Evaluation of the neutralising antibody response in human and hamster sera against SARS-CoV-2 variants up to and including BA.2.86 using an authentic virus neutralisation assay (preprint)

New vaccines, therapeutics and immunity elicited by natural infection create evolutionary pressure on SARS-CoV-2 to evolve and adapt to evade vaccine-induced and infection-elicited immunity. Vaccine and therapeutics developers thus find themselves in an "arms race" with the virus. The ongoing assessment of emerging SARS-CoV-2 variants remains essential as the global community transitions from an emergency response to a long-term management plan. Here, we describe how an authentic virus neutralisation assay using low passage clinical virus isolates has been employed to monitor resistance of emerging virus variants to neutralising antibodies from humans and experimentally infected hamsters. Sera and plasma from people who received three doses of a vaccine as well as those who received a bivalent booster were assessed against SARS-CoV-2 variants, up to and including BA.2.86. Contemporary or recent virus variants showed substantial resistance to neutralisation by antibodies from those who had received three vaccines but were still effectively neutralised by antibodies from individuals who had received a bivalent booster (ancestral/BA.1). Convalescent sera from hamsters that had been experimentally infected with one of seven virus variants (ancestral, BA.1, BA.4, BA.5.2.1, XBB.1.5, XBB.1.16, XBB.2.3) were also tested here. The most contemporary variant, BA.2.86, was effectively neutralised by sera from hamsters infected with XBB.1.5 and XBB.1.16 but it was not neutralised by sera from those infected with BA.5.2.1. These data support the recommendations given by the WHO that a new vaccine was required and should consist of an XBB sub-lineage antigen.

The Omicron Sub-Variant BA.4 Displays a Remarkable Lack of Clinical Signs in a Golden Syrian Hamster Model of SARS-CoV-2 Infection (preprint)

The ongoing emergence of SARS-CoV-2 virus variants remains a source of concern because it is accompanied by the potential for increased virulence as well as evasion of immunity. Here we show that, although having an almost identical spike gene sequence as another Omicron variant (BA.5.2.1), a BA.4 isolate lacked all the typical disease characteristics of other isolates seen in the Golden Syrian hamster model despite replicating almost as effectively. Animals infected with BA.4 had similar viral shedding profiles to that seen with BA.5.2.1 (up to day 6 post infection) but they all failed to lose weight or present with any other significant clinical signs. We hypothesize that this lack of detectable signs of disease during infection with BA.4 was due to a small (nine nucleotide) deletion (∆686-694) in the viral genome (ORF1ab) responsible for production of non-structural protein 1 which resulted in the loss of three amino acids (aa 141-143).

SARS-CoV-2 Disease Severity in the Golden Syrian Hamster Model of Infection is Related to the Volume of Intranasal Inoculum. (preprint)

The Golden Syrian hamster (Mesocricetus auratus) is now commonly used in preclinical research for the study of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and the assessment of vaccines, drugs and therapeutics. Here we show that hamsters inoculated via the intranasal route with the same infectious virus dose of prototypical SARS-CoV-2 administered in a different volume present with different clinical signs, weight loss and viral shedding, with a reduced volume resulting in reduced severity of disease similar to that obtained by a 500-fold reduction in challenge dose. The tissue burden of virus and the severity of pulmonary pathology were also significantly affected by different challenge inoculum volumes. These findings suggest that direct comparison between the severity of SARS-CoV-2 variants or studies assessing the efficacy of treatments determined by hamster studies cannot be made unless both the challenge dose and inoculation volume are matched when using the intranasal route. Additionally, analysis of sub-genomic and total genomic RNA PCR data demonstrated no link between sub-genomic and live viral titres and that sub-genomic analyses do not provide any information beyond that provided by more sensitive total genomic PCR.

Hypoxia inducible factors regulate infectious SARS-CoV-2, epithelial damage and respiratory symptoms in a hamster COVID-19 model. (preprint)

Understanding the host pathways that define susceptibility to SARS-CoV-2 infection and disease are essential for the design of new therapies. Oxygen levels in the microenvironment define the transcriptional landscape, however the influence of hypoxia on virus replication and disease in animal models is not well understood. In this study, we identify a role for the hypoxic inducible factor (HIF) signalling axis to inhibit SARS-CoV-2 infection, epithelial damage and respiratory symptoms in Syrian hamsters. Pharmacological activation of HIF with the prolyl-hydroxylase inhibitor FG-4592 significantly reduced the levels of infectious virus in the upper and lower respiratory tract. Nasal and lung epithelia showed a reduction in SARS-CoV-2 RNA and nucleocapsid expression in treated animals. Transcriptomic and pathological analysis showed reduced epithelial damage and increased expression of ciliated cells. Our study provides new insights on the intrinsic antiviral properties of the HIF signalling pathway in SARS-CoV-2 replication that may be applicable to other respiratory pathogens and identifies new therapeutic opportunities.

Rapid selection of P323L in the SARS-CoV-2 polymerase (NSP12) in humans and non-human primate models and confers a large plaque phenotype (preprint)

The mutational landscape of SARS-CoV-2 varies at both the dominant viral genome sequence and minor genomic variant population. An early change associated with transmissibility was the D614G substitution in the spike protein. This appeared to be accompanied by a P323L substitution in the viral polymerase (NSP12), but this latter change was not under strong selective pressure. Investigation of P323L/D614G changes in the human population showed rapid emergence during the containment phase and early surge phase of wave 1 in the UK. This rapid substitution was from minor genomic variants to become part of the dominant viral genome sequence. A rapid emergence of 323L but not 614G was observed in a non-human primate model of COVID-19 using a starting virus with P323 and D614 in the dominant genome sequence and 323L and 614G in the minor variant population. In cell culture, a recombinant virus with 323L in NSP12 had a larger plaque size than the same recombinant virus with P323. These data suggest that it may be possible to predict the emergence of a new variant based on tracking the distribution and frequency of minor variant genomes at a population level, rather than just focusing on providing information on the dominant viral genome sequence e.g., consensus level reporting. The ability to predict an emerging variant of SARS-CoV-2 in the global landscape may aid in the evaluation of medical countermeasures and non-pharmaceutical interventions.

Convalescence from prototype SARS-CoV-2 protects Syrian hamsters from disease caused by the Omicron variant (preprint)

The mutation profile of the SARS-CoV-2 Omicron variant poses a concern for naturally acquired and vaccine-induced immunity. We investigated the ability of prior infection with an early SARS-CoV-2, 99.99% identical to Wuhan-Hu-1, to protect against disease caused by the Omicron variant. We established that infection with Omicron in naive Syrian hamsters resulted in a less severe disease than a comparable dose of prototype SARS-CoV-2 (Australia/VIC01/2020), with fewer clinical signs and less weight loss. We present data to show that these clinical observations were almost absent in convalescent hamsters challenged with the same dose of Omicron 50 days after an initial infection with Australia/VIC01/2020. The data provide evidence for immunity raised against prototype SARS-CoV-2 being protective against Omicron in the Syrian hamster model. Further investigation is required to conclusively determine whether Omicron is less pathogenic in Syrian hamsters and whether this is predictive of pathogenicity in humans.

A potent SARS-CoV-2 neutralising nanobody shows therapeutic efficacy in the Syrian golden hamster model of COVID-19 (preprint)

SARS-CoV-2 remains a global threat to human health particularly as escape mutants emerge. There is an unmet need for effective treatments against COVID-19 for which neutralizing single domain antibodies (nanobodies) have significant potential. Their small size and stability mean that nanobodies are compatible with respiratory administration. We report four nanobodies (C5, H3, C1, F2) engineered as homotrimers with pmolar affinity for the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Crystal structures show C5 and H3 overlap the ACE2 epitope, whilst C1 and F2 bind to a different epitope. Cryo Electron Microscopy shows C5 binding results in an all down arrangement of the Spike protein. C1, H3 and C5 all neutralize the Victoria strain, and the highly transmissible Alpha (B.1.1.7 first identified in Kent, UK) strain and C1 also neutralizes the Beta (B.1.35, first identified in South Africa). Administration of C5-trimer via the respiratory route showed potent therapeutic efficacy in the Syrian hamster model of COVID-19 and separately effective prophylaxis. The molecule was similarly potent by intraperitoneal injection.

One or two dose regimen of the SARS-CoV-2 synthetic DNA vaccine INO-4800 protects against respiratory tract disease burden in nonhuman primate challenge model (preprint)

Safe and effective vaccines will provide essential medical countermeasures to tackle the COVID-19 pandemic. Here, we evaluate the safety, immunogenicity and efficacy of the intradermal delivery of INO-4800, a synthetic DNA vaccine candidate encoding the SARS-CoV-2 spike protein in the rhesus macaque model. Single and 2 dose vaccination regimens were evaluated. Vaccination induces both binding and neutralizing antibodies, along with IFN-γ-producing T cells against SARS-CoV-2. Upon administration of a high viral dose (5 x 106 pfu) via the intranasal and intratracheal routes we observe significantly reduced virus load in the lung and throat, in the vaccinated animals compared to controls. 2 doses of INO-4800 is associated with more robust vaccine-induced immune responses and improved viral protection. Importantly, histopathological examination of lung tissue provides no indication of vaccine-enhanced disease following SARS-CoV-2 challenge in INO-4800 immunized animals. This vaccine candidate is currently under clinical evaluation as a 2 dose regimen.

Sequential delivery of LAIV and SARS-CoV-2 in the ferret model can reduce SARS-CoV-2 shedding and does not result in enhanced lung pathology. (preprint)

Co-circulation of SARS-CoV-2 and influenza viruses could pose unpredictable risks to health systems globally, with recent studies suggesting more severe disease outcomes in co-infected patients. The lack of a readily available COVID-19 vaccine has reinforced the importance of influenza vaccine programmes during the COVID-19 pandemic. Live Attenuated Influenza Vaccine (LAIV) is an important tool in protecting against influenza, particularly in children. However, it is unknown whether LAIV administration might influence the outcomes of acute SARS-CoV-2 infection or disease. To investigate this, quadrivalent LAIV (QLAIV) was administered to ferrets 3 days pre- or post-SARS-CoV-2 infection. LAIV administration did not exacerbate SARS-CoV-2 disease course or lung pathology with either regimen. Additionally, LAIV administered prior to SARS-CoV-2 infection significantly reduced SARS-CoV-2 replication and shedding in the upper respiratory tract (URT). We conclude that LAIV administration in close proximity to SARS-CoV-2 infection does not exacerbate mild disease and can reduce SARS-CoV-2 shedding.

mRNA vaccine CVnCoV protects non-human primates from SARS-CoV-2 challenge infection (preprint)

The ongoing severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic necessitates the fast development of vaccines to meet a worldwide need. mRNA-based vaccines are the most promising technology for rapid and safe SARS-CoV-2 vaccine development and production. We have designed CVnCoV, a lipid-nanoparticle (LNP) encapsulated, sequence optimised mRNA-based SARS-CoV-2 vaccine that encodes for full length, pre-fusion stabilised Spike protein. Unlike other mRNA-based approaches, CVnCoV exclusively consists of non-chemically modified nucleotides and can be applied at comparatively low doses. Here we demonstrate that CVnCoV induces robust humoral and cellular responses in non-human primates (NHPs). Animals vaccinated with 8 g of CVnCoV were protected from challenge infection with SARS-CoV-2. Comprehensive analyses of pathological changes in challenged animals via lung histopathology and Computed Tomography (CT) scans gave no indication of enhanced disease upon CVnCoV vaccination. These results demonstrate safety, immunogenicity, and protective efficacy of CVnCoV in NHPs that extend our previously published preclinical data and provide strong support for further clinical testing in ongoing phase 2b/3 efficacy studies.

Changes in SARS-CoV-2 before the peak in each country, producing unique variants. (preprint)

The second and third waves of coronavirus disease 2019 (COVID-19) have caused problems worldwide. Those are often thought to have resulted from people's carelessness or people not following restrictions, but in reality, the cause remains unclear. Here, using an objective analytical method, we present the changes in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus causing COVID-19 over time. The virus has mutated in three major directions, with three groups remaining to date. The basic structure of the groups was completed by April and shared across all continents. However, the virus continued to mutate independently in each country after the borders were closed. In particular, the virus mutated before the occurrence of a second or third peak. It seems that the mutations conferred higher infectivity to the virus, because of which the virus overcame previously effective protections. Currently, each country may possess such a unique stronger variant, which may cause another peak in other countries. These viruses could also serve as sources of mutations by exchanging parts of the genome. Such mutations could create a variant with superior infectivity.

The SARS-CoV-2 spike protein disrupts the cooperative function of human cardiac pericytes - endothelial cells through CD147 receptor-mediated signalling: a potential non-infective mechanism of COVID-19 microvascular disease (preprint)

Background: Severe coronavirus disease 2019 (COVID-19) manifests as a life-threatening microvascular syndrome. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses primarily the capsid spike (S) protein to engage with its receptors and infect host cells. To date, it is still not known if the S protein alone, without the other viral elements, is able to trigger vascular cell signalling and provoke cell dysfunction. Methods: We investigated the effects of the recombinant, stabilised S protein on primary human cardiac pericytes (PCs) signalling and function. Endpoints included cell viability, proliferation, migration, cooperation with endothelial cells (ECs) in angiogenesis assays, and release of pro-inflammatory cytokines. Adopting a blocking strategy against the S protein receptors ACE2 and CD147, we explored which receptor mediates the S protein signalling in PCs. Findings: We show, for the first time, that the recombinant S protein alone elicits functional alterations in cardiac PCs. This was documented as: (1) increased migration, (2) reduced ability to support EC network formation on Matrigel, (3) secretion of pro-inflammatory molecules typically involved in the cytokine storm; and (4) production of pro-apoptotic factors responsible for EC death. Furthermore, the S protein stimulates the phosphorylation/activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) through the CD147 receptor, but not ACE2, in cardiac PCs. Accordingly, the neutralization of CD147, using a blocking antibody, prevented the activation of ERK1/2 and partially rescued the PC function in the presence of the S protein. Interpretation: Our findings suggest the new, intriguing hypothesis that the S protein may elicit vascular cell dysfunction, potentially amplifying, or perpetuating, the damage caused by the whole coronavirus. This mechanism may have clinical and therapeutic implication.

Immunological and pathological outcomes of SARS-CoV-2 challenge after formalin-inactivated vaccine immunisation of ferrets and rhesus macaques (preprint)

There is an urgent requirement for safe and effective vaccines to prevent novel coronavirus disease (COVID-19) caused by SARS-CoV-2. A concern for the development of new viral vaccines is the potential to induce vaccine-enhanced disease (VED). This was reported in several preclinical studies with both SARS-CoV-1 and MERS vaccines but has not been reported with SARS-CoV-2 vaccines. We have used ferret and rhesus macaques challenged with SARS-CoV-2 to assess the potential for VED in animals vaccinated with formaldehyde-inactivated SARS-CoV-2 (FIV) formulated with Alhydrogel, compared to a negative control vaccine in ferrets or unvaccinated macaques. We showed no evidence of enhanced disease in ferrets or rhesus macaques given FIV except for mild transient enhanced disease seen at seven days post infection in ferrets. This increased lung pathology was observed early in the infection (day 7) but was resolved by day 15. We also demonstrate that formaldehyde treatment of SARS-CoV-2 reduces exposure of the spike receptor binding domain providing a mechanistic explanation for suboptimal immunity.

Cetylpyridinium chloride-containing mouthwashes reduce in vitro SARS-CoV-2 infectivity (preprint)

Oral mouthwashes decrease the infectivity of several respiratory viruses including SARS-CoV-2. However, the precise agents with antiviral activity present in these oral rinses and their exact mechanism of action remain unknown. Here we show that Cetylpyridinium chloride (CPC), a quaternary ammonium compound present in many oral mouthwashes, reduces SARS-CoV-2 infectivity by inhibiting viral fusion with target cells. We also found that CPC and CPC-containing mouth rinses decreased a thousand times the infectivity of SARS-CoV-2 in vitro, while the corresponding vehicles had no effect. CPC-containing mouth rinses could represent a cost-effective measure to reduce SARS-CoV-2 infectivity in saliva, aiding to reduce viral transmission from infected individuals.

From infection to immunity: understanding the response to SARS-CoV2 through in-silico modeling (preprint)

Background: Immune system conditions of the patient is a key factor in COVID-19 infection survival. A growing number of studies have focused on immunological determinants to develop better biomarkers for therapies. Aim: The dynamics of the insurgence of immunity is at the core of the both SARS-CoV-2 vaccine development and therapies. This paper addresses a fundamental question in the management of the infection: can we describe the insurgence (and the span) of immunity in COVID-19? The in-silico model developed here answers this question at individual (personalized) and population levels. We simulate the immune response to SARS-CoV-2 and analyze the impact of infecting viral load, affinity to the ACE2 receptor and age in the artificially infected population on the course of the disease. Methods: We use a stochastic agent-based immune simulation platform to construct a virtual cohort of infected individuals with age-dependent varying degree of immune competence. We use a parameter setting to reproduce known inter-patient variability and general epidemiological statistics. Results: We reproduce in-silico a number of clinical observations and we identify critical factors in the statistical evolution of the infection. In particular we evidence the importance of the humoral response over the cytotoxic response and find that the antibody titers measured after day 25 from the infection is a prognostic factor for determining the clinical outcome of the infection. Our modeling framework uses COVID-19 infection to demonstrate the actionable effectiveness of simulating the immune response at individual and population levels. The model developed is able to explain and interpret observed patterns of infection and makes verifiable temporal predictions. Within the limitations imposed by the simulated environment, this work proposes in a quantitative way that the great variability observed in the patient outcomes in real life can be the mere result of subtle variability in the infecting viral load and immune competence in the population. In this work we i) show the power of model predictions, ii) identify the clinical end points that could be more suitable for computational modeling of COVID-19 immune response, iii) define the resolution and amount of data required to empower this class of models for translational medicine purposes and, iv) we exemplify how computational modeling of immune response provides an important light to discuss hypothesis and design new experiments.

Binding strength and hydrogen bond numbers between Covid-19 RBD and HVR of antibody (preprint)

The global battle against the Covid-19 pandemic relies strongly on the human defence of antibody, which is assumed to bind the Receptor Binding Domain of the antigen with its Hypervariable Region. Due to the similarity to other viruses such as SARS, however, our understanding of the antibody-virus interaction has been limited to the genomic sequencing, which poses serious challenges to the containment, vaccine exploration and rapid serum testing. Based on the physical/chemical nature of the interaction, infrared spectroscopy was employed to reveal the binding disparity, when unusual temperature dependence was discovered from the 1550cm 1 absorption band, attributed to the hydrogen bonds by carboxyl/amino groups, binding the SARS-CoV-2 spike protein and closely resembled SARS-CoV-2 or SARS-CoV-1 antibodies. The infrared absorption intensity, associated with the number of hydrogen bonds, was found to increase sharply between 27C and 31C, with the relative absorbance matches at 37C the hydrogen bonding numbers of the two antibody types (19 vs 12). As a result, the specificity of the SARS-CoV-2 antibody will be more conclusive beyond 31C, instead of at the usual room temperature of 20C - 25C, when the vaccine research and antibody diagnosis would likely be undermined.

Predicting COVID-19 cases with unknown homogeneous or heterogeneous resistance to infectivity (preprint)

This article constructs a restricted infection rate inverse binomial-based approach to predict COVID-19 cases after a family gathering. The traditional inverse binomial (IB) model is unqualified to match the reality of COVID-19, because the data contradicts the models requirement that variance should be greater than expected value. A refined version of the IB model is a necessity to predict COVID-19 cases after family gatherings. Our refined version of an IB model is more appropriate and versatile, as it accommodates all potential data scenarios: equal, lesser, or greater variance than expected value. Application of the approach is based on a restricted infectivity rate and methodology on Fan et al.s COVID-19 data, which exhibits two clusters of infectivity. Cluster 1 has a smaller number of primary cases and exhibits larger variance than the expected cases with a negative correlation of 28%, implying that the number of secondary cases is lesser when the number of primary cases increases and vice versa. The traditional inverse binomial (IB) model is appropriate for Cluster 1. The probability of contracting COVID-19 is estimated to be 0.13 among the primary, but is 0.75 among the secondary in Cluster 1, with a wider gap. Conversely, Cluster 2, exhibits smaller variance than the expected cases with a correlation of 79%, implying the number of primary and secondary cases increase or decrease together. Cluster 2 disqualifies the traditional IB model and demands its refined version. Probability of contracting COVID-19 is estimated to be 0.74 among the primary, but is 0.72 among the secondary in Cluster 2, with a narrower gap. The models ability to estimate the communitys health system memory for future policies to be developed is an asset of this approach. The current hazard level to be infected with COVID-19 among the primary and secondary groups are estimable and interpretable. Author SummaryCurrent statistical models are not able to accurately predict disease infection spread in the COVID-19 pandemic. We have applied a widely-used inverse binomial method to predict rates of infection after small gatherings, going from primary (original) cases to secondary (later) cases after family gatherings or social events, using the data from the Wuhan and Gansu provinces in China, where the virus first spread. The advantages of the proposed approach include that the models ability to estimate the communitys health system memory for future policies to be developed, as such policies might reduce COVIDs spread if not its control. In our approach, as demonstrated, the current hazard level of becoming infected with COVID-19 and the odds of contracting COVID-19 among the primary in comparison to the secondary groups are estimable and interpretable. We hope the proposed approach will be used in future epidemics.

In vitro characterization of engineered red blood cells as potent viral traps against HIV-1 and SARS-CoV-2 (preprint)

Engineered red blood cells (RBCs) expressing viral receptors could be used therapeutically as viral traps as RBCs lack nuclei and other organelles required for viral replication. Here we show that the combination of a powerful erythroid-specific expression system and transgene codon optimization yields high expression levels of the HIV-1 receptors CD4 and CCR5, as well as a CD4-glycophorin A (CD4-GpA) fusion protein on enucleated RBCs. Engineered RBCs expressing CD4 and CCR5 were efficiently infected by HIV-1, but CD4 or CD4-GpA expression in the absence of CCR5 was sufficient to potently neutralize HIV-1 in vitro. To facilitate continuous large-scale production of engineered RBCs, we generated erythroblast cell lines stably expressing CD4-GpA or ACE2-GpA fusion proteins, which produced potent RBC viral traps against HIV-1 and SARS-CoV-2. Our results suggest that this approach warrants further investigation as a potential treatment against viral infections.

A recombinant protein SARS-CoV-2 candidate vaccine elicits high-titer neutralizing antibodies in macaques. (preprint)

Vaccines that generate robust and long-lived protective immunity against SARS-CoV-2 infection are urgently required. We assessed the potential of vaccine candidates based on the SARS-CoV-2 spike in cynomolgus macaques (M. fascicularis) by examining their ability to generate spike binding antibodies with neutralizing activity. Antigens were derived from two distinct regions of the spike S1 subunit, either the N-terminal domain (NTD) or an extended C-terminal domain containing the receptor-binding domain (RBD) and were fused to the human IgG1 Fc domain. Three groups of 2 animals each were immunized with either each antigen, alone or in combination. The development of antibody responses was evaluated through 20 weeks post-immunization. A robust IgG response to the spike protein was detected as early as 2 weeks after immunization with either protein and was maintained for over 20 weeks. Sera from animals immunized with antigens derived from the RBD were able to prevent binding of soluble spike proteins to the ACE2 receptor, shown by in vitro binding assays, while sera from animals immunized with the NTD alone lacked this activity. Crucially, sera from animals immunized with the RBD but not the NTD had potent neutralizing activity against SARS-CoV-2 pseudotyped virus, with titers in excess of 10,000, greatly exceeding that typically found in convalescent humans. Neutralizing activity persisted for more than 20 weeks. These data support the utility of spike subunit-based antigens as a vaccine for use in humans.

In vitro measurements of protein-protein interactions show that antibody affinity governs the inhibition of SARS-CoV-2 spike/ACE2 binding in convalescent serum (preprint)

The humoral immune response plays a key role in suppressing the pathogenesis of SARS-CoV-2. The molecular determinants underlying the neutralization of the virus remain, however, incompletely understood. Here, we show that the ability of antibodies to disrupt the binding of the viral spike protein to the angiotensin-converting enzyme 2 (ACE2) receptor on the cell, the key molecular event initiating SARS-CoV-2 entry into host cells, is controlled by the affinity of these antibodies to the viral antigen. By using microfluidic antibody-affinity profiling, we were able to quantify the serum-antibody mediated inhibition of ACE2-spike binding in two SARS-CoV-2 seropositive individuals. Measurements to determine the affinity, concentration, and neutralization potential of antibodies were performed directly in human serum. Using this approach, we demonstrate that the level of inhibition in both samples can be quantitatively described using the binding energies of the binary interactions between the ACE2 receptor and the spike protein, and the spike protein and the neutralizing antibody. These experiments represent a new type of in-solution receptor binding competition assay, which has further potential areas of application ranging from decisions on donor selection for convalescent plasma therapy, to identification of lead candidates in therapeutic antibody development, and vaccine development.