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Resistance to influenza antibodies Broadly neutralizing human antibodies (bnAbs) to the stem of hemagglutinin (HA), a trimeric glycoprotein found on the surface of influenza viruses, are valuable therapeutics and can guide the development of universal influenza vaccines. For their use in therapy development, it is important to understand the extent to which HA stem variants with resistance to bnAbs can develop. Wu et al. used saturation mutagenesis combined with next-generation sequencing to systematically search for resistance mutations to prototypic bnAbs in two influenza subtypes, H3 and H1. They found that the genetic barrier to resistance to stem bnAbs was low for the H3 subtype but higher for the H1 subtype. The ability of H3 to develop resistance to bnAbs presents a challenge in the development of a universal influenza vaccine. Science , this issue p. 1335

SUMMARY Antibodies targeting the receptor binding site (RBS) of the influenza virus hemagglutinin (HA) protein are usually not broadly reactive because their footprints are typically large and extend to nearby variable HA residues. Here, we identify several human H3N2 HA RBS-targeting monoclonal antibodies (mAbs) that are sensitive to substitutions in conventional antigenic sites and are therefore not broadly reactive. However, we also identify an H3N2 HA RBS-targeting mAb that is exceptionally broadly reactive despite being sensitive to substitutions in residues outside of the RBS. We show that similar antibodies are present at measurable levels in the sera of some individuals but that they are inefficiently elicited by conventional vaccines. Our data indicate that HA RBS-targeting antibodies can be effective against variable viral strains even when they are somewhat sensitive to substitutions in HA residues adjacent to the RBS., Graphical Abstract, In Brief Zost et al. show that most antibodies targeting the RBS of the H3N2 HAs are not broadly reactive. They identify one broadly reactive H3 HA RBS antibody that is tolerant of substitutions in adjacent antigenic sites but show that these types of antibodies are not efficiently elicited by vaccination.

Airborne transmission of seasonal and pandemic influenza viruses is the reason for their epidemiological success and public health burden in humans. Efficient airborne transmission of the H1N1 influenza virus relies on the receptor specificity and pH of fusion of the surface glycoprotein hemagglutinin (HA). In this study, we examined the role of HA pH of fusion on transmissibility of a cell-culture-adapted H3N2 virus. Mutations in the HA head at positions 78 and 212 of A/Perth/16/2009 (H3N2), which were selected after cell culture adaptation, decreased the acid stability of the virus from pH 5.5 (WT) to pH 5.8 (mutant). In addition, the mutant H3N2 virus replicated to higher titers in cell culture but had reduced airborne transmission in the ferret model. These data demonstrate that, like H1N1 HA, the pH of fusion for H3N2 HA is a determinant of efficient airborne transmission. Surprisingly, noncoding regions of the NA segment can impact the pH of fusion of mutant viruses. Taken together, our data confirm that HA acid stability is an important characteristic of epidemiologically successful human influenza viruses and is influenced by HA/NA balance.

Background High morbidity and mortality have been observed in patients with cancer and coronavirus disease 2019 (COVID-19); however, there are limited data on antimicrobial use, coinfections, and viral shedding. Methods We conducted a retrospective cohort study of adult patients at the Seattle Cancer Care Alliance diagnosed with COVID-19 between February 28, 2020 and June 15, 2020 to characterize antimicrobial use, coinfections, viral shedding, and outcomes within 30 days after diagnosis. Cycle threshold values were used as a proxy for viral load. We determined viral clearance, defined as 2 consecutive negative results using severe acute respiratory syndrome coronavirus 2 reverse-transcription polymerase chain reaction results through July 30, 2020. Results Seventy-one patients were included with a median age of 61 years; 59% had a solid tumor. Only 3 patients had documented respiratory bacterial coinfection. Empiric antibiotics for pneumonia were prescribed more frequently early in the study period (February 29–March 28, 2020; 12/34) compared to the later period (March 29–June 15, 2020; 2/36) (P = .002). The median number of days from symptom onset to viral clearance was 37 days with viral load rapidly declining in the first 7–10 days after symptom onset. Within 30 days of diagnosis, 29 (41%) patients were hospitalized and 12 (17%) died. Each additional comorbidity was associated with 45% lower odds of days alive and out of hospital in the month following diagnosis in adjusted models. Conclusions Patients at a cancer center, particularly those with multiple comorbidities, are at increased risk of poor outcomes from COVID-19. Prolonged viral shedding is frequently observed among cancer patients, and its implications on transmission and treatment strategies warrant further study.

In the past decade, the discovery and characterization of broadly neutralizing antibodies (bnAbs) to the highly conserved stem region of influenza hemagglutinin (HA) have provided valuable insights for development of a universal influenza vaccine. However, the genetic barrier for resistance to stem bnAbs has not been thoroughly evaluated. Here, we performed a series of deep mutational scanning experiments to probe for resistance mutations. We found that the genetic barrier to resistance to stem bnAbs is generally very low for the H3 subtype but substantially higher for the H1 subtype. Several resistance mutations in H3 cannot be neutralized by stem bnAbs at the highest concentration tested, do not reducein vitroviral fitness andin vivopathogenicity, and are often present in circulating strains as minor variants. Thus, H3 HAs have a higher propensity than H1 HAs to escape major stem bnAbs and creates a potential challenge in the development of abona fideuniversal influenza vaccine.ONE SENTENCE SUMMARYAcquisition of resistance by influenza virus to broadly neutralizing hemagglutinin stem antibodies varies tremendously depending on subtype.

Influenza virus can escape most antibodies with single mutations. However, rare antibodies broadly neutralize many viral strains. It is unclear how easily influenza virus might escape such antibodies if there was strong pressure to do so. Here, we map all single amino-acid mutations that increase resistance to broad antibodies to H1 hemagglutinin. Our approach not only identifies antigenic mutations but also quantifies their effect sizes. All antibodies select mutations, but the effect sizes vary widely. The virus can escape a broad antibody to hemagglutinin’s receptor-binding site the same way it escapes narrow strain-specific antibodies: via single mutations with huge effects. In contrast, broad antibodies to hemagglutinin’s stalk only select mutations with small effects. Therefore, among the antibodies we examine, breadth is an imperfect indicator of the potential for viral escape via single mutations. Antibodies targeting the H1 hemagglutinin stalk are quantifiably harder to escape than the other antibodies tested here., Influenza A virus can escape antibodies, but it is unclear how the ease of escape depends on the epitope targeted by an antibody. Here, the authors show that neutralization breadth is an imperfect indicator of the ease of viral escape by single mutations, and that antibodies targeting the stalk of hemagglutinin are harder to escape.

Background IDSA/SHEA guidelines recommend that antimicrobial stewardship programs support providers in antibiotic decisions for end of life care. Washington State Physician Orders for Life-Sustaining Treatment (POLST) forms allow patients to indicate antimicrobial use preferences. We sought to characterize antimicrobial use in the last 30 days of life for cancer patients by presence of a POLST and antimicrobial use preferences. Methods We performed a single-center, retrospective cohort study of cancer patient deaths from January 1, 2016 - June 30, 3018. Patient demographics, clinical characteristics, POLST, and antimicrobial use within 30 days before death were extracted from electronic records. To test for an association between POLST completed at least 30 days before death and inpatient antimicrobial days of therapy (DOT) in the 30 days before death, we used negative binomial models adjusted for age, sex, race, and service line (hematologic versus solid malignancy); model estimates are presented as incidence rate ratios (IRR) with 95% confidence intervals (CI) Results Of 1796 patients, 406 (23%) had a POLST. 177/406 (44%) were completed less than 30 days before death, and 58/177 (32.8%) specified limited antibiotic use; 40/177 (23%) did not specify any antimicrobial use preference (Fig 1). Of 1295 patients with at least 1 inpatient day in the 30 days before death, 1070 (83%) received at least 1 inpatient antimicrobial with median DOT of 1077 per 1000 inpatient days (Tab 1). There was no difference in DOT among patients with and without a POLST > /= 30 days before death (IRR 0.92, CI 0.77, 1.10). Patients with a POLST specifying limited antibiotic use had significantly lower inpatient IV antimicrobial DOT compared to those without a POLST (IRR 0.64, CI 0.42–0.97) (Fig 2). Figure 1. Classification of Patients by Presence of POLST, Timing, and Antimicrobial Preference Content of POLST. Numbers shown represent the number of patients (percentage). Full antibiotic use refers to the selection “Use antibiotics for prolongation of life.” Limited antibiotic use refers to the selection “Do not use antibiotics except when needed for symptom management.” Table 1: Antimicrobial use for all patients and by advance directive group Figure 2. Forest plot of model estimates, represented as incidence rate ratios (IRR) with 95% confidence intervals (CI), for associations between POLST antimicrobial specifications completed at least 30 days before death and inpatient antibiotic days of therapy (DOT) in the 30 days before death. Estimates represent comparisons between each POLST category and no POLST completed at least 30 days before death. Dots represent the IRR and brackets extend to the lower and upper limit of the 95% CI. Blue estimates are for the inpatient antibiotic DOT outcome and red estimates are for the inpatient IV antibiotic DOT outcome. Conclusion POLST completion is rare > /= 30 days before death, with few POLSTs specifying antimicrobial use. Compared to those with no POLST in this time frame, patients who indicated that antibiotics should be used only for symptom management received significantly fewer inpatient IV antimicrobials. Early discussion of advance directives including POLST with specification of antimicrobial use preferences may promote more thoughtful use of antimicrobials near the end of life in a compassionate, patient-centered way. Disclosures Steven A. Pergam, MD, MPH, Chimerix, Inc (Scientific Research Study Investigator)Global Life Technologies, Inc. (Research Grant or Support)Merck & Co. (Scientific Research Study Investigator)Sanofi-Aventis (Other Financial or Material Support, Participate in clinical trial sponsored by NIAID (U01-AI132004); vaccines for this trial are provided by Sanofi-Aventis)

SUMMARYAntibodies targeting the receptor binding site (RBS) of the influenza virus hemagglutinin (HA) protein are usually not broadly-reactive because their footprints are typically large and extend to nearby variable HA residues. Here, we identified several human H3N2 HA RBS-targeting monoclonal antibodies (mAbs) that were sensitive to substitutions in conventional antigenic sites and were not broadly-reactive. However, we also identified one H3N2 HA RBS-targeting mAb that was exceptionally broadly reactive despite being sensitive to substitutions in residues outside of the RBS. We determined that similar antibodies are present at measurable levels in the sera of some individuals but that they are inefficiently elicited by conventional vaccines. Our data indicate that some HA RBS-targeting antibodies can be surprisingly effective against variable viral strains even if they are somewhat sensitive to substitutions in HA residues adjacent to the RBS.

A longstanding question is how influenza virus evolves to escape human immunity, which is polyclonal and can target many distinct epitopes. Here, we map how all amino-acid mutations to influenza’s major surface protein affect viral neutralization by polyclonal human sera. The serum of some individuals is so focused that it selects single mutations that reduce viral neutralization by over an order of magnitude. However, different viral mutations escape the sera of different individuals. This individual-to-individual variation in viral escape mutations is not present among ferrets that have been infected just once with a defined viral strain. Our results show how different single mutations help influenza virus escape the immunity of different members of the human population, a phenomenon that could shape viral evolution and disease susceptibility., eLife digest The human immune system protects the body from repeat attacks by remembering past infections. However, a typical person comes down with the flu every five to seven years. This is because flu viruses rapidly evolve to bypass our defenses. So, after a few years, the viruses look so different that the immune system no longer recognizes them. The immune system recognizes flu viruses by producing proteins known as antibodies, which can bind to the virus and prevent it from infecting cells. Many of these antibodies bind to a protein on the surface of the virus called hemagglutinin, but each anti-flu antibody recognizes only a small region of the protein. This means that to escape recognition by a single antibody, all the virus needs to do is wait for a lucky mutation to change the part of hemagglutinin recognized by that antibody. But humans make many different antibodies. To escape them all, flu viruses would need lots of lucky mutations. So how do flu viruses keep winning the evolutionary lottery? To answer this question, Lee et al. made all the possible individual mutations to the hemagglutinin protein of a human flu virus. A pool of these viruses was then exposed to the full mix of antibodies present in human serum (the liquid component of blood). Lee et al. then checked which mutations helped the virus survive contact with the antibodies. For most human serum samples, a single mutation was enough to allow the virus to escape most of one person’s anti-flu antibodies. This suggests that the immune response to flu is so focused on a small region of hemagglutinin that a mutation in this region can enable the virus to take a huge step towards evading immune detection. Even more surprising was what happened when Lee et al. looked at serum from different people. A mutation that helped the virus to escape immune detection in one person often had little or no effect on escape from another person’s immunity. In other words, the lucky mutation that the virus needed to escape differed from one person to the next. Every year there are many related flu viruses that infect humans. The results of Lee et al. suggest that people could be susceptible to different forms of the virus. Understanding how flu viruses escape immune detection in different people could help us identify which version of the virus different people are more susceptible to, and perhaps eventually better predict how the virus will evolve and spread.

Significance A key goal in the study of influenza virus evolution is to forecast which viral strains will persist and which ones will die out. Here we experimentally measure the effects of all amino acid mutations to the hemagglutinin protein from a human H3N2 influenza strain on viral growth in cell culture. We show that these measurements have utility for distinguishing among viral strains that do and do not succeed in nature. Overall, our work suggests that new high-throughput experimental approaches may be useful for understanding virus evolution in nature., 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 virus can completely escape most antibodies with single mutations. However, rare antibodies broadly neutralize many viral strains. It is unclear how easily influenza virus might escape such antibodies if it was under strong pressure to do so. Here we map all single amino-acid mutations that increase resistance to broad antibodies targeting an H1 hemagglutinin. Crucially, our approach not only identifies antigenic mutations but also quantifies their effect sizes. All antibodies select mutations, but the effect sizes vary widely. The virus can escape a broad antibody that targets residues in hemagglutinin’s receptor-binding site the same way it escapes narrow strain-specific antibodies: via single mutations with huge effects. In contrast, broad antibodies targeting hemagglutinin’s stalk only select mutations with small effects. Therefore, among the antibodies we have examined, breadth is an imperfect indicator of the potential for viral escape via single mutations. Broadly neutralizing antibodies targeting the H1 hemagglutinin stalk are quantifiably harder to escape than the other antibodies tested here.

Human mitochondrial DNLZ/HEP regulates the catalytic activity and solubility of the mitochondrial hsp70 chaperone HSPA9. Here, we investigate the role that the DNLZ zinc-binding and C-terminal subdomains play in regulating HSPA9. We show that truncations lacking portions of the zinc-binding subdomain (ZBS) do not affect the solubility of HSPA9 or its ATPase domain, whereas those containing the ZBS and at least 10 residues following this subdomain enhance chaperone solubility. Binding measurements further show that DNLZ requires its ZBS to form a stable complex with the HSPA9 ATPase domain, and ATP hydrolysis measurements reveal that the ZBS is critical for full stimulation of HSPA9 catalytic activity. We also examined if DNLZ is active in vivo. We found that DNLZ partially complements the growth of Δzim17 Saccharomyces cerevisiae, and we discovered that a Zim17 truncation lacking a majority of the C-terminal subdomain strongly complements growth like full-length Zim17. These findings provide direct evidence that human DNLZ is a functional ortholog of Zim17. In addition, they implicate the pair of antiparallel β-strands that coordinate zinc in Zim17/DNLZ-type proteins as critical for binding and regulating hsp70 chaperones.

Traumatic brain injury (TBI) involves the elaboration of oxidative stress that causes cerebrovascular dysfunction, including impairment of autoregulation of cerebral blood flow. Currently, there is no clinically effective antioxidant treatment for these pathologies. Most currently available antioxidants act through mechanisms in which the antioxidant either transfers the radical or requires regeneration, both of which are impaired in the toxic post-TBI environment. We previously reported that single-walled carbon nanotubes (SWCNTs) and ultrashort SWCNTs possess antioxidant activity, and their characteristics suggest that radical annihilation is the major mechanism. We have now developed a biologically compatible class of carbon-based nanovectors, poly(ethylene glycol)-functionalized hydrophilic carbon clusters (PEG-HCCs) that can be further functionalized with antibodies, and hence show promise as targeted drug delivery platforms. Here we report that PEG-HCCs possess innate antioxidant activity and can be rapidly targeted via an antibody to the P-selectin antigen in a model of injured cultured brain endothelial cells. One immediate application of this therapy is to vascular dysfunction that accompanies TBI and worsens outcome in the face of systemic hypotension. These in vitro results support the need for further investigation in animal models.