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Antigenic diversity shapes immunity in distinct and unexpected ways. This is particularly true of the humoral response generated against influenza A viruses. While it is known that immunological memory developed against previously-encountered influenza A virus strains impacts the outcome of subsequent infections, exactly how sequential exposures to antigenically variant viruses shape the humoral immune response in humans remains poorly understood. To address this important question, a longitudinal analysis of antibody titers against various pandemic and seasonal strains of influenza virus spanning a 20-year period (1987–2008) was performed using samples from 40 individuals (d.o.b. 1917–1952) obtained from the Framingham Heart Study. Longitudinal increases in neutralizing antibody titers were observed against previously-encountered pandemic H2N2, H3N2 and H1N1 influenza A virus strains. Antibody titers against seasonal strains encountered later in life also increased longitudinally at a rate similar to that against their pandemic predecessors. Titers of cross-reactive antibodies specific to the hemagglutinin stalk domain were also investigated, since they are known to be influenced by exposure to antigenically diverse influenza A viruses. These titers rose modestly over time, even in the absence of major antigenic shifts. No sustained increase in neutralizing antibody titers against an antigenically more stable virus (human cytomegalovirus) was observed. The results herein describe a role for antigenic variation in shaping the humoral immune compartment, and provide a rational basis for the hierarchical nature of antibody titers against influenza A viruses in humans.

Significance Virus infections must be combated at a cellular level. The strategies used to inhibit virus differ dramatically when comparing plants and insects to mammals. Here, we identify an evolutionary conserved antiviral response that is independent of these known defenses. We demonstrate that an RNA nuclease called Drosha is repurposed during virus infection to cleave viral RNA and modulate the cellular environment as a means of inhibiting virus replication.

In contrast to the DNA-based viruses in prokaryotes, the emergence of eukaryotes provided the necessary compartmentalization and membranous environment for RNA viruses to flourish, creating the need for an RNA-targeting antiviral system1,2. Present day eukaryotes employ at least two main defense strategies that emerged as a result of this viral shift, namely antiviral RNA interference (RNAi) and the interferon (IFN) system2. Here, we demonstrate that Drosha and related RNase III ribonucleases from all three domains of life, also elicit RNA-targeting antiviral activity. Systemic evolution of ligands by exponential enrichment (SELEX) on this class of proteins illustrates the recognition of unbranched RNA stem loops. Biochemical analyses reveal that in this context, Drosha functions as an antiviral clamp, conferring steric hindrance on the RNA dependent RNA polymerases (RdRps) of diverse positive stranded RNA viruses. We present evidence for cytoplasmic translocation of RNase III nucleases in response to virus in diverse eukaryotes including: plants, arthropods, invertebrate chordates, and fish. These data implicate RNase III recognition of viral RNA as an antiviral defense that is independent of, and possibly predates, other known eukaryotic antiviral systems.

SUMMARY With the capacity to fine-tune protein expression via sequence-specific interactions, microRNAs (miRNAs) help regulate cell maintenance and differentiation. While some studies have also implicated miRNAs as regulators of the antiviral response, others have found that the RISC complex that facilitates miRNA-mediated silencing is rendered non-functional during cellular stress, including virus infection. To determine the global role of miRNAs in the cellular response to virus infection, we generated a vector that rapidly eliminates total cellular miRNA populations in terminally differentiated primary cultures. Loss of miRNAs has a negligible impact on both innate sensing of and immediate response to acute viral infection. In contrast, miRNA depletion specifically enhances cytokine expression, providing a post-translational mechanism for immune cell activation during cellular stress. This work highlights the physiological role of miRNAs during the antiviral response and suggests their contribution is limited to chronic infections and the acute activation of the adaptive immune response.

Over one year after its inception, the coronavirus disease-2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) remains difficult to control despite the availability of several excellent vaccines. Progress in controlling the pandemic is slowed by the emergence of variants that appear to be more transmissible and more resistant to antibodies. Here we report on a cohort of 63 COVID-19-convalescent individuals assessed at 1.3, 6.2 and 12 months after infection, 41% of whom also received mRNA vaccines. In the absence of vaccination antibody reactivity to the receptor binding domain (RBD) of SARS-CoV-2, neutralizing activity and the number of RBD-specific memory B cells remain relatively stable from 6 to 12 months. Vaccination increases all components of the humoral response, and as expected, results in serum neutralizing activities against variants of concern that are comparable to or greater than neutralizing activity against the original Wuhan Hu-1 achieved by vaccination of naive individuals. The mechanism underlying these broad-based responses involves ongoing antibody somatic mutation, memory B cell clonal turnover, and development of monoclonal antibodies that are exceptionally resistant to SARS-CoV-2 RBD mutations, including those found in variants of concern. In addition, B cell clones expressing broad and potent antibodies are selectively retained in the repertoire over time and expand dramatically after vaccination. The data suggest that immunity in convalescent individuals will be very long lasting and that convalescent individuals who receive available mRNA vaccines will produce antibodies and memory B cells that should be protective against circulating SARS-CoV-2 variants. Should memory responses evolve in a similar manner in vaccinated individuals, additional appropriately timed boosting with available vaccines could cover most circulating variants of concern.

Every living entity requires the capacity to defend against viruses in some form. From bacteria to plants to arthropods, cells retain the capacity to capture genetic material, process it in a variety of ways, and subsequently use it to generate pathogen-specific small RNAs. These small RNAs can then be used to provide specificity to an otherwise non-specific nuclease, generating a potent antiviral system. While small RNA-based defenses in chordates are less utilized, the protein-based antiviral invention in this phylum appears to have derived from components of the same ancestral small RNA machinery. Based on recent evidence, it would seem that RNase III nucleases have been reiteratively repurposed over billions of years to provide cells with the capacity to recognize and destroy unwanted genetic material. Here we describe an overview of what is known on this subject and provide a model for how these defenses may have evolved.

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