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.

Human seasonal coronavirus neutralization and COVID-19 severity.

The virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for the global coronavirus disease-2019 (COVID-19) pandemic, spread rapidly around the world causing high morbidity and mortality. However, there are four known, endemic seasonal coronaviruses in humans (HCoVs), and whether antibodies for these HCoVs play a role in severity of COVID-19 disease has generated a lot of interest. Of these seasonal viruses NL63 is of particular interest as it uses the same cell entry receptor as SARS-CoV-2. We use functional, neutralizing assays to investigate cross-reactive antibodies and their relationship with COVID-19 severity. We analyzed the neutralization of SARS-CoV-2, NL63, HKU1, and 229E in 38 COVID-19 patients and 62 healthcare workers, and a further 182 samples to specifically study the relationship between SARS-CoV-2 and NL63. We found that although HCoV neutralization was very common there was little evidence that these antibodies neutralized SARS-CoV-2. Despite no evidence in cross-neutralization, levels of NL63 neutralizing antibodies become elevated after exposure to SARS-CoV-2 through infection or following vaccination.

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.

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.

A double pedigree reveals genetic but not cultural inheritance of cooperative personalities in wild banded mongooses.

Personality traits, such as the propensity to cooperate, are often inherited from parents to offspring, but the pathway of inheritance is unclear. Traits could be inherited via genetic or parental effects, or culturally via social learning from role models. However, these pathways are difficult to disentangle in natural systems as parents are usually the source of all of these effects. Here, we exploit natural 'cross fostering' in wild banded mongooses to investigate the inheritance of cooperative behaviour. Our analysis of 800 adult helpers over 21 years showed low but significant genetic heritability of cooperative personalities in males but not females. Cross fostering revealed little evidence of cultural heritability: offspring reared by particularly cooperative helpers did not become more cooperative themselves. Our results demonstrate that cooperative personalities are not always highly heritable in wild, and that the basis of behavioural traits can vary within a species (here, by sex).

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.

Plasticity-Led Evolution and Human Culture.

Some human traits arise via organic evolution while others are acquired from the prevailing culture via a process of social learning. A mainstream interpretation is that evolution amounts to a change in the relative frequency of gene variants in a population and that culture coevolves at arm's length. Matters look different if one starts instead from the view that organisms are modified during evolution because of changes in gene expression as much as changes in the relative frequency of gene variants. Gene expression, i.e. generation of the product encoded by a gene, is not under genetic control, for it requires location- and time-specific triggers, which cannot be provided by genes. The genes present in an individual are present in every cell, hence at all locations in the individual's body and at all times during the individual's life. The necessary location- and time-specific triggers are provided internally by developmental events and conditions, or externally by environmental events and conditions, i.e. non-genetically. Socially-learned traits, having no special connection with genes, may nevertheless influence evolution, as for any trait. Like organic traits generally, socially-learned traits can be positively or negatively selected, for they similarly influence survival and reproduction. Like learned traits generally, they can play an important role in evolution by providing repeated selective pressure. The resulting evolutionary change typically affects an associated trait (e.g. adult ability to digest the sugar contained in milk), not the socially-learned trait itself (e.g. dairying), which continues under the influence of cultural processes of change.

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.

Inbreeding depresses altruism in a cooperative society.

In some animal species, individuals regularly breed with relatives, including siblings and parents. Given the high fitness costs of inbreeding, evolutionary biologists have found it challenging to understand the persistence of these inbred societies in nature. One appealing but untested explanation is that early life care may create a benign environment that offsets inbreeding depression, allowing inbred societies to evolve. We test this possibility using 21 years of data from a wild cooperatively breeding mammal, the banded mongoose, a species where almost one in ten young result from close inbreeding. We show that care provided by parents and alloparents mitigates inbreeding depression for early survival. However, as adults, inbred individuals provide less care, reducing the amount of help available to the next generation. Our results suggest that inbred cooperative societies are rare in nature partly because the protective care that enables elevated levels of inbreeding can be reduced by inbreeding depression.

Decoupling of Genetic and Cultural Inheritance in a Wild Mammal.

Cultural inheritance, the transmission of socially learned information across generations, is a non-genetic, "second inheritance system" capable of shaping phenotypic variation in humans and many non-human animals [1-3]. Studies of wild animals show that conformity [4, 5] and biases toward copying particular individuals [6, 7] can result in the rapid spread of culturally transmitted behavioral traits and a consequent increase in behavioral homogeneity within groups and populations [8, 9]. These findings support classic models of cultural evolution [10, 11], which predict that many-to-one or one-to-many transmission erodes within-group variance in culturally inherited traits. However, classic theory [10, 11] also predicts that within-group heterogeneity is preserved when offspring each learn from an exclusive role model. We tested this prediction in a wild mammal, the banded mongoose (Mungos mungo), in which offspring are reared by specific adult carers that are not their parents, providing an opportunity to disentangle genetic and cultural inheritance of behavior. We show using stable isotope analysis that young mongooses inherit their adult foraging niche from cultural role models, not from their genetic parents. As predicted by theory, one-to-one cultural transmission prevented blending inheritance and allowed the stable coexistence of distinct behavioral traditions within the same social groups. Our results confirm that cultural inheritance via role models can promote rather than erode behavioral heterogeneity in natural populations.

A high-quality pedigree and genetic markers both reveal inbreeding depression for quality but not survival in a cooperative mammal.

Inbreeding depression, the reduced fitness of offspring of closely related parents, is commonplace in both captive and wild populations and has important consequences for conservation and mating system evolution. However, because of the difficulty of collecting pedigree and life-history data from wild populations, relatively few studies have been able to compare inbreeding depression for traits at different points in the life cycle. Moreover, pedigrees give the expected proportion of the genome that is identical by descent (IBDg ) whereas in theory with enough molecular markers realized IBDg can be quantified directly. We therefore investigated inbreeding depression for multiple life-history traits in a wild population of banded mongooses using pedigree-based inbreeding coefficients (fped ) and standardized multilocus heterozygosity (sMLH) measured at 35-43 microsatellites. Within an information theoretic framework, we evaluated support for either fped or sMLH as inbreeding terms and used sequential regression to determine whether the residuals of sMLH on fped explain fitness variation above and beyond fped . We found no evidence of inbreeding depression for survival, either before or after nutritional independence. By contrast, inbreeding was negatively associated with two quality-related traits, yearling body mass and annual male reproductive success. Yearling body mass was associated with fped but not sMLH, while male annual reproductive success was best explained by both fped and residual sMLH. Thus, our study not only uncovers variation in the extent to which different traits show inbreeding depression, but also reveals trait-specific differences in the ability of pedigrees and molecular markers to explain fitness variation and suggests that for certain traits, genetic markers may capture variation in realized IBDg above and beyond the pedigree expectation.

Male brush-turkeys attempt sexual coercion in unusual circumstances.

Sexual coercion by males is generally understood to have three forms: forced copulation, harassment and intimidation. We studied Australian brush-turkeys, Alectura lathami, to determine whether some male behaviours towards females at incubation mounds could be classified as aggressive, whether males were attempting sexual coercion and, if so, whether the coercion was successful. We found that some male behaviours towards females were significantly more likely to be followed by the cessation of female mound activity, and hence could be classified as aggressive, while others were significantly more likely to be followed by the commencement of female mound activity, and hence could be classified as enticing. Copulation was preceded by higher rates of male enticement and by higher rates of certain types of male aggression. It therefore seemed that males were attempting sexual coercion. There was little evidence, however, that this combination of coercion and enticement was successful in obtaining copulations. While forced copulation did occur, it was infrequent, and no evidence could be found for intimidation. We conclude that harassment is the primary form of sexual coercion by male brush-turkeys. Although sexual coercion is understood to be a sub-optimal tactic, brush-turkey sexual coercion was employed as a primary tactic by dominant males who owned incubation mounds. One possible explanation for this apparent paradox is that aggression is the default solution for social conflicts in this species, and hence can be interpreted as a behavioural syndrome.