivalent booster immunization in 48 MM patients. Spike binding IgG antibody levels were measured by SARS-CoV-2 spike binding ELISA and neutralization capacity was assessed by a SARS-CoV-2 multi-cycle microneutralization assay. We measured spike specific T cell function using the QuantiFERON SARS-CoV-2 (Qiagen) assay as well as flow cytometry-based T cell assays quantifying interferon-gamma (IFNg) production. In a subset of 38 patients, immune profiling was performed using high-dimensional flow cytometry. The frequencies of dendritic cells (DCs), B cells, Natural Killer (NK) cells and T follicular helper cells (TFH) were compared between responders and non-responders. Results: The serological effect of the bivalent vaccination in MM patients is nuanced with the overall cohort not demonstrating a significant increase post bivalent vaccination (median 196 AU/mL prior to bivalent to median 276 post bivalent p=0.11). The lack of antibody response was driven by patients undergoing anti-CD38 and anti-BCMA antibody therapy as compared to MM patients undergoing other therapies who demonstrated a significant increase in antibody levels post vaccination (p=0.021). Patients on anti-CD38 and anti-BCMA antibody therapy also did not significantly increase neutralizing capacity to WA1 (p=0.42) or BA.5 omicron strain (p=0.48) while MM patients on other therapies benefitted significantly, as illustrated by the increased neutralizing capacity of WA1 (p=0.024) and BA.5 (p=0.0055 Figure 1A). In addition to quantifying antibody responses, we profiled T cell responses using our published flow cytometry method using WA1 anti-S peptides as well as the QuantiFERON SARS-CoV-2 RUO assay (Qiagen). Spike reactive T cell responses significantly correlated with anti-Spike IgG levels in MM patients (r 2=0.49, p&amp;lt;0.001), WA1 neutralizing ID50 (R 2 = 0.6, p&amp;lt;0.01) and BA.5 neutralizing ID50 (R 2= 0.54, p=0.01) reinforcing the fact that T cell compensation is lacking in MM patients. We separated the lowest quantile (patients with Anti-S IgG &amp;lt;156 AU/mL) to delineate suboptimal producing patients compared to the remaining MM anti-S producing patients. Non-responders exhibited deficiencies in immune populations involved in robust antibody production including lower frequency of CD1c+ DCs (p=0.02 Figure 1B), B Cells (p&amp;lt;0.01 Figure 1B) and circulating CXCR3-CCR4+TFH cells (p&amp;lt;0.01 Figure 1B). Furthermore, immature NK cells were the predominant population in non-responders, while responders had more cytotoxic mature NK phenotype (p&amp;lt;0.01 Figure 1B) after bivalent vaccination. Conclusions: Our study highlights the varying immune responses observed in MM patients after receiving bivalent COVID-19 vaccination. Specifically, a subgroup of MM patients undergoing anti-CD38 and anti-BCMA therapy experience impairments in immune cells such DCs, B cells, NK cells and TFH cells, leading to an inability to generate adequate antibody and T cell responses to vaccinations. Our results indicate that the QuantiFERON SARS-CoV-2 assay could be deployed for clinical use bridging the need for suitable laboratory tests to measure SARS-CoV-2 specific T cell responses. Ongoing research will further investigate whether the compromised immune machinery and diminished T cell activity identified in this study can serve as discriminators for identifying MM patients at high risk of infectious complications stemming from anti-BCMA therapies. Such insights will aid in developing targeted strategies to address these challenges and improve patient outcomes.

Cellular mechanisms associated with sub-optimal immune responses to SARS-CoV-2 bivalent booster vaccination in patients with Multiple Myeloma

The real-world impact of bivalent vaccines for wild type (WA.1) and Omicron variant (BA.5) is largely unknown in immunocompromised patients with Multiple Myeloma (MM). We characterize the humoral and cellular immune responses in patients with MM before and after receiving the bivalent booster, including neutralizing assays to identify patterns associated with continuing vulnerability to current variants (XBB1.16, EG5) in the current post-pandemic era. We studied the humoral and cellular immune responses before and after bivalent booster immunization in 48MM patients. Spike binding IgG antibody levels were measured by SARS-CoV-2 spike binding ELISA and neutralization capacity was assessed by a SARS-CoV-2 multi-cycle microneutralization assays to assess inhibition of live virus. We measured spike specific T-cell function using the QuantiFERON SARS-CoV-2 (Qiagen) assay as well as flow-cytometry based T-cell. In a subset of 38 patients, high-dimensional flow cytometry was performed to identify immune cell subsets associated with lack of humoral antibodies. We find that bivalent vaccination provides significant boost in protection to the omicron variant in our MM patients, in a treatment specific manner. MM patients remain vulnerable to newer variants with mutations in the spike portion. Anti-CD38 and anti-BCMA therapies affect the immune machinery needed to produce antibodies. Our study highlights varying immune responses observed in MM patients after receiving bivalent COVID-19 vaccination. Specifically, a subgroup of MM patients undergoing anti-CD38 and anti-BCMA therapy experience impairment in immune cells such DCs, B cells, NK cells and TFH cells, leading to an inability to generate adequate humoral and cellular responses to vaccination. National Cancer Institute (National Institutes of Health), National Institute of Allergy and Infectious Diseases (National Institutes of Health), NCI Serological Sciences Network for COVID-19 (SeroNet) and The Icahn School of Medicine at Mount Sinai.

Superior immunogenicity of mRNA over adenoviral vectored COVID-19 vaccines reflects B cell dynamics independent of anti-vector immunity: Implications for future pandemic vaccines

Both vector and mRNA vaccines were an important part of the response to the COVID-19 pandemic and may be required in future outbreaks and pandemics. The aim of this study was to validate whether immunogenicity differs for adenoviral vectored (AdV) versus mRNA vaccines against SARS-CoV-2, and to investigate how anti-vector immunity and B cell dynamics modulate immunogenicity. We enrolled SARS-CoV-2 infection-naïve health care workers who had received two doses of either AdV AZD1222 (n = 184) or mRNA BNT162b2 vaccine (n = 274) between April and October 2021. Blood was collected at least once, 10–48 days after vaccine dose 2 for antibody and B cell analyses. Median ages were 42 and 39 years, for AdV and mRNA vaccinees, respectively. Surrogate virus neutralization test (sVNT) and spike binding antibody titres were a median of 4.2 and 2.2 times lower, respectively, for AdV compared to mRNA vaccinees (p < 0.001). Median percentages of memory B cells that recognized fluorescent-tagged spike and RBD were 2.9 and 8.3 times lower, respectively for AdV compared to mRNA vaccinees. Titres of IgG reactive with human adenovirus type 5 hexon protein rose a median of 2.2-fold after AdV vaccination but were not correlated with anti-spike antibody titres. Together the results show that mRNA induced substantially more sVNT antibody than AdV vaccine, which reflected greater B cell expansion and targeting of the RBD rather than an attenuating effect of anti-vector antibodies.ClinicalTrials.gov Identifier: NCT05110911.

Circularized Nanodiscs for Multivalent Mosaic Display of SARS-CoV-2 Spike Protein Antigens

The emergence of vaccine-evading SARS-CoV-2 variants urges the need for vaccines that elicit broadly neutralizing antibodies (bnAbs). Here, we assess covalently circularized nanodiscs decorated with recombinant SARS-CoV-2 spike glycoproteins from several variants for eliciting bnAbs with vaccination. Cobalt porphyrin–phospholipid (CoPoP) was incorporated into the nanodisc to allow for anchoring and functional orientation of spike trimers on the nanodisc surface through their His-tag. Monophosphoryl-lipid (MPLA) and QS-21 were incorporated as immunostimulatory adjuvants to enhance vaccine responses. Following optimization of nanodisc assembly, spike proteins were effectively displayed on the surface of the nanodiscs and maintained their conformational capacity for binding with human angiotensin-converting enzyme 2 (hACE2) as verified using electron microscopy and slot blot assay, respectively. Six different formulations were prepared where they contained mono antigens; four from the year 2020 (WT, Beta, Lambda, and Delta) and two from the year 2021 (Omicron BA.1 and BA.2). Additionally, we prepared a mosaic nanodisc displaying the four spike proteins from year 2020. Intramuscular vaccination of CD-1 female mice with the mosaic nanodisc induced antibody responses that not only neutralized matched pseudo-typed viruses, but also neutralized mismatched pseudo-typed viruses corresponding to later variants from year 2021 (Omicron BA.1 and BA.2). Interestingly, sera from mosaic-immunized mice did not effectively inhibit Omicron spike binding to human ACE-2, suggesting that some of the elicited antibodies were directed towards conserved neutralizing epitopes outside the receptor binding domain. Our results show that mosaic nanodisc vaccine displaying spike proteins from 2020 can elicit broadly neutralizing antibodies that can neutralize mismatched viruses from a following year, thus decreasing immune evasion of new emerging variants and enhancing healthcare preparedness.

The emergence of SARS-CoV-2 lineages and associated saliva antibody responses among asymptomatic individuals in a large university community.

SARS-CoV-2 (CoV2) infected, asymptomatic individuals are an important contributor to COVID transmission. CoV2-specific immunoglobulin (Ig)-as generated by the immune system following infection or vaccination-has helped limit CoV2 transmission from asymptomatic individuals to susceptible populations (e.g. elderly). Here, we describe the relationships between COVID incidence and CoV2 lineage, viral load, saliva Ig levels (CoV2-specific IgM, IgA and IgG), and ACE2 binding inhibition capacity in asymptomatic individuals between January 2021 and May 2022. These data were generated as part of a large university COVID monitoring program in Ohio, United States of America, and demonstrate that COVID incidence among asymptomatic individuals occurred in waves which mirrored those in surrounding regions, with saliva CoV2 viral loads becoming progressively higher in our community until vaccine mandates were established. Among the unvaccinated, infection with each CoV2 lineage (pre-Omicron) resulted in saliva Spike-specific IgM, IgA, and IgG responses, the latter increasing significantly post-infection and being more pronounced than N-specific IgG responses. Vaccination resulted in significantly higher Spike-specific IgG levels compared to unvaccinated infected individuals, and uninfected vaccinees' saliva was more capable of inhibiting Spike function. Vaccinees with breakthrough Delta infections had Spike-specific IgG levels comparable to those of uninfected vaccinees; however, their ability to inhibit Spike binding was diminished. These data are consistent with COVID vaccines having achieved hoped-for effects in our community, including the generation of mucosal antibodies that inhibit Spike and lower community viral loads, and suggest breakthrough Delta infections were not due to an absence of vaccine-elicited Ig, but instead limited Spike binding activity in the face of high community viral loads.

SARS-CoV-2 vaccination, booster, and infection in pregnant population enhances passive immunity in neonates

The effects of heterogeneous infection, vaccination and boosting histories prior to and during pregnancy have not been extensively studied and are likely important for protection of neonates. We measure levels of spike binding antibodies in 4600 patients and their neonates with different vaccination statuses, with and without history of SARS-CoV-2 infection. We investigate neutralizing antibody activity against different SARS-CoV-2 variant pseudotypes in a subset of 259 patients and determined correlation between IgG levels and variant neutralizing activity. We further study the ability of maternal antibody and neutralizing measurements to predict neutralizing antibody activity in the umbilical cord blood of neonates. In this work, we show SARS-CoV-2 vaccination and boosting, especially in the setting of previous infection, leads to significant increases in antibody levels and neutralizing activity even against the recent omicron BA.1 and BA.5 variants in both pregnant patients and their neonates.