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The effectiveness of the annual influenza vaccine has declined in recent years, especially for the H3N2 component, and is a concern for global public health. A major cause for this lack in effectiveness has been attributed to the egg-based vaccine production process. Substitutions on the hemagglutinin glycoprotein (HA) often arise during virus passaging that change its antigenicity and hence vaccine effectiveness. Here, we characterize the effect of a prevalent substitution, L194P, in egg-passaged H3N2 viruses. X-ray structural analysis reveals that this substitution surprisingly increases the mobility of the 190-helix and neighboring regions in antigenic site B, which forms one side of the receptor binding site (RBS) and is immunodominant in recent human H3N2 viruses. Importantly, the L194P substitution decreases binding and neutralization by an RBS-targeted broadly neutralizing antibody by three orders of magnitude and significantly changes the HA antigenicity as measured by binding of human serum antibodies. The receptor binding mode and specificity are also altered to adapt to avian receptors during egg passaging. Overall, these findings help explain the low effectiveness of the seasonal vaccine against H3N2 viruses, and suggest that alternative approaches should be accelerated for producing influenza vaccines as well as isolating clinical isolates.

The ideal vaccine against viruses such as influenza and SARS-CoV-2 must provide a robust, durable and broad immune protection against multiple viral variants. However, antibody responses to current vaccines often lack robust cross-reactivity. Here we describe a polymeric Toll-like receptor 7 agonist nanoparticle (TLR7-NP) adjuvant, which enhances lymph node targeting, and leads to persistent activation of immune cells and broad immune responses. When mixed with alum-adsorbed antigens, this TLR7-NP adjuvant elicits cross-reactive antibodies for both dominant and subdominant epitopes and antigen-specific CD8+ T-cell responses in mice. This TLR7-NP-adjuvanted influenza subunit vaccine successfully protects mice against viral challenge of a different strain. This strategy also enhances the antibody response to a SARS-CoV-2 subunit vaccine against multiple viral variants that have emerged. Moreover, this TLR7-NP augments antigen-specific responses in human tonsil organoids. Overall, we describe a nanoparticle adjuvant to improve immune responses to viral antigens, with promising implications for developing broadly protective vaccines.A nanoparticle-based adjuvant incorporating a Toll-like receptor 7 agonist elicits cross-reactive antibodies for both dominant and subdominant epitopes and enhances immune responses against multiple variants of influenza and SARS-CoV-2.

Egg-adaptive mutations in influenza hemagglutinin (HA) often emerge during the production of egg-based seasonal influenza vaccines, which contribute to the largest share in the global influenza vaccine market. While some egg-adaptive mutations have minimal impact on the HA antigenicity (e.g. G186V), others can alter it (e.g. L194P). Here, we show that the preference of egg-adaptive mutation in human H3N2 HA is strain-dependent. In particular, Thr160 and Asn190, which are found in many recent H3N2 strains, restrict the emergence of L194P but not G186V. Our results further suggest that natural amino acid variants at other HA residues also play a role in determining the preference of egg-adaptive mutation. Consistently, recent human H3N2 strains from different clades acquire different mutations during egg passaging. Overall, these results demonstrate that natural mutations in human H3N2 HA can influence the preference of egg-adaptation mutation, which has important implications in seed strain selection for egg-based influenza vaccine.

Human influenza virus rapidly accumulates mutations in its major surface protein hemagglutinin (HA). The evolutionary success of influenza virus lineages depends on how these mutations affect HA’s functionality and antigenicity. Here we experimentally measure the effects on viral growth in cell culture of all single amino acid mutations to the HA from a recent human H3N2 influenza virus strain. We show that mutations that are measured to be more favorable for viral growth are enriched in evolutionarily successful H3N2 viral lineages relative to mutations that are measured to be less favorable for viral growth. Therefore, despite the well-known caveats about cell-culture measurements of viral fitness, such measurements can still be informative for understanding evolution in nature. We also compare our measurements for H3 HA to similar data previously generated for a distantly related H1 HA and find substantial differences in which amino acids are preferred at many sites. For instance, the H3 HA has less disparity in mutational tolerance between the head and stalk domains than the H1 HA. Overall, our work suggests that experimental measurements of mutational effects can be leveraged to help understand the evolutionary fates of viral lineages in nature—but only when the measurements are made on a viral strain similar to the ones being studied in nature.

Influenza viruses routinely acquire mutations in their hemagglutinin (HA) and neuraminidase (NA) glycoproteins that abrogate binding of pre-existing antibodies in a process known as antigenic drift. Most human antibodies against HA and NA are directed against epitopes that are hypervariable and not against epitopes that are conserved among different influenza virus strains. Universal influenza vaccines are currently being developed to elicit protective responses against functionally conserved sites on influenza proteins where viral escape mutations can result in large fitness costs [1]. Universal vaccine targets include the highly conserved HA stem domain [2–12], the less conserved HA receptor-binding site (RBS) [13–16], as well as conserved sites on NA [17–19]. One central challenge of universal vaccine efforts is to steer human antibody responses away from immunodominant, variable epitopes and towards subdominant, functionally conserved sites. Overcoming this challenge will require further understanding of the structural basis of broadly neutralizing HA and NA antibody binding epitopes and factors that influence immunodominance hierarchies of human antibody responses.

The structure of fitness landscapes is critical for understanding adaptive protein evolution. Previous empirical studies on fitness landscapes were confined to either the neighborhood around the wild type sequence, involving mostly single and double mutants, or a combinatorially complete subgraph involving only two amino acids at each site. In reality, the dimensionality of protein sequence space is higher (20L) and there may be higher-order interactions among more than two sites. Here we experimentally characterized the fitness landscape of four sites in protein GB1, containing 204 = 160,000 variants. We found that while reciprocal sign epistasis blocked many direct paths of adaptation, such evolutionary traps could be circumvented by indirect paths through genotype space involving gain and subsequent loss of mutations. These indirect paths alleviate the constraint on adaptive protein evolution, suggesting that the heretofore neglected dimensions of sequence space may change our views on how proteins evolve. Proteins can evolve over time by changing their component parts, which are called amino acids. These changes usually happen one at a time and natural selection tends to preserve those changes that make the protein more efficient at its specific tasks, while discarding those that impair the protein’s activity. However the effect of each change depends on the protein as a whole, and so two changes that separately make the protein worse can make it much better if they occur together. This phenomenon is called epistasis and in some cases it can trap proteins in a sub-optimal form and prevent them from improving further. Proteins are made from twenty different kinds of amino acid, and there are millions of different combinations of amino acids that could, in theory, make a protein of a given length. Studying protein evolution involves making variants of the same protein, each with just a few changes, and comparing how efficient, or “fit”, they are. Previous studies only measured the fitness of a few variants and showed that epistasis could block protein evolution by requiring the protein to lose some fitness before it could improve further. However, new techniques have now made it easier to study protein evolution by testing many more protein variants. Wu, Dai et al. focused on four amino acids in part of a protein called GB1 and tested the efficiency of every possible combination of these four amino acids, a total of 160,000 (204) variants. Contrary to expectations, the results suggested that the protein could evolve quickly to maximise fitness despite there being epistasis between the four amino acids. Overcoming epistasis typically involved making a change to one amino acid that paved the way for further changes while avoiding the need to lose fitness. The original change could then be reversed once the epistasis was overcome. The complexity of this solution means it can only be seen by studying a large number of protein variants that represent many alternative sequences of protein changes. Wu, Dai et al. conclude that proteins are able to achieve a higher level of fitness through evolution by exploring a large number of changes. There are many possible changes for each protein and it is this variety that, despite epistasis, allows proteins to become naturally optimised for the tasks that they perform. While the full complexity of protein evolution cannot be explored at the moment, as technology advances it will become possible to study more protein variants. Such advances would therefore hopefully allow researchers to discover even more about the natural mechanisms of protein evolution.

Antigenic drift of influenza virus hemagglutinin (HA) is enabled by facile evolvability. However, HA antigenic site B, which has become immunodominant in recent human H3N2 influenza viruses, is also evolutionarily constrained by its involvement in receptor binding. Here, we employ deep mutational scanning to probe the local fitness landscape of HA antigenic site B in six different human H3N2 strains spanning from 1968 to 2016. We observe that the fitness landscape of HA antigenic site B can be very different between strains. Sequence variants that exhibit high fitness in one strain can be deleterious in another, indicating that the evolutionary constraints of antigenic site B have changed over time. Structural analysis suggests that the local fitness landscape of antigenic site B can be reshaped by natural mutations via modulation of the receptor-binding mode. Overall, these findings elucidate how influenza virus continues to explore new antigenic space despite strong functional constraints. Antigenic site B in influenza A virus hemagglutinin (HA) is immunodominant in circulating human H3N2 strains. Using deep mutational scanning, Wu et al. here define the local fitness landscapes of HA antigenic site B in six human H3N2 strains, providing insights into evolvability of influenza antigenicity.

Hemagglutinin (HA) glycoprotein is an important focus of influenza research due to its role in antigenic drift and shift, as well as its receptor binding and membrane fusion functions, which are indispensable for viral entry. Over the past four decades, X-ray crystallography has greatly facilitated our understanding of HA receptor binding, membrane fusion, and antigenicity. The recent advances in cryo-EM have further deepened our comprehension of HA biology. Since influenza HA constantly evolves in natural circulating strains, there are always new questions to be answered. The incessant accumulation of knowledge on the structural biology of HA over several decades has also facilitated the design and development of novel therapeutics and vaccines. This review describes the current status of the field of HA structural biology, how we got here, and what the next steps might be.

Virus propagation methods generally use transformed cell lines to grow viruses from clinical specimens, which may force viruses to rapidly adapt to cell culture conditions, a process facilitated by high viral mutation rates. Upon propagation in VeroE6 cells, SARS-CoV-2 may mutate or delete the multibasic cleavage site (MBCS) in the spike protein. Previously, we showed that the MBCS facilitates serine protease-mediated entry into human airway cells (Mykytyn et al., 2021). Here, we report that propagating SARS-CoV-2 on the human airway cell line Calu-3 – that expresses serine proteases – prevents cell culture adaptations in the MBCS and directly adjacent to the MBCS (S686G). Similar results were obtained using a human airway organoid-based culture system for SARS-CoV-2 propagation. Thus, in-depth knowledge on the biology of a virus can be used to establish methods to prevent cell culture adaptation.

Physiology can underlie movement, including short-term activity, exploration of unfamiliar environments, and larger scale dispersal, and thereby influence species distributions in an environmentally sensitive manner. We conducted meta-analyses of the literature to establish, firstly, whether physiological traits underlie activity, exploration, and dispersal by individuals (88 studies), and secondly whether physiological characteristics differed between range core and edges of distributions (43 studies). We show that locomotor performance and metabolism influenced individual movement with varying levels of confidence. Range edges differed from cores in traits that may be associated with dispersal success, including metabolism, locomotor performance, corticosterone levels, and immunity, and differences increased with increasing time since separation. Physiological effects were particularly pronounced in birds and amphibians, but taxon-specific differences may reflect biased sampling in the literature, which also focussed primarily on North America, Europe, and Australia. Hence, physiology can influence movement, but undersampling and bias currently limits general conclusions. Physiological constraints can influence multiple aspects of an animal’s fitness for life and adaptation. Here, a meta-analytical approach demonstrates how physiology can influence animal dispersal ecology via limitations on the ability to move.