Positive-strand RNA viruses-a Keystone Symposia report.

Positive-strand RNA viruses have been the cause of several recent outbreaks and epidemics, including the Zika virus epidemic in 2015, the SARS outbreak in 2003, and the ongoing SARS-CoV-2 pandemic. On June 18-22, 2022, researchers focusing on positive-strand RNA viruses met for the Keystone Symposium "Positive-Strand RNA Viruses" to share the latest research in molecular and cell biology, virology, immunology, vaccinology, and antiviral drug development. This report presents concise summaries of the scientific discussions at the symposium.

IgM antibodies derived from memory B cells are potent cross-variant neutralizers of SARS-CoV-2.

Humoral immunity to SARS-CoV-2 can be supplemented with polyclonal sera from convalescent donors or an engineered monoclonal antibody (mAb) product. While pentameric IgM antibodies are responsible for much of convalescent sera's neutralizing capacity, all available mAbs are based on the monomeric IgG antibody subtype. We now show that IgM mAbs derived from immune memory B cell receptors are potent neutralizers of SARS-CoV-2. IgM mAbs outperformed clonally identical IgG antibodies across a range of affinities and SARS-CoV-2 receptor-binding domain epitopes. Strikingly, efficacy against SARS-CoV-2 viral variants was retained for IgM but not for clonally identical IgG. To investigate the biological role for IgM memory in SARS-CoV-2, we also generated IgM mAbs from antigen-experienced IgM+ memory B cells in convalescent donors, identifying a potent neutralizing antibody. Our results highlight the therapeutic potential of IgM mAbs and inform our understanding of the role for IgM memory against a rapidly mutating pathogen.

Imprinted SARS-CoV-2-specific memory lymphocytes define hybrid immunity.

Immune memory is tailored by cues that lymphocytes perceive during priming. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic created a situation in which nascent memory could be tracked through additional antigen exposures. Both SARS-CoV-2 infection and vaccination induce multifaceted, functional immune memory, but together, they engender improved protection from disease, termed hybrid immunity. We therefore investigated how vaccine-induced memory is shaped by previous infection. We found that following vaccination, previously infected individuals generated more SARS-CoV-2 RBD-specific memory B cells and variant-neutralizing antibodies and a distinct population of IFN-γ and IL-10-expressing memory SARS-CoV-2 spike-specific CD4+ T cells than previously naive individuals. Although additional vaccination could increase humoral memory in previously naive individuals, it did not recapitulate the distinct CD4+ T cell cytokine profile observed in previously infected subjects. Thus, imprinted features of SARS-CoV-2-specific memory lymphocytes define hybrid immunity.

Functional SARS-CoV-2-Specific Immune Memory Persists after Mild COVID-19.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus is causing a global pandemic, and cases continue to rise. Most infected individuals experience mildly symptomatic coronavirus disease 2019 (COVID-19), but it is unknown whether this can induce persistent immune memory that could contribute to immunity. We performed a longitudinal assessment of individuals recovered from mild COVID-19 to determine whether they develop and sustain multifaceted SARS-CoV-2-specific immunological memory. Recovered individuals developed SARS-CoV-2-specific immunoglobulin (IgG) antibodies, neutralizing plasma, and memory B and memory T cells that persisted for at least 3 months. Our data further reveal that SARS-CoV-2-specific IgG memory B cells increased over time. Additionally, SARS-CoV-2-specific memory lymphocytes exhibited characteristics associated with potent antiviral function: memory T cells secreted cytokines and expanded upon antigen re-encounter, whereas memory B cells expressed receptors capable of neutralizing virus when expressed as monoclonal antibodies. Therefore, mild COVID-19 elicits memory lymphocytes that persist and display functional hallmarks of antiviral immunity.

SARS-CoV-2 Serologic Assays in Control and Unknown Populations Demonstrate the Necessity of Virus Neutralization Testing.

BACKGROUND: To determine how serologic antibody testing outcome links with virus neutralization of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), we evaluated individuals for SARS-CoV-2 antibody level and viral neutralization. METHODS: We compared serum Ig levels across platforms of viral antigens and antibodies with 15 positive and 30 negative SARS-CoV-2 controls followed by viral neutralization assessment. We then applied these platforms to a clinically relevant cohort of 114 individuals with unknown histories of SARS-CoV-2 infection. RESULTS: In controls, the best-performing virus-specific antibody detection platforms were SARS-CoV-2 receptor binding domain (RBD) IgG (sensitivity 87%, specificity 100%, positive predictive value [PPV] 100%, negative predictive value [NPV] 94%), spike IgG3 (sensitivity 93%, specificity 97%, PPV 93%, NPV 97%), and nucleocapsid protein (NP) IgG (sensitivity 93%, specificity 97%, PPV 93%, NPV 97%). Neutralization of positive and negative control sera showed 100% agreement. Twenty individuals with unknown history had detectable SARS-CoV-2 antibodies with 16 demonstrating virus neutralization. Spike IgG3 provided the highest accuracy for predicting serologically positive individuals with virus neutralization activity (misidentified 1/20 unknowns compared to 2/20 for RBD and NP IgG). CONCLUSIONS: The coupling of virus neutralization analysis to a spike IgG3 antibody test is optimal to categorize patients for correlates of SARS-CoV-2 immune protection status.

Functional SARS-CoV-2-specific immune memory persists after mild COVID-19.

The recently emerged SARS-CoV-2 virus is currently causing a global pandemic and cases continue to rise. The majority of infected individuals experience mildly symptomatic coronavirus disease 2019 (COVID-19), but it is unknown whether this can induce persistent immune memory that might contribute to herd immunity. Thus, we performed a longitudinal assessment of individuals recovered from mildly symptomatic COVID-19 to determine if they develop and sustain immunological memory against the virus. We found that recovered individuals developed SARS-CoV-2-specific IgG antibody and neutralizing plasma, as well as virus-specific memory B and T cells that not only persisted, but in some cases increased numerically over three months following symptom onset. Furthermore, the SARS-CoV-2-specific memory lymphocytes exhibited characteristics associated with potent antiviral immunity: memory T cells secreted IFN-γ and expanded upon antigen re-encounter, while memory B cells expressed receptors capable of neutralizing virus when expressed as antibodies. These findings demonstrate that mild COVID-19 elicits memory lymphocytes that persist and display functional hallmarks associated with antiviral protective immunity.

Functional SARS-CoV-2-specific immune memory persists after mild COVID-19.

The recently emerged SARS-CoV-2 virus is currently causing a global pandemic and cases continue to rise. The majority of infected individuals experience mildly symptomatic coronavirus disease 2019 (COVID-19), but it is unknown whether this can induce persistent immune memory that might contribute to herd immunity. Thus, we performed a longitudinal assessment of individuals recovered from mildly symptomatic COVID-19 to determine if they develop and sustain immunological memory against the virus. We found that recovered individuals developed SARS-CoV-2-specific IgG antibody and neutralizing plasma, as well as virus-specific memory B and T cells that not only persisted, but in some cases increased numerically over three months following symptom onset. Furthermore, the SARS-CoV-2-specific memory lymphocytes exhibited characteristics associated with potent antiviral immunity: memory T cells secreted IFN-γ and expanded upon antigen re-encounter, while memory B cells expressed receptors capable of neutralizing virus when expressed as antibodies. These findings demonstrate that mild COVID-19 elicits memory lymphocytes that persist and display functional hallmarks associated with antiviral protective immunity.

Type I interferon response impairs differentiation potential of pluripotent stem cells.

Upon virus infection, pluripotent stem cells neither induce nor respond to canonical type I interferons (IFN-I). To better understand this biology, we characterized induced pluripotent stem cells (iPSCs) as well as their differentiated parental or rederived counterparts. We confirmed that only iPSCs failed to respond to viral RNA, IFN-I, or viral infection. This lack of response could be phenocopied in fibroblasts with the expression of a reprogramming factor which repressed the capacity to induce canonical antiviral pathways. To ascertain the consequences of restoring the antiviral response in the context of pluripotency, we engineered a system to engage these defenses in iPSCs. Inducible expression of a recombinant virus-activated transcription factor resulted in the successful reconstitution of antiviral defenses through the direct up-regulation of IFN-I-stimulated genes. Induction of the antiviral signature in iPSCs, even for a short duration, resulted in the dysregulation of genes associated with all three germ layers despite maintaining pluripotency markers. Trilineage differentiation of these same cells showed that engagement of the antiviral defenses compromised ectoderm and endoderm formation and dysregulated the development of mesodermal sublineages. In all, these data suggest that the temporal induction of the antiviral response primes iPSCs away from pluripotency and induces numerous aberrant gene products upon differentiation. Together these results suggest that the IFN-I system and pluripotency may be incompatible with each other and thus explain why stem cells do not utilize the canonical antiviral system.

Diversity of Influenza A(H5N1) Viruses in Infected Humans, Northern Vietnam, 2004-2010.

Influenza viruses exist in each host as a collection of genetically diverse variants, which might enhance their adaptive potential. To assess the genetic and functional diversity of highly pathogenic avian influenza A(H5N1) viruses within infected humans, we used deep-sequencing methods to characterize samples obtained from infected patients in northern Vietnam during 2004-2010 on different days after infection, from different anatomic sites, or both. We detected changes in virus genes that affected receptor binding, polymerase activity, or interferon antagonism, suggesting that these factors could play roles in influenza virus adaptation to humans. However, the frequency of most of these mutations remained low in the samples tested, implying that they were not efficiently selected within these hosts. Our data suggest that adaptation of influenza A(H5N1) viruses is probably stepwise and depends on accumulating combinations of mutations that alter function while maintaining fitness.