A potent pan-sarbecovirus neutralizing antibody resilient to epitope diversification.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evolution has resulted in viral escape from clinically authorized monoclonal antibodies (mAbs), creating a need for mAbs that are resilient to epitope diversification. Broadly neutralizing coronavirus mAbs that are sufficiently potent for clinical development and retain activity despite viral evolution remain elusive. We identified a human mAb, designated VIR-7229, which targets the viral receptor-binding motif (RBM) with unprecedented cross-reactivity to all sarbecovirus clades, including non-ACE2-utilizing bat sarbecoviruses, while potently neutralizing SARS-CoV-2 variants since 2019, including the recent EG.5, BA.2.86, and JN.1. VIR-7229 tolerates extraordinary epitope variability, partly attributed to its high binding affinity, receptor molecular mimicry, and interactions with RBM backbone atoms. Consequently, VIR-7229 features a high barrier for selection of escape mutants, which are rare and associated with reduced viral fitness, underscoring its potential to be resilient to future viral evolution. VIR-7229 is a strong candidate to become a next-generation medicine.

A pseudovirus system enables deep mutational scanning of the full SARS-CoV-2 spike.

A major challenge in understanding SARS-CoV-2 evolution is interpreting the antigenic and functional effects of emerging mutations in the viral spike protein. Here, we describe a deep mutational scanning platform based on non-replicative pseudotyped lentiviruses that directly quantifies how large numbers of spike mutations impact antibody neutralization and pseudovirus infection. We apply this platform to produce libraries of the Omicron BA.1 and Delta spikes. These libraries each contain ∼7,000 distinct amino acid mutations in the context of up to ∼135,000 unique mutation combinations. We use these libraries to map escape mutations from neutralizing antibodies targeting the receptor-binding domain, N-terminal domain, and S2 subunit of spike. Overall, this work establishes a high-throughput and safe approach to measure how ∼105 combinations of mutations affect antibody neutralization and spike-mediated infection. Notably, the platform described here can be extended to the entry proteins of many other viruses.

Antibody escape of SARS-CoV-2 Omicron BA.4 and BA.5 from vaccine and BA.1 serum.

The Omicron lineage of SARS-CoV-2, which was first described in November 2021, spread rapidly to become globally dominant and has split into a number of sublineages. BA.1 dominated the initial wave but has been replaced by BA.2 in many countries. Recent sequencing from South Africa's Gauteng region uncovered two new sublineages, BA.4 and BA.5, which are taking over locally, driving a new wave. BA.4 and BA.5 contain identical spike sequences, and although closely related to BA.2, they contain further mutations in the receptor-binding domain of their spikes. Here, we study the neutralization of BA.4/5 using a range of vaccine and naturally immune serum and panels of monoclonal antibodies. BA.4/5 shows reduced neutralization by the serum from individuals vaccinated with triple doses of AstraZeneca or Pfizer vaccine compared with BA.1 and BA.2. Furthermore, using the serum from BA.1 vaccine breakthrough infections, there are, likewise, significant reductions in the neutralization of BA.4/5, raising the possibility of repeat Omicron infections.

Circulating SARS-CoV-2 spike N439K variants maintain fitness while evading antibody-mediated immunity.

SARS-CoV-2 can mutate and evade immunity, with consequences for efficacy of emerging vaccines and antibody therapeutics. Here, we demonstrate that the immunodominant SARS-CoV-2 spike (S) receptor binding motif (RBM) is a highly variable region of S and provide epidemiological, clinical, and molecular characterization of a prevalent, sentinel RBM mutation, N439K. We demonstrate N439K S protein has enhanced binding affinity to the hACE2 receptor, and N439K viruses have similar in vitro replication fitness and cause infections with similar clinical outcomes as compared to wild type. We show the N439K mutation confers resistance against several neutralizing monoclonal antibodies, including one authorized for emergency use by the US Food and Drug Administration (FDA), and reduces the activity of some polyclonal sera from persons recovered from infection. Immune evasion mutations that maintain virulence and fitness such as N439K can emerge within SARS-CoV-2 S, highlighting the need for ongoing molecular surveillance to guide development and usage of vaccines and therapeutics.

Deep mutational scanning reveals functional constraints and antibody-escape potential of Lassa virus glycoprotein complex.

Lassa virus is estimated to cause thousands of human deaths per year, primarily due to spillovers from its natural host, Mastomys rodents. Efforts to create vaccines and antibody therapeutics must account for the evolutionary variability of the Lassa virus's glycoprotein complex (GPC), which mediates viral entry into cells and is the target of neutralizing antibodies. To map the evolutionary space accessible to GPC, we used pseudovirus deep mutational scanning to measure how nearly all GPC amino-acid mutations affected cell entry and antibody neutralization. Our experiments defined functional constraints throughout GPC. We quantified how GPC mutations affected neutralization with a panel of monoclonal antibodies. All antibodies tested were escaped by mutations that existed among natural Lassa virus lineages. Overall, our work describes a biosafety-level-2 method to elucidate the mutational space accessible to GPC and shows how prospective characterization of antigenic variation could aid the design of therapeutics and vaccines.

Molecular Modeling Predicts Novel Antibody Escape Mutations in the Respiratory Syncytial Virus Fusion Glycoprotein.

Monoclonal antibodies are increasingly used for the prevention and/or treatment of viral infections. One caveat of their use is the ability of viruses to evolve resistance to antibody binding and neutralization. Computational strategies to identify viral mutations that may disrupt antibody binding would leverage the wealth of viral genomic sequence data to monitor for potential antibody-resistant mutations. The respiratory syncytial virus is an important pathogen for which monoclonal antibodies against the fusion (F) protein are used to prevent severe disease in high-risk infants. In this study, we used an approach that combines molecular dynamics simulations with FoldX to estimate changes in free energy in F protein folding and binding to the motavizumab antibody upon each possible amino acid change. We systematically selected 8 predicted escape mutations and tested them in an infectious clone. Consistent with our F protein stability predictions, replication-effective viruses were observed for each selected mutation. Six of the eight variants showed increased resistance to neutralization by motavizumab. Flow cytometry was used to validate the estimated (model-predicted) effects on antibody binding to F. Using surface plasmon resonance, we determined that changes in the on-rate of motavizumab binding were associated with the reduced affinity for two novel escape mutations. Our study empirically validated the accuracy of our molecular modeling approach and emphasized the role of biophysical protein modeling in predicting viral resistance to antibody-based therapeutics that can be used to monitor the emergence of resistant viruses and to design improved therapeutic antibodies. IMPORTANCE Respiratory syncytial virus (RSV) causes severe disease in young infants, particularly those with heart or lung diseases or born prematurely. Because no vaccine is currently available, monoclonal antibodies are used to prevent severe RSV disease in high-risk infants. While it is known that RSV evolves to avoid recognition by antibodies, screening tools that can predict which changes to the virus may lead to antibody resistance are greatly needed.

SARS-CoV-2 Variant Exposures Elicit Antibody Responses With Differential Cross-Neutralization of Established and Emerging Strains Including Delta and Omicron.

The wide spectrum of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with phenotypes impacting transmission and antibody sensitivity necessitates investigation of immune responses to different spike protein versions. Here, we compare neutralization of variants of concern, including B.1.617.2 (delta) and B.1.1.529 (omicron), in sera from individuals exposed to variant infection, vaccination, or both. We demonstrate that neutralizing antibody responses are strongest against variants sharing certain spike mutations with the immunizing exposure, and exposure to multiple spike variants increases breadth of variant cross-neutralization. These findings contribute to understanding relationships between exposures and antibody responses and may inform booster vaccination strategies.

Decoding the effects of spike receptor binding domain mutations on antibody escape abilities of omicron variants.

Recent times witnessed an upsurge in the number of COVID19 cases which is primarily attributed to the emergence of several omicron variants, although there is substantial population vaccination coverage across the globe. Currently, many therapeutic antibodies have been approved for emergency usage. The present study critically evaluates the effect of mutations observed in several omicron variants on the binding affinities of different classes of RBD-specific antibodies using a combined approach of immunoinformatics and binding free energy calculations. Our binding affinity data clearly show that omicron variants achieve antibody escape abilities by incorporating mutations at the immunogenic hotspot residues for each specific class of antibody. K417N and Y505H point mutations are primarily accountable for the loss of class I antibody binding affinities. The K417N/Q493R/Q498R/Y505H combined mutant significantly reduces binding affinities for all the class I antibodies. E484A single mutation, on the other hand, drastically reduces binding affinities for most of the class II antibodies. E484A and E484A/Q493R double mutations cause a 33-38% reduction in binding affinity for an approved therapeutic monoclonal antibody. The Q498R RBD mutation observed across all the omicron variants can reduce ∼12% binding affinity for REGN10987, a class III therapeutic antibody, and the L452R/Q498R double mutation causes a ∼6% decrease in binding affinities for another class III therapeutic antibody, LY-CoV1404. Our data suggest that achieving the immune evasion abilities appears to be the selection pressure behind the emergence of omicron variants.

SARS-CoV-2 B.1.617 Mutations L452R and E484Q Are Not Synergistic for Antibody Evasion.

The SARS-CoV-2 B.1.617 variant emerged in the Indian state of Maharashtra in late 2020. There have been fears that 2 key mutations seen in the receptor-binding domain, L452R and E484Q, would have additive effects on evasion of neutralizing antibodies. We report that spike bearing L452R and E484Q confers modestly reduced sensitivity to BNT162b2 mRNA vaccine-elicited antibodies following either first or second dose. The effect is similar in magnitude to the loss of sensitivity conferred by L452R or E484Q alone. These data demonstrate reduced sensitivity to vaccine-elicited neutralizing antibodies by L452R and E484Q but lack of synergistic loss of sensitivity.

Hepatitis C Virus Escape Studies of Human Antibody AR3A Reveal a High Barrier to Resistance and Novel Insights on Viral Antibody Evasion Mechanisms.

Yearly, ∼2 million people become hepatitis C virus (HCV) infected, resulting in an elevated lifetime risk for severe liver-related chronic illnesses. Characterizing epitopes of broadly neutralizing antibodies (NAbs), such as AR3A, is critical to guide vaccine development. Previously identified alanine substitutions that can reduce AR3A binding to expressed H77 envelope were introduced into chimeric cell culture-infectious HCV recombinants (HCVcc) H77(core-NS2)/JFH1. Substitutions G523A, G530A, and D535A greatly reduced fitness, and S424A, P525A, and N540A, although viable, conferred only low-level AR3A resistance. Using highly NAb-sensitive hypervariable region 1 (HVR1)-deleted HCVcc, H77/JFH1ΔHVR1 and J6(core-NS2)/JFH1ΔHVR1, we previously reported a low barrier to developing AR5A NAb resistance substitutions. Here, we cultured Huh7.5 cells infected with H77/JFH1, H77/JFH1ΔHVR1, or J6/JFH1ΔHVR1 with AR3A. We identified the resistance envelope substitutions M345T in H77/JFH1, L438S and F442Y in H77/JFH1ΔHVR1, and D431G in J6/JFH1ΔHVR1 M345T increased infectivity and conferred low-level AR3A resistance to H77/JFH1 but not H77/JFH1ΔHVR1 L438S and F442Y conferred high-level AR3A resistance to H77/JFH1ΔHVR1 but abrogated the infectivity of H77/JFH1. D431G conferred AR3A resistance to J6/JFH1ΔHVR1 but not J6/JFH1. This was possibly because D431G conferred broadly increased neutralization sensitivity to J6/JFH1D431G but not J6/JFH1ΔHVR1/D431G while decreasing scavenger receptor class B type I coreceptor dependency. Common substitutions at positions 431 and 442 did not confer high-level resistance in other genotype 2a recombinants [JFH1 or T9(core-NS2)/JFH1]. Although the data indicate that AR3A has a high barrier to resistance, our approach permitted identification of low-level resistance substitutions. Also, the HVR1-dependent effects on AR3A resistance substitutions suggest a complex role of HVR1 in virus escape and receptor usage, with important implications for HCV vaccine development.IMPORTANCE Hepatitis C virus (HCV) is a leading cause of liver-related mortality, and limited treatment accessibility makes vaccine development a high priority. The vaccine-relevant cross-genotype-reactive antibody AR3A has shown high potency, but the ability of the virus to rapidly escape by mutating the AR3A epitope (barrier to resistance) remains unexplored. Here, we succeeded in inducing only low-level AR3A resistance, indicating a higher barrier to resistance than what we have previously reported for AR5A. Furthermore, we identify AR3A resistance substitutions that have hypervariable region 1 (HVR1)-dependent effects on HCV viability and on broad neutralization sensitivity. One of these substitutions increased envelope breathing and decreased scavenger receptor class B type I HCV coreceptor dependency, both in an HVR1-dependent fashion. Thus, we identify novel AR3A-specific resistance substitutions and the role of HVR1 in protecting HCV from AR3-targeting antibodies. These viral escape mechanisms should be taken into consideration in future HCV vaccine development.

Recombinant Receptor-Binding Domains of Multiple Middle East Respiratory Syndrome Coronaviruses (MERS-CoVs) Induce Cross-Neutralizing Antibodies against Divergent Human and Camel MERS-CoVs and Antibody Escape Mutants.

Middle East respiratory syndrome coronavirus (MERS-CoV) binds to cellular receptor dipeptidyl peptidase 4 (DPP4) via the spike (S) protein receptor-binding domain (RBD). The RBD contains critical neutralizing epitopes and serves as an important vaccine target. Since RBD mutations occur in different MERS-CoV isolates and antibody escape mutants, cross-neutralization of divergent MERS-CoV strains by RBD-induced antibodies remains unknown. Here, we constructed four recombinant RBD (rRBD) proteins with single or multiple mutations detected in representative human MERS-CoV strains from the 2012, 2013, 2014, and 2015 outbreaks, respectively, and one rRBD protein with multiple changes derived from camel MERS-CoV strains. Like the RBD of prototype EMC2012 (EMC-RBD), all five RBDs maintained good antigenicity and functionality, the ability to bind RBD-specific neutralizing monoclonal antibodies (MAbs) and the DPP4 receptor, and high immunogenicity, able to elicit S-specific antibodies. They induced potent neutralizing antibodies cross-neutralizing 17 MERS pseudoviruses expressing S proteins of representative human and camel MERS-CoV strains identified during the 2012-2015 outbreaks, 5 MAb escape MERS-CoV mutants, and 2 live human MERS-CoV strains. We then constructed two RBDs mutated in multiple key residues in the receptor-binding motif (RBM) of RBD and demonstrated their strong cross-reactivity with anti-EMC-RBD antibodies. These RBD mutants with diminished DPP4 binding also led to virus attenuation, suggesting that immunoevasion after RBD immunization is accompanied by loss of viral fitness. Therefore, this study demonstrates that MERS-CoV RBD is an important vaccine target able to induce highly potent and broad-spectrum neutralizing antibodies against infection by divergent circulating human and camel MERS-CoV strains. IMPORTANCE: MERS-CoV was first identified in June 2012 and has since spread in humans and camels. Mutations in its spike (S) protein receptor-binding domain (RBD), a key vaccine target, have been identified, raising concerns over the efficacy of RBD-based MERS vaccines against circulating human and camel MERS-CoV strains. Here, we constructed five vaccine candidates, designated 2012-RBD, 2013-RBD, 2014-RBD, 2015-RBD, and Camel-RBD, containing single or multiple mutations in the RBD of representative human and camel MERS-CoV strains during the 2012-2015 outbreaks. These RBD-based vaccine candidates maintained good functionality, antigenicity, and immunogenicity, and they induced strong cross-neutralizing antibodies against infection by divergent pseudotyped and live MERS-CoV strains, as well as antibody escape MERS-CoV mutants. This study provides impetus for further development of a safe, highly effective, and broad-spectrum RBD-based subunit vaccine to prevent MERS-CoV infection.

Maturation of secreted HCV particles by incorporation of secreted ApoE protects from antibodies by enhancing infectivity.

BACKGROUND & AIMS: Hepatitis C virus (HCV) evades humoral immunity and establishes chronic infections. Virus particles circulate in complex with lipoproteins facilitating antibody escape. Apolipoprotein E (ApoE) is essential for intracellular HCV assembly and for HCV cell entry. We aimed to explore if ApoE released from non-infected cells interacts with and modulates secreted HCV particles. METHODS: ApoE secreted from non-infected cells was incubated with HCV from primary human hepatocytes or Huh-7.5 cells. Co-immunoprecipitation, viral infectivity and neutralization experiments were conducted. RESULTS: Physiological levels of secreted ApoE (10-60µg/ml) enhanced the infectivity of HCV up to 8-fold across all genotypes, which indirectly decreased virus neutralization by antibodies targeting E1 or E2 up to 10-fold. Infection enhancement was observed for particles produced in primary human hepatocytes and Huh-7.5 cells. Selective depletion of ApoE ablated infection enhancement. Addition of HA-tagged ApoE to HCV particles permitted co-precipitation of HCV virions. Serum ApoE levels ranged between 10-60µg/ml, which is ca 100-fold higher than in Huh-7.5 conditioned cell culture fluids. Serum-derived HCV particles carried much higher amounts of ApoE than cell culture-derived HCV particles. Serum ApoE levels correlated with efficiency of co-precipitation of HCV upon exogenous addition of HA-ApoE. ApoE-dependent infection enhancement was independent of the hypervariable region 1 and SR-B1, but was dependent on heparan sulfate proteoglycans (HSPGs). CONCLUSIONS: Physiological quantities of secreted ApoE stimulate HCV infection and increase antibody escape, by incorporating into virus particles and enhancing particle interactions with cellular HSPGs. Thus, secreted particles undergo ApoE-dependent maturation to enhance infectivity and to facilitate evasion from neutralizing antibodies. Lay summary: This study shows that HCV particle infectivity is remodeled by secreted ApoE after particle release from cells. Fluctuation of the availability of ApoE likely influences HCV infectivity, antibody escape and transmission.

SARS-CoV-2 Variants from Long-Term, Persistently Infected Immunocompromised Patients Have Altered Syncytia Formation, Temperature-Dependent Replication, and Serum Neutralizing Antibody Escape.

SARS-CoV-2 infection of immunocompromised individuals often leads to prolonged detection of viral RNA and infectious virus in nasal specimens, presumably due to the lack of induction of an appropriate adaptive immune response. Mutations identified in virus sequences obtained from persistently infected patients bear signatures of immune evasion and have some overlap with sequences present in variants of concern. We characterized virus isolates obtained greater than 100 days after the initial COVID-19 diagnosis from two COVID-19 patients undergoing immunosuppressive cancer therapy, wand compared them to an isolate from the start of the infection. Isolates from an individual who never mounted an antibody response specific to SARS-CoV-2 despite the administration of convalescent plasma showed slight reductions in plaque size and some showed temperature-dependent replication attenuation on human nasal epithelial cell culture compared to the virus that initiated infection. An isolate from another patient-who did mount a SARS-CoV-2 IgM response-showed temperature-dependent changes in plaque size as well as increased syncytia formation and escape from serum-neutralizing antibodies. Our results indicate that not all virus isolates from immunocompromised COVID-19 patients display clear signs of phenotypic change, but increased attention should be paid to monitoring virus evolution in this patient population.

SARS-CoV-2 omicron spike simulations: broad antibody escape, weakened ACE2 binding, and modest furin cleavage.

The recent emergence of the omicron variant of the SARS-CoV-2 virus with large numbers of mutations has raised concern about a potential new surge in infections. Here we use molecular dynamics to study the biophysics of the interface of the BA1 and BA2 omicron spike protein binding to (i) the ACE2 receptor protein, (ii) antibodies from all known binding regions, and (iii) the furin binding domain. Our simulations suggest that while there is a significant reduction of antibody (Ab) binding strength corresponding to escape, the omicron spikes pay a cost in terms of weaker receptor binding as measured by interfacial hydrogen bonds (H-bond). The furin cleavage domain (FCD) is the same or weaker binding than the delta variant, suggesting lower fusogenicity resulting in less viral load and disease intensity than the delta variant. IMPORTANCE The BA1 and BA2 and closely related BA2.12.2 and BA.5 omicron variants of SARS-CoV-2 dominate the current global infection landscape. Given the high number of mutations, particularly those which will lead to antibody escape, it is important to establish accurate methods that can guide developing health policy responses that identify at a fundamental level whether omicron and its variants are more threatening than its predecessors, especially delta. The importance of our work is to demonstrate that simple in silico simulations can predict biochemical binding details of the omicron spike protein that have epidemiological consequences, especially for binding to the cells and for fusing the viral membrane with the cells. In each case, we predicted weaker binding of the omicron spike, which agreed with subsequent experimental results. Future virology experiments will be needed to test these predictions further.

Evolution of a globally unique SARS-CoV-2 Spike E484T monoclonal antibody escape mutation in a persistently infected, immunocompromised individual.

Prolonged infections in immunocompromised individuals may be a source for novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants, particularly when both the immune system and antiviral therapy fail to clear the infection and enable within-host evolution. Here we describe a 486-day case of SARS-CoV-2 infection in an immunocompromised individual. Following monotherapy with the monoclonal antibody Bamlanivimab, the individual's virus acquired resistance, likely via the earliest known occurrence of Spike amino acid variant E484T. Recently, E484T has arisen again as a derivative of E484A in the Omicron Variant of Concern, supporting the hypothesis that prolonged infections can give rise to novel variants long before they become prevalent in the human population.

The landscape of antibody binding affinity in SARS-CoV-2 Omicron BA.1 evolution.

The Omicron BA.1 variant of SARS-CoV-2 escapes convalescent sera and monoclonal antibodies that are effective against earlier strains of the virus. This immune evasion is largely a consequence of mutations in the BA.1 receptor binding domain (RBD), the major antigenic target of SARS-CoV-2. Previous studies have identified several key RBD mutations leading to escape from most antibodies. However, little is known about how these escape mutations interact with each other and with other mutations in the RBD. Here, we systematically map these interactions by measuring the binding affinity of all possible combinations of these 15 RBD mutations (215=32,768 genotypes) to 4 monoclonal antibodies (LY-CoV016, LY-CoV555, REGN10987, and S309) with distinct epitopes. We find that BA.1 can lose affinity to diverse antibodies by acquiring a few large-effect mutations and can reduce affinity to others through several small-effect mutations. However, our results also reveal alternative pathways to antibody escape that does not include every large-effect mutation. Moreover, epistatic interactions are shown to constrain affinity decline in S309 but only modestly shape the affinity landscapes of other antibodies. Together with previous work on the ACE2 affinity landscape, our results suggest that the escape of each antibody is mediated by distinct groups of mutations, whose deleterious effects on ACE2 affinity are compensated by another distinct group of mutations (most notably Q498R and N501Y).

Structure and Mutations of SARS-CoV-2 Spike Protein: A Focused Overview.

The spike protein (S-protein) of SARS-CoV-2, the protein that enables the virus to infect human cells, is the basis for many vaccines and a hotspot of concerning virus evolution. Here, we discuss the outstanding progress in structural characterization of the S-protein and how these structures facilitate analysis of virus function and evolution. We emphasize the differences in reported structures and that analysis of structure-function relationships is sensitive to the structure used. We show that the average residue solvent exposure in nearly complete structures is a good descriptor of open vs closed conformation states. Because of structural heterogeneity of functionally important surface-exposed residues, we recommend using averages of a group of high-quality protein structures rather than a single structure before reaching conclusions on specific structure-function relationships. To illustrate these points, we analyze some significant chemical tendencies of prominent S-protein mutations in the context of the available structures. In the discussion of new variants, we emphasize the selectivity of binding to ACE2 vs prominent antibodies rather than simply the antibody escape or ACE2 affinity separately. We note that larger chemical changes, in particular increased electrostatic charge or side-chain volume of exposed surface residues, are recurring in mutations of concern, plausibly related to adaptation to the negative surface potential of human ACE2. We also find indications that the fixated mutations of the S-protein in the main variants are less destabilizing than would be expected on average, possibly pointing toward a selection pressure on the S-protein. The richness of available structures for all of these situations provides an enormously valuable basis for future research into these structure-function relationships.

T cell deficiency precipitates antibody evasion and emergence of neurovirulent polyomavirus.

JC polyomavirus (JCPyV) causes progressive multifocal leukoencephalopathy (PML), a life-threatening brain disease in immunocompromised patients. Inherited and acquired T cell deficiencies are associated with PML. The incidence of PML is increasing with the introduction of new immunomodulatory agents, several of which target T cells or B cells. PML patients often carry mutations in the JCPyV VP1 capsid protein, which confer resistance to neutralizing VP1 antibodies (Ab). Polyomaviruses (PyV) are tightly species-specific; the absence of tractable animal models has handicapped understanding PyV pathogenesis. Using mouse polyomavirus (MuPyV), we found that T cell deficiency during persistent infection, in the setting of monospecific VP1 Ab, was required for outgrowth of VP1 Ab-escape viral variants. CD4 T cells were primarily responsible for limiting polyomavirus infection in the kidney, a major reservoir of persistent infection by both JCPyV and MuPyV, and checking emergence of these mutant viruses. T cells also provided a second line of defense by controlling the outgrowth of VP1 mutant viruses that evaded Ab neutralization. A virus with two capsid mutations, one conferring Ab-escape yet impaired infectivity and a second compensatory mutation, yielded a highly neurovirulent variant. These findings link T cell deficiency and evolution of Ab-escape polyomavirus VP1 variants with neuropathogenicity.

An antibody-escape estimator for mutations to the SARS-CoV-2 receptor-binding domain.

A key goal of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) surveillance is to rapidly identify viral variants with mutations that reduce neutralization by polyclonal antibodies elicited by vaccination or infection. Unfortunately, direct experimental characterization of new viral variants lags their sequence-based identification. Here we help address this challenge by aggregating deep mutational scanning data into an 'escape estimator' that estimates the antigenic effects of arbitrary combinations of mutations to the virus's spike receptor-binding domain. The estimator can be used to intuitively visualize how mutations impact polyclonal antibody recognition and score the expected antigenic effect of combinations of mutations. These scores correlate with neutralization assays performed on SARS-CoV-2 variants and emphasize the ominous antigenic properties of the recently described Omicron variant. An interactive version of the estimator is at https://jbloomlab.github.io/SARS2_RBD_Ab_escape_maps/escape-calc/ (last accessed 11 March 2022), and we provide a Python module for batch processing. Currently the calculator uses primarily data for antibodies elicited by Wuhan-Hu-1-like vaccination or infection and so is expected to work best for calculating escape from such immunity for mutations relative to early SARS-CoV-2 strains.

Understanding polyomavirus CNS disease - a perspective from mouse models.

JC polyomavirus (JCPyV), a ubiquitous human pathogen, causes several devastating brain diseases in immune-compromised individuals. The most notable of these JCPyV-associated CNS diseases is the frequently fatal demyelinating brain disease progressive multifocal leukoencephalopathy (PML). PML, an AIDS-defining disease in the pre-cART epoch, has emerged as a life-threatening complication in patients receiving immunomodulatory agents for autoimmune and inflammatory disorders and treatment for certain hematological malignancies. Among the rapidly expanding list of PML-associated biologics, natalizumab (Tysabri®) has the highest incidence and is an ominous sequela for multiple sclerosis (MS) patients who otherwise benefit from dramatic reductions in relapses using this immunomodulatory agent. Drug withdrawal, the only therapeutic option for PML, is often complicated by a high-mortality cerebral inflammatory reaction. No anti-JCPyV agents are available. Lack of a tractable animal model of polyomavirus-induced central nervous system (CNS) disease is an acknowledged bottleneck to elucidating PML pathogenesis, immunological mechanisms that control JCPyV, in vivo evaluation of agents that inhibit polyomavirus replication in tissue culture, and uncovering early events that presage JCPyV-associated neuropathology. The natural virus-host mouse polyomavirus (MuPyV) model has recently been developed to explore mechanisms of polyomavirus-associated CNS disease. In this review, we will cover the benefits of using the MuPyV model to answer fundamental questions about innate and adaptive immune control of JCPyV, the impact of immunomodulation on JCPyV pathogenesis, and how this MuPyV CNS infection model will help improve criteria for identifying patients at risk for JCPyV-associated CNS diseases before the development of irreversible lesions.