C1q enables influenza hemagglutinin stem binding antibodies to block viral attachment and broadens the antibody escape repertoire.

Antigenic drift, the gradual accumulation of amino acid substitutions in the influenza virus hemagglutinin (HA) receptor protein, enables viral immune evasion. Antibodies (Abs) specific for the drift-resistant HA stem region are a promising universal influenza vaccine target. Although anti-stem Abs are not believed to block viral attachment, here we show that complement component 1q (C1q), a 460-kilodalton protein with six Ab Fc-binding domains, confers attachment inhibition to anti-stem Abs and enhances their fusion and neuraminidase inhibition. As a result, virus neutralization activity in vitro is boosted up to 30-fold, and in vivo protection from influenza PR8 infection in mice is enhanced. These effects reflect increased steric hindrance and not increased Ab avidity. C1q greatly expands the anti-stem Ab viral escape repertoire to include residues throughout the HA, some of which cause antigenic alterations in the globular region or modulate HA receptor avidity. We also show that C1q enhances the neutralization activity of non-receptor binding domain anti-SARS-CoV-2 spike Abs, an effect dependent on spike density on the virion surface. These findings demonstrate that C1q can greatly expand Ab function and thereby contribute to viral evolution and immune escape.

Monoclonal Antibodies as SARS-CoV-2 Serology Standards: Experimental Validation and Broader Implications for Correlates of Protection.

COVID-19 has highlighted challenges in the measurement quality and comparability of serological binding and neutralization assays. Due to many different assay formats and reagents, these measurements are known to be highly variable with large uncertainties. The development of the WHO international standard (WHO IS) and other pool standards have facilitated assay comparability through normalization to a common material but does not provide assay harmonization nor uncertainty quantification. In this paper, we present the results from an interlaboratory study that led to the development of (1) a novel hierarchy of data analyses based on the thermodynamics of antibody binding and (2) a modeling framework that quantifies the probability of neutralization potential for a given binding measurement. Importantly, we introduced a precise, mathematical definition of harmonization that separates the sources of quantitative uncertainties, some of which can be corrected to enable, for the first time, assay comparability. Both the theory and experimental data confirmed that mAbs and WHO IS performed identically as a primary standard for establishing traceability and bridging across different assay platforms. The metrological anchoring of complex serological binding and neuralization assays and fast turn-around production of an mAb reference control can enable the unprecedented comparability and traceability of serological binding assay results for new variants of SARS-CoV-2 and immune responses to other viruses.

Mapping the Antibody Repertoires in Ferrets with Repeated Influenza A/H3 Infections: Is Original Antigenic Sin Really "Sinful"?

The influenza-specific antibody repertoire is continuously reshaped by infection and vaccination. The host immune response to contemporary viruses can be redirected to preferentially boost antibodies specific for viruses encountered early in life, a phenomenon called original antigenic sin (OAS) that is suggested to be responsible for diminished vaccine effectiveness after repeated seasonal vaccination. Using a new computational tool called Neutralization Landscapes, we tracked the progression of hemagglutination inhibition antibodies within ferret antisera elicited by repeated influenza A/H3 infections and deciphered the influence of prior exposures on the de novo antibody response to evolved viruses. The results indicate that a broadly neutralizing antibody signature can nevertheless be induced by repeated exposures despite OAS induction. Our study offers a new way to visualize how immune history shapes individual antibodies within a repertoire, which may help to inform future universal influenza vaccine design.

Enhanced virulence and waning vaccine-elicited antibodies account for breakthrough infections caused by SARS-CoV-2 delta and beyond.

Here we interrogate the factors responsible for SARS-CoV-2 breakthrough infections in a K18-hACE2 transgenic mouse model. We show that Delta and the closely related Kappa variant cause viral pneumonia and severe lung lesions in K18-hACE2 mice. Human COVID-19 mRNA post-vaccination sera after the 2nd dose are significantly less efficient in neutralizing Delta/Kappa than early 614G virus in vitro and in vivo. By 5 months post-vaccination, ≥50% of donors lack detectable neutralizing antibodies against Delta and Kappa and all mice receiving 5-month post-vaccination sera die after the lethal challenges. Although a 3rd vaccine dose can boost antibody neutralization against Delta in vitro and in vivo, the mean log neutralization titers against the latest Omicron subvariants are 1/3-1/2 of those against the original 614D virus. Our results suggest that enhanced virulence, greater immune evasion, and waning of vaccine-elicited protection account for SARS-CoV-2 variants caused breakthrough infections.

Functional and structural modifications of influenza antibodies during pregnancy.

Pregnancy represents a unique tolerogenic immune state which may alter susceptibility to infection and vaccine response. Here, we characterized humoral immunity to seasonal influenza vaccine strains in pregnant and non-pregnant women. Although serological responses to influenza remained largely intact during late pregnancy, distinct modifications were observed. Pregnant women had reduced hemagglutinin subtype-1 (H1)- IgG, IgG1, IgG2, and IgG3, hemagglutination inhibition, and group 1 and 2 stem IgG titers. Intriguingly, H1-specific avidity and FcγR1 binding increased, and influenza antibodies had distinct Fc and Fab glycans characterized by increased di-galactosylation and di-sialylation. H1-specific Fc-functionality (i.e. monocyte phagocytosis and complement deposition) was moderately reduced in pregnancy. Multivariate antibody analysis revealed two distinct populations (pregnant vs. non-pregnant) segregated by H1 FcγR1 binding, H1-IgG levels, and Fab and Fc glycosylation. Our results demonstrated a structural and functional modulation of influenza humoral immunity during pregnancy that was antigen-specific and consistent with reduced inflammation and efficient placental transport.

SARS-CoV-2 B.1.1.7 (alpha) and B.1.351 (beta) variants induce pathogenic patterns in K18-hACE2 transgenic mice distinct from early strains.

SARS-CoV-2 variants of concern (VOC) B.1.1.7 (alpha) and B.1.351 (beta) show increased transmissibility and enhanced antibody neutralization resistance. Here we demonstrate in K18-hACE2 transgenic mice that B.1.1.7 and B.1.351 are 100-fold more lethal than the original SARS-CoV-2 bearing 614D. B.1.1.7 and B.1.351 cause more severe organ lesions in K18-hACE2 mice than early SARS-CoV-2 strains bearing 614D or 614G, with B.1.1.7 and B.1.351 infection resulting in distinct tissue-specific cytokine signatures, significant D-dimer depositions in vital organs and less pulmonary hypoxia signaling before death. However, K18-hACE2 mice with prior infection of early SARS-CoV-2 strains or intramuscular immunization of viral spike or receptor binding domain are resistant to the lethal reinfection of B.1.1.7 or B.1.351, despite having reduced neutralization titers against these VOC than early strains. Our results thus distinguish pathogenic patterns in K18-hACE2 mice caused by B.1.1.7 and B.1.351 infection from those induced by early SARS-CoV-2 strains, and help inform potential medical interventions for combating COVID-19.

SARS-CoV-2 show no infectivity at later stages in a prolonged COVID-19 patient despite positivity in RNA testing.

Inpatient coronavirus disease 2019 (COVID-19) cases present enormous costs to patients and health systems in the United States. Many hospitalized patients may continue testing COVID-19 positive even after the resolution of symptoms. Thus, a pressing concern for clinicians is the safety of discharging these asymptomatic patients if they have any remaining infectivity. This case report explores the viral viability in a patient with persistent COVID-19 over the course of a 2-month hospitalization. Positive nasopharyngeal swab samples were collected and isolated in the laboratory and analyzed by quantitative reverse-transcription polymerase chain reactions (qRT-PCR), and serology was tested for neutralizing antibodies throughout the hospitalization period. The patient experienced waning symptoms by hospital day 40 and had no viable virus growth by hospital day 41, suggesting no risk of infectivity, despite positive RT-PCR results which prolonged his hospital stay. Notably, this case showed infectivity for at least 24 days after disease onset, which is longer than the discontinuation of transmission-based precautions recommended by the Center for Disease Control and Prevention. Thus, our findings suggest that the timeline for discontinuing transmission-based precautions may need to be extended for patients with severe and prolonged COVID-19 disease. Additional large-scale studies are needed to draw definitive conclusions on the appropriate clinical management for these patients. ​.

Highly pathogenic avian influenza A/Guangdong/17SF003/2016 is immunogenic and induces cross-protection against antigenically divergent H7N9 viruses.

Avian influenza A(H7N9) epidemics have a fatality rate of approximately 40%. Previous studies reported that low pathogenic avian influenza (LPAI)-derived candidate vaccine viruses (CVVs) are poorly immunogenic. Here, we assess the immunogenicity and efficacy of a highly pathogenic avian influenza (HPAI) A/Guangdong/17SF003/2016 (GD/16)-extracted hemagglutinin (eHA) vaccine. GD/16 eHA induces robust H7-specific antibody responses in mice with a marked adjuvant antigen-sparing effect. Mice immunized with adjuvanted GD/16 eHA are protected from the lethal LPAI and HPAI H7N9 challenges, in stark contrast to low antibody titers and high mortality in mice receiving adjuvanted LPAI H7 eHAs. The protection correlates well with the magnitude of the H7-specific antibody response (IgG and microneutralization) or HA group 2 stem-specific IgG. Inclusion of adjuvanted GD/16 eHA in heterologous prime-boost improves the immunogenicity and protection of LPAI H7 HAs in mice. Our findings support the inclusion of GD/16-derived CVV in the pandemic preparedness vaccine stockpile.

Reduced Influenza B-Specific Postvaccination Antibody Cross-reactivity in the B/Victoria Lineage-Predominant 2019/20 Season.

BACKGROUND: The influenza activity of the 2019/20 season remained high and widespread in the United States with type B viruses predominating the early season. The majority of B viruses characterized belonged to B/Victoria (B/Vic) lineage and contained a triple deletion of amino acid (aa) 162-164 in hemagglutinin (3DEL). These 3DEL viruses are antigenically distinct from B/Colorado/06/2017 (CO/06)-the B/Vic vaccine component of the 2018/19 and 2019/20 seasons representing the viruses with a double deletion of aa 162-163 in hemagglutinin (2DEL). METHODS: We performed molecular characterization and phylogenetic analysis of circulating B/Vic viruses. We also conducted hemagglutination inhibition (HAI) assay using archived human postvaccination sera collected from healthy subjects administered with different types of 2018/19 or 2019/20 seasonal vaccines. Their HAI cross-reactivity to representative 3DEL viruses was analyzed. RESULTS: The CO/06-specific human postvaccination sera, after being adjusted for vaccine type, had significantly reduced HAI cross-reactivity toward representative 3DEL viruses, especially the 136E+150K subgroup. The geometric mean titers against 3DEL viruses containing 136E+150K mutations were 1.6-fold lower in all populations (P = .051) and 1.9-fold lower in adults (P = .016) compared with those against the 136E+150N viruses. CONCLUSIONS: Our results indicate that postvaccination antibodies induced by the B/Vic vaccine component of the 2019/20 influenza season had reduced HAI cross-reactivity toward predominant 3DEL viruses in the United States. A close monitoring of the 3DEL 136E+150K subgroup is warranted should this subgroup return and predominate the 2020/21 influenza season.

Pregnancy level of estradiol attenuated virus-specific humoral immune response in H5N1-infected female mice despite inducing anti-inflammatory protection.

Estradiol, a major female steroid produced during pregnancy, has been reported to protect ovariectomized animals against H1N1 influenza infections via its anti-inflammatory effects. However, it remains unclear why pregnant women with high gestational estradiol levels are highly susceptible to influenza infections. This study was aimed to investigate the effects of pregnancy level of estradiol on female immunity against H5N1 infection in Balb/c mice. A sex-dependent susceptibility to H5N1 infection (higher morbidity and higher mortality) was observed in both pregnant and non-pregnant female mice as compared to male mice. Subcutaneous implantation of estradiol pellets increased serum estradiol concentrations of non-pregnant female mice to the pregnancy level. These mice were protected from H5N1 infection through downregulation of pulmonary pro-inflammatory cytokines. However, the production of virus-specific antibodies after infection was significantly delayed in estradiol-implanted mice when compared to placebos. Virus-specific IgG-secreting and IL-4-secreting cells were also reduced in estradiol-implanted mice. Similarly, lower antibody titers to seasonal vaccine antigens were found in pregnant women as compared to non-pregnant females without hormone usage. Our results indicate that estradiol levels equivalent to those found during pregnancy have divergent effects on female immunity against influenza, highlighting the importance of vaccination during pregnancy to prevent severe influenza infections.

Neuraminidase inhibition contributes to influenza A virus neutralization by anti-hemagglutinin stem antibodies.

Broadly neutralizing antibodies (Abs) that bind the influenza virus hemagglutinin (HA) stem may enable universal influenza vaccination. Here, we show that anti-stem Abs sterically inhibit viral neuraminidase (NA) activity against large substrates, with activity inversely proportional to the length of the fibrous NA stalk that supports the enzymatic domain. By modulating NA stalk length in recombinant IAVs, we show that anti-stem Abs inhibit virus release from infected cells by blocking NA, accounting for their in vitro neutralization activity. NA inhibition contributes to anti-stem Ab protection in influenza-infected mice, likely due at least in part to NA-mediated inhibition of FcγR-dependent activation of innate immune cells by Ab bound to virions. Food and Drug Administration-approved NA inhibitors enhance anti-stem-based Fc-dependent immune cell activation, raising the possibility of therapeutic synergy between NA inhibitors and anti-stem mAb treatment in humans.

Comprehensive analysis of N-glycans in IgG purified from ferrets with or without influenza A virus infection.

Influenza viruses cause contagious respiratory infections, resulting in significant economic burdens to communities. Production of influenza-specific Igs, specifically IgGs, is one of the major protective immune mechanisms against influenza viruses. In humans, N-glycosylation of IgGs plays a critical role in antigen binding and effector functions. The ferret is the most commonly used animal model for studying influenza pathogenesis, virus transmission, and vaccine development, but its IgG structure and functions remain largely undefined. Here we show that ferret IgGs are N-glycosylated and that their N-glycan structures are diverse. Using a comprehensive strategy based on MS and ultra-HPLC analyses in combination with exoglycosidase digestions, we assigned 42 N-glycan structures in ferret IgGs. We observed that N-glycans of ferret IgGs consist mainly of complex-type glycans, including some high-mannose and hybrid glycans, similar to those observed in human IgG. The complex-type glycans of ferret IgGs were primarily core-fucosylated. Furthermore, a fraction of N-glycans carried bisecting GlcNAc. Ferret IgGs also had a minor fraction of glycans carrying α2-6Neu5Ac(s). We noted that, unlike human IgG, ferret IgGs have αGal epitopes on some N-glycans. Interestingly, influenza A infection caused prominent changes in the N-glycans of ferret IgG, mainly because of an increase in bisecting GlcNAc and F1A2G0 and a corresponding decrease in F1A2G1. This suggests that the glycosylation of virus-specific IgG may play a role in its functionality. Our study highlights the need to further elucidate the structure-function relationships of IgGs in universal influenza vaccine development.

Correction: Influenza A virus hemagglutinin glycosylation compensates for antibody escape fitness costs.

[This corrects the article DOI: 10.1371/journal.ppat.1006796.].

Imprinting of Repeated Influenza A/H3 Exposures on Antibody Quantity and Antibody Quality: Implications for Seasonal Vaccine Strain Selection and Vaccine Performance.

Background: Reduced seasonal influenza vaccine effectiveness (VE) was observed in individuals who received repeated annual vaccinations. Preexisting influenza antibody levels were also found inversely correlated with postvaccination titers. These reports suggest that preexisting immunity may affect contemporary seasonal vaccine performance. Methods: Influenza A/H3 specific cross-reactivity of postvaccination sera from humans with or without preexisting immunity was assessed by hemagglutination inhibition (HAI) assay. Ferret antisera induced by repeated H3 exposures were also subjected to HAI, antibody affinity, and antibody avidity analyses. Results: Human postvaccination sera derived from subjects with or without preexisting immunity showed different cross-reactivity against H3 variant viruses. Similarly, the breadth of cross-reactive ferret antibodies induced by repeated H3 exposures was also broadened. Antigenic differences between H3 viruses characterized by ferret antisera became smaller as the number of exposures increased. Although repeated H3 exposures induced "original antigenic sin" phenomena in HAI titers against later exposed viruses, resultant ferret antibodies showed gradually enhanced avidity for different H3/hemagglutinin. Increased antibody avidity was found to be inversely correlated with decreased antigenic differences among H3 viruses characterized. Conclusions: Our results suggest that repeated H3 exposures imprinted not only antibody quantity but also antibody quality. The "naive" ferret model currently used for vaccine strain selection does not recapitulate the complexity of human preexisting immunity. Vaccine strains identified hereby may not provide coverage sufficient for those who were frequently infected and/or vaccinated, leading to the reduced VE observed.

Influenza A virus hemagglutinin glycosylation compensates for antibody escape fitness costs.

Rapid antigenic evolution enables the persistence of seasonal influenza A and B viruses in human populations despite widespread herd immunity. Understanding viral mechanisms that enable antigenic evolution is critical for designing durable vaccines and therapeutics. Here, we utilize the primerID method of error-correcting viral population sequencing to reveal an unexpected role for hemagglutinin (HA) glycosylation in compensating for fitness defects resulting from escape from anti-HA neutralizing antibodies. Antibody-free propagation following antigenic escape rapidly selected viruses with mutations that modulated receptor binding avidity through the addition of N-linked glycans to the HA globular domain. These findings expand our understanding of the viral mechanisms that maintain fitness during antigenic evolution to include glycan addition, and highlight the immense power of high-definition virus population sequencing to reveal novel viral adaptive mechanisms.

Maintaining pH-dependent conformational flexibility of M1 is critical for efficient influenza A virus replication.

The M gene segment of influenza A virus has been shown to be a contributing factor to the high growth phenotype. However, it remains largely unknown why matrix protein 1 (M1), the major structural protein encoded by M gene, exhibits pH-dependent conformational changes during virus replication. Understanding the mechanisms underlying efficient virus replication can help to develop strategies not only to combat influenza infections but also to improve vaccine supplies. M(NLS-88R) and M(NLS-88E) are two M1 mutants differing by only a single amino acid: G88R vs G88E. G88R but not G88E was the compensatory mutation naturally selected by the virus after its nuclear localization signal was disrupted. Our study shows that, compared with M(NLS-88E) M1, M(NLS-88R) M1 dissociated quickly from viral ribonucleoproteins (vRNPs) at higher pH and took less time to dissemble in vitro, despite forming thicker matrix layer and having stronger association with vRNP in assembled virions. Correspondingly, M(NLS-88R) replicated more efficiently and was genetically more stable than M(NLS-88E). Crystallographic analysis indicated that M(NLS-88R) M1, like wild-type M1, is able to switch from a face-to-back-oriented conformation to a face-to-face-oriented conformation when pH drops from neutral to acidic, whereas G88E mutation causes M(NLS-88E) M1 to be trapped in a face-to-face-arranged conformation regardless of environmental pH. Our results suggest that maintaining M1 pH-dependent conformational flexibility is critical for efficient virus replication, and position 88 is a key residue controlling M1 pH-dependent conformational changes. Our findings provide insights into developing M1-based antiviral agents.

Solid-Phase Chemical Modification for Sialic Acid Linkage Analysis: Application to Glycoproteins of Host Cells Used in Influenza Virus Propagation.

Differentiation between the sialyl linkages is often critical to understanding biological consequence. Here we present a facile method for determining these linkages in glycans. Analysis of sialic acids is challenging due to their labile nature during sample preparation and ionization. Derivatization is often required via chemical reaction. Amidation derivatizes all sialic acids regardless of linkage, while esterification enables differentiation between α2,3-linked and α2,6-linked sialic acids. Reactions have been primarily performed on free glycans in solution but have been recently adapted to solid-phase providing unique advantages such as simplified sample preparation, improved yield, and high throughput applications. Here, we immobilized glycoproteins on resin via reductive amination, modified α2,6-linked sialic acids through ethyl esterification, and α2,3-linked sialic acids via amidation. N-glycans and O-glycans were released via enzyme and chemical reactions. The method was applied for analysis of three different MDCK cell lines used for influenza propagation and where distributions of α2,3 and α2,6 sialic acids are critical for cell performance. Linkage specific distribution of these sialic acids was quantitatively determined and unique for each cell line. Our study demonstrates that protein sialylation can be reliably and quantitatively characterized in terms of sialic acid linkage of each glycan using the solid-phase esterification/amidation strategy.

Different Repeat Annual Influenza Vaccinations Improve the Antibody Response to Drifted Influenza Strains.

Seasonal influenza vaccine formulas change almost every year yet information about how this affects the antibody repertoire of vaccine recipients is inadequate. New vaccine virus strains are selected, replacing older strains to better match the currently circulating strains. But even while the vaccine is being manufactured the circulating strains can evolve. The ideal response to a seasonal vaccine would maintain antibodies toward existing strains that might continue to circulate, and to generate cross-reactive antibodies, particularly towards conserved influenza epitopes, potentially limiting infections caused by newly evolving strains. Here we use the hemagglutination inhibition assay to analyze the antibody repertoire in subjects vaccinated two years in a row with either identical vaccine virus strains or with differing vaccine virus strains. The data indicates that changing the vaccine formulation results in an antibody repertoire that is better able to react with strains emerging after the vaccine virus strains are selected. The effect is observed for both influenza A and B strains in groups of subjects vaccinated in three different seasons. Analyses include stratification by age and sex.

Differential Effects of Prior Influenza Exposures on H3N2 Cross-reactivity of Human Postvaccination Sera.

BACKGROUND: Effectiveness of seasonal influenza vaccines mainly depends upon how well vaccine strains represent circulating viruses; mismatched strains can lead to reduced protection. Humans have complex influenza exposure histories that increase with age, which may lead to different postvaccination responses to emerging influenza variants. Recent observational studies also suggest that prior vaccination may influence the performance of current seasonal vaccines. METHODS: To elucidate the effects of age and influenza preexposures on cross-reactivity of vaccination-induced human antibodies, we generated antigenic maps based on postvaccination hemagglutination inhibition titers against representative H3 variants circulating during the 2015-2016, 2014-2015, and 2012-2013 influenza seasons. RESULTS: Antigenic maps determined using sera from subjects 18-64 and ≥65 years of age correlated well with each other but poorly with those determined using sera from children. Antigenic maps derived from human postvaccination sera with H1 influenza preexposure also correlated poorly with those derived from sera with neither H1 nor type B influenza preexposure, and the correlation lessened considerably over time. In contrast, antigenic maps derived from human postvaccination sera with only type B influenza preexposure consistently showed good correlation with those derived from sera with neither H1 nor type B influenza preexposure. CONCLUSIONS: Our results suggest an age-specific difference in human postvaccination responses. Our findings also suggest that prior exposure to H1 or type B influenza may differentially affect cross-reactivity of vaccination-induced H3-specific hemagglutination inhibition antibody responses, and consequently might affect vaccine effectiveness. Our study highlights the need to study the impact of prior exposure on influenza vaccine performance.

The ubiquitination of the influenza A virus PB1-F2 protein is crucial for its biological function.

The aim of the present study was to identify what influences the short half-life of the influenza A virus PB1-F2 protein and whether a prolonged half-life affects the properties of this molecule. We hypothesized that the short half-life of PB1-F2 could conceal the phenotype of the protein. Because proteasome degradation might be involved in PB1-F2 degradation, we focused on ubiquitination, a common label for proteasome targeting. A cluster of lysine residues was demonstrated as an ubiquitination acceptor site in evolutionary and functionally distinct proteins. The PB1-F2 sequence alignment revealed a cluster of lysines on the carboxy terminal end of PB1-F2 in almost all of the GenBank sequences available to date. Using a proximity ligation assay, we identified ubiquitination as a novel posttranslational modification of PB1-F2. Changing the lysines at positions 73, 78, and 85 to arginines suppressed the ubiquitination of A/Puerto Rico/8/1934 (H1N1)-derived PB1-F2. The mutation of the C-terminal lysine residue cluster positively affected the overall expression levels of avian A/Honk Kong/156/1997 (H5N1)- and mammalian A/Puerto Rico/8/1934 (H1N1)-derived PB1-F2. Moreover, increased PB1-F2 copy numbers strengthened the functions of this virus in the infected cells. The results of a minigenome luciferase reporter assay revealed an enhancement of viral RNA-dependent RNA polymerase activity in the presence of stabilized PB1-F2, regardless of viral origin. IFNß antagonism was enhanced in 293T cells transfected with a plasmid expressing stabilized K→R mutant variants of PB1-F2. Compared with PB1-F2 wt, the loss of ubiquitination enhanced the antibody response after DNA vaccination. In summary, we revealed that PB1-F2 is an ubiquitinated IAV protein, and this posttranslational modification plays a central role in the regulation of the biological functions of this protein.