Inhibition of the Lectin Pathway of Complement Activation Reduces Acute Respiratory Distress Syndrome Severity in a Mouse Model of SARS-CoV-2 Infection.

Most patients with COVID-19 in the intensive care unit develop an acute respiratory distress syndrome characterized by severe hypoxemia, decreased lung compliance, and high vascular permeability. Activation of the complement system is a hallmark of moderate and severe COVID-19, with abundant deposition of complement proteins in inflamed tissue and on the endothelium during COVID-19. Using a transgenic mouse model of SARS-CoV-2 infection, we assessed the therapeutic utility of an inhibitory antibody (HG4) targeting MASP-2, a key enzyme in the lectin pathway. Treatment of infected mice with HG4 reduced the disease severity score and improved survival vs mice that received an isotype control antibody. Administration of HG4 significantly reduced the lung injury score, including alveolar inflammatory cell infiltration, alveolar edema, and alveolar hemorrhage. The ameliorating effect of MASP-2 inhibition on the severity of COVID-19 pathology is reflected by a significant reduction in the proinflammatory activation of brain microglia in HG4-treated mice.

The SARS-CoV-2 protein ORF3c is a mitochondrial modulator of innate immunity.

The SARS-CoV-2 genome encodes a multitude of accessory proteins. Using comparative genomic approaches, an additional accessory protein, ORF3c, has been predicted to be encoded within the ORF3a sgmRNA. Expression of ORF3c during infection has been confirmed independently by ribosome profiling. Despite ORF3c also being present in the 2002-2003 SARS-CoV, its function has remained unexplored. Here we show that ORF3c localizes to mitochondria, where it inhibits innate immunity by restricting IFN-ß production, but not NF-κB activation or JAK-STAT signaling downstream of type I IFN stimulation. We find that ORF3c is inhibitory after stimulation with cytoplasmic RNA helicases RIG-I or MDA5 or adaptor protein MAVS, but not after TRIF, TBK1 or phospho-IRF3 stimulation. ORF3c co-immunoprecipitates with the antiviral proteins MAVS and PGAM5 and induces MAVS cleavage by caspase-3. Together, these data provide insight into an uncharacterized mechanism of innate immune evasion by this important human pathogen.

Tetherin antagonism by SARS-CoV-2 ORF3a and spike protein enhances virus release.

The antiviral restriction factor, tetherin, blocks the release of several different families of enveloped viruses, including the Coronaviridae. Tetherin is an interferon-induced protein that forms parallel homodimers between the host cell and viral particles, linking viruses to the surface of infected cells and inhibiting their release. We demonstrate that SARS-CoV-2 infection causes tetherin downregulation and that tetherin depletion from cells enhances SARS-CoV-2 viral titres. We investigate the potential viral proteins involved in abrogating tetherin function and find that SARS-CoV-2 ORF3a reduces tetherin localisation within biosynthetic organelles where Coronaviruses bud, and increases tetherin localisation to late endocytic organelles via reduced retrograde recycling. We also find that expression of Spike protein causes a reduction in cellular tetherin levels. Our results confirm that tetherin acts as a host restriction factor for SARS-CoV-2 and highlight the multiple distinct mechanisms by which SARS-CoV-2 subverts tetherin function.

Differential T-cell and antibody responses induced by mRNA versus adenoviral vectored COVID-19 vaccines in patients with immunodeficiencies.

Background: Immunodeficient patients (IDPs) are at higher risk of contracting severe coronavirus disease 2019 (COVID-19). Targeted vaccination strategies have been implemented to enhance vaccine-induced protection. In this population, however, clinical effectiveness is variable and the duration of protection unknown. Objective: We sought to better understand the cellular and humoral immune responses to mRNA and adenoviral vectored COVID-19 vaccines in patients with immunodeficiency. Methods: Immune responses to severe acute respiratory syndrome coronavirus 2 spike were assessed after 2 doses of homologous ChAdOx1-nCoV-19 or BNT162b2 vaccines in 112 infection-naive IDPs and 131 healthy health care workers as controls. Predictors of vaccine responsiveness were investigated. Results: Immune responses to vaccination were low, and virus neutralization by antibody was not detected despite high titer binding responses in many IDPs. In those exhibiting response, the frequency of specific T-cell responses in IDPs was similar to controls, while antibody responses were lower. Sustained vaccine specific differences were identified: T-cell responses were greater in ChAdOx1-nCoV-19- compared to BNT162b2-immunized IDPs, and antibody binding and neutralization were greater in all cohorts immunized with BNT162b2. The positive correlation between T-cell and antibody responses was weak and increased with subsequent vaccination. Conclusion: Immunodeficient patients have impaired immune responses to mRNA and viral vector COVID-19 vaccines that appear to be influenced by vaccine formulation. Understanding the relative roles of T-cell- and antibody-mediated protection as well as the potential of heterologous prime and boost immunization protocols is needed to optimize the vaccination approach in these high-risk groups.

Glycan masking of a non-neutralising epitope enhances neutralising antibodies targeting the RBD of SARS-CoV-2 and its variants.

The accelerated development of the first generation COVID-19 vaccines has saved millions of lives, and potentially more from the long-term sequelae of SARS-CoV-2 infection. The most successful vaccine candidates have used the full-length SARS-CoV-2 spike protein as an immunogen. As expected of RNA viruses, new variants have evolved and quickly replaced the original wild-type SARS-CoV-2, leading to escape from natural infection or vaccine induced immunity provided by the original SARS-CoV-2 spike sequence. Next generation vaccines that confer specific and targeted immunity to broadly neutralising epitopes on the SARS-CoV-2 spike protein against different variants of concern (VOC) offer an advance on current booster shots of previously used vaccines. Here, we present a targeted approach to elicit antibodies that neutralise both the ancestral SARS-CoV-2, and the VOCs, by introducing a specific glycosylation site on a non-neutralising epitope of the RBD. The addition of a specific glycosylation site in the RBD based vaccine candidate focused the immune response towards other broadly neutralising epitopes on the RBD. We further observed enhanced cross-neutralisation and cross-binding using a DNA-MVA CR19 prime-boost regime, thus demonstrating the superiority of the glycan engineered RBD vaccine candidate across two platforms and a promising candidate as a broad variant booster vaccine.

Influenza A (N1-N9) and Influenza B (B/Victoria and B/Yamagata) Neuraminidase Pseudotypes as Tools for Pandemic Preparedness and Improved Influenza Vaccine Design.

To better understand how inhibition of the influenza neuraminidase (NA) protein contributes to protection against influenza, we produced lentiviral vectors pseudotyped with an avian H11 hemagglutinin (HA) and the NA of all influenza A (N1-N9) subtypes and influenza B (B/Victoria and B/Yamagata). These NA viral pseudotypes (PV) possess stable NA activity and can be utilized as target antigens in in vitro assays to assess vaccine immunogenicity. Employing these NA PV, we developed an enzyme-linked lectin assay (pELLA) for routine serology to measure neuraminidase inhibition (NI) titers of reference antisera, monoclonal antibodies and post-vaccination sera with various influenza antigens. We also show that the pELLA is more sensitive than the commercially available NA-Fluor™ in detecting NA inhibition in these samples. Our studies may lead to establishing the protective NA titer that contributes to NA-based immunity. This will aid in the design of superior, longer lasting and more broadly protective vaccines that can be employed together with HA-targeted vaccines in a pre-pandemic approach.

Fatal COVID-19 outcomes are associated with an antibody response targeting epitopes shared with endemic coronaviruses.

The role of immune responses to previously seen endemic coronavirus epitopes in severe acute respiratory coronavirus 2 (SARS-CoV-2) infection and disease progression has not yet been determined. Here, we show that a key characteristic of fatal outcomes with coronavirus disease 2019 (COVID-19) is that the immune response to the SARS-CoV-2 spike protein is enriched for antibodies directed against epitopes shared with endemic beta-coronaviruses and has a lower proportion of antibodies targeting the more protective variable regions of the spike. The magnitude of antibody responses to the SARS-CoV-2 full-length spike protein, its domains and subunits, and the SARS-CoV-2 nucleocapsid also correlated strongly with responses to the endemic beta-coronavirus spike proteins in individuals admitted to an intensive care unit (ICU) with fatal COVID-19 outcomes, but not in individuals with nonfatal outcomes. This correlation was found to be due to the antibody response directed at the S2 subunit of the SARS-CoV-2 spike protein, which has the highest degree of conservation between the beta-coronavirus spike proteins. Intriguingly, antibody responses to the less cross-reactive SARS-CoV-2 nucleocapsid were not significantly different in individuals who were admitted to an ICU with fatal and nonfatal outcomes, suggesting an antibody profile in individuals with fatal outcomes consistent with an "original antigenic sin" type response.

Neutralisation Hierarchy of SARS-CoV-2 Variants of Concern Using Standardised, Quantitative Neutralisation Assays Reveals a Correlation With Disease Severity; Towards Deciphering Protective Antibody Thresholds.

The rise of SARS-CoV-2 variants has made the pursuit to define correlates of protection more troublesome, despite the availability of the World Health Organisation (WHO) International Standard for anti-SARS-CoV-2 Immunoglobulin sera, a key reagent used to standardise laboratory findings into an international unitage. Using pseudotyped virus, we examine the capacity of convalescent sera, from a well-defined cohort of healthcare workers (HCW) and Patients infected during the first wave from a national critical care centre in the UK to neutralise B.1.1.298, variants of interest (VOI) B.1.617.1 (Kappa), and four VOCs, B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.617.2 (Delta), including the B.1.617.2 K417N, informally known as Delta Plus. We utilised the WHO International Standard for anti-SARS-CoV-2 Immunoglobulin to report neutralisation antibody levels in International Units per mL. Our data demonstrate a significant reduction in the ability of first wave convalescent sera to neutralise the VOCs. Patients and HCWs with more severe COVID-19 were found to have higher antibody titres and to neutralise the VOCs more effectively than individuals with milder symptoms. Using an estimated threshold for 50% protection, 54 IU/mL, we found most asymptomatic and mild cases did not produce titres above this threshold.

SARS-CoV-2 nucleocapsid protein adheres to replication organelles before viral assembly at the Golgi/ERGIC and lysosome-mediated egress.

Despite being the target of extensive research efforts due to the COVID-19 (coronavirus disease 2019) pandemic, relatively little is known about the dynamics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication within cells. We investigate and characterize the tightly orchestrated virus assembly by visualizing the spatiotemporal dynamics of the four structural SARS-CoV-2 proteins at high resolution. The nucleoprotein is expressed first and accumulates around folded endoplasmic reticulum (ER) membranes in convoluted layers that contain viral RNA replication foci. We find that, of the three transmembrane proteins, the membrane protein appears at the Golgi apparatus/ER-to-Golgi intermediate compartment before the spike and envelope proteins. Relocation of a lysosome marker toward the assembly compartment and its detection in transport vesicles of viral proteins confirm an important role of lysosomes in SARS-CoV-2 egress. These data provide insights into the spatiotemporal regulation of SARS-CoV-2 assembly and refine the current understanding of SARS-CoV-2 replication.

Tetherin antagonism by SARS-CoV-2 enhances virus release: multiple mechanisms including ORF3a-mediated defective retrograde traffic.

The antiviral restriction factor, tetherin, blocks the release of several different families of enveloped viruses, including the Coronaviridae. Tetherin is an interferon-induced protein that forms parallel homodimers between the host cell and viral particles, linking viruses to the surface of infected cells and inhibiting their release. We demonstrated that SARS-CoV-2 infection causes tetherin downregulation, and that tetherin depletion from cells enhances SARS-CoV-2 viral titres. We investigated the potential viral proteins involved in abrogating tetherin function and found that SARS-CoV-2 ORF3a reduces tetherin localisation within biosynthetic organelles via reduced retrograde recycling and increases tetherin localisation to late endocytic organelles. By removing tetherin from the Coronavirus budding compartments, ORF3a enhances virus release. We also found expression of Spike protein caused a reduction in cellular tetherin levels. Our results confirm that tetherin acts as a host restriction factor for SARS-CoV-2 and highlight the multiple distinct mechanisms by which SARS-CoV-2 subverts tetherin function.

Analysis of Serological Biomarkers of SARS-CoV-2 Infection in Convalescent Samples From Severe, Moderate and Mild COVID-19 Cases.

Precision monitoring of antibody responses during the COVID-19 pandemic is increasingly important during large scale vaccine rollout and rise in prevalence of Severe Acute Respiratory Syndrome-related Coronavirus-2 (SARS-CoV-2) variants of concern (VOC). Equally important is defining Correlates of Protection (CoP) for SARS-CoV-2 infection and COVID-19 disease. Data from epidemiological studies and vaccine trials identified virus neutralising antibodies (Nab) and SARS-CoV-2 antigen-specific (notably RBD and S) binding antibodies as candidate CoP. In this study, we used the World Health Organisation (WHO) international standard to benchmark neutralising antibody responses and a large panel of binding antibody assays to compare convalescent sera obtained from: a) COVID-19 patients; b) SARS-CoV-2 seropositive healthcare workers (HCW) and c) seronegative HCW. The ultimate aim of this study is to identify biomarkers of humoral immunity that could be used to differentiate severe from mild or asymptomatic SARS-CoV-2 infections. Some of these biomarkers could be used to define CoP in further serological studies using samples from vaccination breakthrough and/or re-infection cases. Whenever suitable, the antibody levels of the samples studied were expressed in International Units (IU) for virus neutralisation assays or in Binding Antibody Units (BAU) for ELISA tests. In this work we used commercial and non-commercial antibody binding assays; a lateral flow test for detection of SARS-CoV-2-specific IgG/IgM; a high throughput multiplexed particle flow cytometry assay for SARS-CoV-2 Spike (S), Nucleocapsid (N) and Receptor Binding Domain (RBD) proteins); a multiplex antigen semi-automated immuno-blotting assay measuring IgM, IgA and IgG; a pseudotyped microneutralisation test (pMN) and an electroporation-dependent neutralisation assay (EDNA). Our results indicate that overall, severe COVID-19 patients showed statistically significantly higher levels of SARS-CoV-2-specific neutralising antibodies (average 1029 IU/ml) than those observed in seropositive HCW with mild or asymptomatic infections (379 IU/ml) and that clinical severity scoring, based on WHO guidelines was tightly correlated with neutralisation and RBD/S antibodies. In addition, there was a positive correlation between severity, N-antibody assays and intracellular virus neutralisation.

Exploiting Pan Influenza A and Pan Influenza B Pseudotype Libraries for Efficient Vaccine Antigen Selection.

We developed an influenza hemagglutinin (HA) pseudotype library encompassing Influenza A subtypes HA1-18 and Influenza B subtypes (both lineages) to be employed in influenza pseudotype microneutralization (pMN) assays. The pMN is highly sensitive and specific for detecting virus-specific neutralizing antibodies against influenza viruses and can be used to assess antibody functionality in vitro. Here we show the production of these viral HA pseudotypes and their employment as substitutes for wildtype viruses in influenza neutralization assays. We demonstrate their utility in detecting serum responses to vaccination with the ability to evaluate cross-subtype neutralizing responses elicited by specific vaccinating antigens. Our findings may inform further preclinical studies involving immunization dosing regimens in mice and may help in the creation and selection of better antigens for vaccine design. These HA pseudotypes can be harnessed to meet strategic objectives that contribute to the strengthening of global influenza surveillance, expansion of seasonal influenza prevention and control policies, and strengthening pandemic preparedness and response.

SARS-CoV-2 Spike Protein Stabilized in the Closed State Induces Potent Neutralizing Responses.

The majority of SARS-CoV-2 vaccines in use or advanced development are based on the viral spike protein (S) as their immunogen. S is present on virions as prefusion trimers in which the receptor binding domain (RBD) is stochastically open or closed. Neutralizing antibodies have been described against both open and closed conformations. The long-term success of vaccination strategies depends upon inducing antibodies that provide long-lasting broad immunity against evolving SARS-CoV-2 strains. Here, we have assessed the results of immunization in a mouse model using an S protein trimer stabilized in the closed state to prevent full exposure of the receptor binding site and therefore interaction with the receptor. We compared this with other modified S protein constructs, including representatives used in current vaccines. We found that all trimeric S proteins induced a T cell response and long-lived, strongly neutralizing antibody responses against 2019 SARS-CoV-2 and variants of concern P.1 and B.1.351. Notably, the protein binding properties of sera induced by the closed spike differed from those induced by standard S protein constructs. Closed S proteins induced more potent neutralizing responses than expected based on the degree to which they inhibit interactions between the RBD and ACE2. These observations suggest that closed spikes recruit different, but equally potent, immune responses than open spikes and that this is likely to include neutralizing antibodies against conformational epitopes present in the closed conformation. We suggest that closed spikes, together with their improved stability and storage properties, may be a valuable component of refined, next-generation vaccines. IMPORTANCE Vaccines in use against SARS-CoV-2 induce immune responses against the spike protein. There is intense interest in whether the antibody response induced by vaccines will be robust against new variants, as well as in next-generation vaccines for use in previously infected or immunized individuals. We assessed the use as an immunogen of a spike protein engineered to be conformationally stabilized in the closed state where the receptor binding site is occluded. Despite occlusion of the receptor binding site, the spike induces potently neutralizing sera against multiple SARS-CoV-2 variants. Antibodies are raised against a different pattern of epitopes to those induced by other spike constructs, preferring conformational epitopes present in the closed conformation. Closed spikes, or mRNA vaccines based on their sequence, can be a valuable component of next-generation vaccines.

Nanobodies mapped to cross-reactive and divergent epitopes on A(H7N9) influenza hemagglutinin using yeast display.

Influenza H7N9 virus continues to cause infections in humans and represents a significant pandemic risk. During the most recent 5th epidemic wave in 2016/17 two distinct lineages with increased human infections and wider geographical spread emerged. In preparation for any future adaptations, broadly reactive antibodies against H7N9 are required for surveillance, therapy and prophylaxis. In this study we have isolated a panel of nanobodies (Nbs) with broad reactivity across H7 influenza strains, including H7N9 strains between 2013 and 2017. We also describe Nbs capable of distinguishing between the most recent high and low pathogenicity Yangtze River Delta lineage H7N9 strains. Nanobodies were classified into 5 distinct groups based on their epitope footprint determined using yeast display and mutational scanning. The epitope footprint of Nbs capable of distinguishing high pathogenic (HP) A/Guangdong/17SF003/2016 from low pathogenic (LP) A/Hong Kong/125/2017 (H7N9) were correlated to natural sequence divergence in the head domain at lysine 164. Several Nbs binding to the head domain were capable of viral neutralisation. The potency of one nanobody NB7-14 could be increased over 1000-fold to 113 pM by linking two Nbs together. Nbs specific for distinct epitopes on H7N9 may be useful for surveillance or therapy in human or veterinary settings.

Correlation of Influenza B Haemagglutination Inhibiton, Single-Radial Haemolysis and Pseudotype-Based Microneutralisation Assays for Immunogenicity Testing of Seasonal Vaccines.

Influenza B is responsible for a significant proportion of the global morbidity, mortality and economic loss caused by influenza-related disease. Two antigenically distinct lineages co-circulate worldwide, often resulting in mismatches in vaccine coverage when vaccine predictions fail. There are currently operational issues with gold standard serological assays for influenza B, such as lack of sensitivity and requirement for specific antigen treatment. This study encompasses the gold standard assays with the more recent Pseudotype-based Microneutralisation assay in order to study comparative serological outcomes. Haemagglutination Inhibition, Single Radial Haemolysis and Pseudotype-based Microneutralisation correlated strongly for strains in the Yamagata lineage; however, it correlated with neither gold standard assays for the Victoria lineage.

AutoPlate: Rapid Dose-Response Curve Analysis for Biological Assays.

The emergence of COVID-19 has emphasised that biological assay data must be analysed quickly to develop safe, effective and timely vaccines/therapeutics. For viruses such as SARS-CoV-2, the primary way of measuring immune correlates of protection is through assays such as the pseudotype microneutralisation (pMN) assay, thanks to its safety and versatility. However, despite the presence of existing tools for data analysis such as PRISM and R the analysis of these assays remains cumbersome and time-consuming. We introduce an open-source R Shiny web application and R library (AutoPlate) to accelerate data analysis of dose-response curve immunoassays. Using example data from influenza studies, we show that AutoPlate improves on available analysis software in terms of ease of use, flexibility and speed. AutoPlate (https://philpalmer.shinyapps.io/AutoPlate/) is a tool for the use of laboratories and wider scientific community to accelerate the analysis of biological assays in the development of viral vaccines and therapeutics.

Cross-Reactive and Lineage-Specific Single Domain Antibodies against Influenza B Hemagglutinin.

Influenza B virus (IBV) circulates in the human population and causes considerable disease burden worldwide, each year. Current IBV vaccines can struggle to mount an effective cross-reactive immune response, as strains become mismatched, due to constant antigenic changes. Additional strategies which use monoclonal antibodies, with broad reactivity, are of considerable interest, both, as diagnostics and as immunotherapeutics. Alternatives to conventional monoclonal antibodies, such as single domain antibodies (NanobodiesTM) with well-documented advantages for applications in infectious disease, have been emerging. In this study we have isolated single domain antibodies (sdAbs), specific to IBV, using alpacas immunised with recombinant hemagglutinin (HA) from two representative viruses, B/Florida/04/2006 (B/Yamagata lineage) and B/Brisbane/60/2008 (B/Victoria lineage). Using phage display, we have isolated a panel of single domain antibodies (sdAbs), with both cross-reactive and lineage-specific binding. Several sdAbs recognise whole virus antigens, corresponding to influenza B strains included in vaccines spanning over 20 years, and were capable of neutralising IBV pseudotypes corresponding to prototype strains from both lineages. Lineage-specific sdAbs recognised the head domain, whereas, sdAbs identified as cross-reactive could be classified as either head binding or stem binding. Using yeast display, we were able to correlate lineage specificity with naturally occurring sequence divergence, at residue 122 in the highly variable 120 loop of the HA1 domain. The single domain antibodies described, might have applications in IBV diagnostics, vaccine potency testing and as immunotherapeutics.

A naturally protective epitope of limited variability as an influenza vaccine target.

Current antigenic targets for influenza vaccine development are either highly immunogenic epitopes of high variability or conserved epitopes of low immunogenicity. This requires continuous update of the variable epitopes in the vaccine formulation or boosting of immunity to invariant epitopes of low natural efficacy. Here we identify a highly immunogenic epitope of limited variability in the head domain of the H1 haemagglutinin protein. We show that a cohort of young children exhibit natural immunity to a set of historical influenza strains which they could not have previously encountered and that this is partially mediated through the epitope. Furthermore, vaccinating mice with these epitope conformations can induce immunity to human H1N1 influenza strains that have circulated since 1918. The identification of epitopes of limited variability offers a mechanism by which a universal influenza vaccine can be created; these vaccines would also have the potential to protect against newly emerging influenza strains.