Extremely potent human monoclonal antibodies from COVID-19 convalescent patients.

Human monoclonal antibodies are safe, preventive, and therapeutic tools that can be rapidly developed to help restore the massive health and economic disruption caused by the coronavirus disease 2019 (COVID-19) pandemic. By single-cell sorting 4,277 SARS-CoV-2 spike protein-specific memory B cells from 14 COVID-19 survivors, 453 neutralizing antibodies were identified. The most potent neutralizing antibodies recognized the spike protein receptor-binding domain, followed in potency by antibodies that recognize the S1 domain, the spike protein trimer, and the S2 subunit. Only 1.4% of them neutralized the authentic virus with a potency of 1-10 ng/mL. The most potent monoclonal antibody, engineered to reduce the risk of antibody-dependent enhancement and prolong half-life, neutralized the authentic wild-type virus and emerging variants containing D614G, E484K, and N501Y substitutions. Prophylactic and therapeutic efficacy in the hamster model was observed at 0.25 and 4 mg/kg respectively in absence of Fc functions.

Human neutralizing antibodies against SARS-CoV-2 require intact Fc effector functions for optimal therapeutic protection.

SARS-CoV-2 has caused the global COVID-19 pandemic. Although passively delivered neutralizing antibodies against SARS-CoV-2 show promise in clinical trials, their mechanism of action in vivo is incompletely understood. Here, we define correlates of protection of neutralizing human monoclonal antibodies (mAbs) in SARS-CoV-2-infected animals. Whereas Fc effector functions are dispensable when representative neutralizing mAbs are administered as prophylaxis, they are required for optimal protection as therapy. When given after infection, intact mAbs reduce SARS-CoV-2 burden and lung disease in mice and hamsters better than loss-of-function Fc variant mAbs. Fc engagement of neutralizing antibodies mitigates inflammation and improves respiratory mechanics, and transcriptional profiling suggests these phenotypes are associated with diminished innate immune signaling and preserved tissue repair. Immune cell depletions establish that neutralizing mAbs require monocytes and CD8+ T cells for optimal clinical and virological benefit. Thus, potently neutralizing mAbs utilize Fc effector functions during therapy to mitigate lung infection and disease.

Isolation and characterization of IgG3 glycan-targeting antibodies with exceptional cross-reactivity for diverse viral families.

Broadly reactive antibodies that target sequence-diverse antigens are of interest for vaccine design and monoclonal antibody therapeutic development because they can protect against multiple strains of a virus and provide a barrier to evolution of escape mutants. Using LIBRA-seq (linking B cell receptor to antigen specificity through sequencing) data for the B cell repertoire of an individual chronically infected with human immunodeficiency virus type 1 (HIV-1), we identified a lineage of IgG3 antibodies predicted to bind to HIV-1 Envelope (Env) and influenza A Hemagglutinin (HA). Two lineage members, antibodies 2526 and 546, were confirmed to bind to a large panel of diverse antigens, including several strains of HIV-1 Env, influenza HA, coronavirus (CoV) spike, hepatitis C virus (HCV) E protein, Nipah virus (NiV) F protein, and Langya virus (LayV) F protein. We found that both antibodies bind to complex glycans on the antigenic surfaces. Antibody 2526 targets the stem region of influenza HA and the N-terminal domain (NTD) region of SARS-CoV-2 spike. A crystal structure of 2526 Fab bound to mannose revealed the presence of a glycan-binding pocket on the light chain. Antibody 2526 cross-reacted with antigens from multiple pathogens and displayed no signs of autoreactivity. These features distinguish antibody 2526 from previously described glycan-reactive antibodies. Further study of this antibody class may aid in the selection and engineering of broadly reactive antibody therapeutics and can inform the development of effective vaccines with exceptional breadth of pathogen coverage.

Distinct Functional Humoral Immune Responses Are Induced after Live Attenuated and Inactivated Seasonal Influenza Vaccination.

Influenza viruses infect 5-30% of the world's population annually, resulting in millions of incidents of hospitalization and thousands of mortalities worldwide every year. Although annual vaccination has significantly reduced hospitalization rates in vulnerable populations, the current vaccines are estimated to offer a wide range of protection from 10 to 60% annually. Such incomplete immunity may be related to both poor antigenic coverage of circulating strains, as well as to the insufficient induction of protective immunity. Beyond the role of hemagglutinin (HA) and neuraminidase (NA), vaccine-induced Abs have the capacity to induce a broader array of Ab effector functions, including Ab-dependent cellular cytotoxicity, that has been implicated in universal immunity against influenza viruses. However, whether different vaccine platforms can induce functional humoral immunity in a distinct manner remains incompletely defined. In this study, we compared vaccine-induced humoral immune responses induced by two seasonal influenza vaccines in Homo sapiens, the i.m. inactivated vaccine (IIV/Fluzone) and the live attenuated mucosal vaccine (LAIV/FluMist). Whereas the inactivated influenza vaccine induced superior Ab titers and FcγR binding capacity to diverse HA and NA Ags, the live attenuated influenza mucosal vaccine induced a more robust functional humoral immune response against both the HA and NA domains. Multivariate Ab analysis further highlighted the significantly different overall functional humoral immune profiles induced by the two vaccines, marked by differences in IgG titers, FcR binding, and both NK cell-recruiting and opsonophagocytic Ab functions. These results highlight the striking differences in Ab Fc-effector profiles induced systemically by two distinct influenza vaccine platforms.

Multiplexed Antibody Sequencing and Profiling of the Human Hemagglutinin-specific Memory B Cell Response following Influenza Vaccination.

Influenza virus is a highly contagious respiratory pathogen causing between 9.4 and 41 million infections per year in the United States in the last decade. Annual vaccination is recommended by the World Health Organization, with the goal to reduce influenza severity and transmission. Ag-specific single B cell sequencing methodologies have opened up new avenues into the dissection of the Ab response to influenza virus. The improvement of these methodologies is pivotal to reduce the associated costs and optimize the operational workflow and throughput, especially in the context of multiple samples. In this study, PBMCs and serum samples were collected longitudinally from eight influenza vaccinees either vaccinated yearly for four consecutive influenza seasons or once for one season. Following the serological and B cell profiling of their polyclonal Ab response to a panel of historical, recent, and next-generation influenza vaccine hemagglutinin (HA) and virus strains, a single multiplexed Ag-specific single B cell sequencing run allowed to capture HA-specific memory B cells that were analyzed for preferential Ig H chain/L chain pairing, isotype/subclass usage, and the presence of public BCR clonotypes across participants. Binding and functional profiles of representative private and public clonotypes confirmed their HA specificity, and their overall binding and functional activity were consistent with those observed at the polyclonal level. Collectively, this high-resolution and multiplexed Ab repertoire analysis demonstrated the validity of this optimized methodology in capturing Ag-specific BCR clonotypes, even in the context of a rare B cell population, such as in the case of the peripheral Ag-specific memory B cells.

Intranasal administration of octavalent next-generation influenza vaccine elicits protective immune responses against seasonal and pre-pandemic viruses.

Development of next-generation influenza virus vaccines is crucial to improve protection against circulating and emerging viruses. Current vaccine formulations have to be updated annually due to mutations in seasonal strains and do not offer protection against strains with pandemic potential. Computationally optimized broadly reactive antigen (COBRA) methodology has been utilized by our group to generate broadly reactive immunogens for individual influenza subtypes, which elicit protective immune responses against a broad range of strains over numerous seasons. Octavalent mixtures of COBRA hemagglutinin (HA) (H1, H2, H3, H5, H7, and influenza B virus) plus neuraminidase (NA) (N1 and N2) recombinant proteins mixed with c-di-AMP adjuvant were administered intranasally to naive or pre-immune ferrets in prime-boost fashion. Four weeks after final vaccination, collected sera were analyzed for breadth of antibody response, and the animals were challenged with seasonal or pre-pandemic strains. The octavalent COBRA vaccine elicited antibodies that recognized a broad panel of strains representing different subtypes, and these vaccinated animals were protected against influenza virus challenges. Overall, this study demonstrated that the mixture of eight COBRA HA/NA proteins mixed with an intranasal adjuvant is a promising candidate for a universal influenza vaccine. IMPORTANCE: Influenza is a respiratory virus which infects around a billion people globally every year, with millions experiencing severe illness. Commercial vaccine efficacy varies year to year and can be low due to mismatch of circulating virus strains. Thus, the formulation of current vaccines has to be adapted accordingly every year. The development of a broadly reactive influenza vaccine would lessen the global economic and public health burden caused by the different types of influenza viruses. The significance of our research is producing a promising universal vaccine candidate which provides protection against a wider range of virus strains over a wider range of time.

rAAV expressing a COBRA-designed influenza hemagglutinin generates a protective and durable adaptive immune response with a single dose.

Influenza remains a worldwide public health threat. Although seasonal influenza vaccines are currently the best means of preventing severe disease, the standard-of-care vaccines require frequent updating due to antigenic drift and can have low efficacy, particularly in vulnerable populations. Here, we demonstrate that a single administration of a recombinant adenovirus-associated virus (rAAV) vector expressing a computationally optimized broadly reactive antigen (COBRA)-derived influenza H1 hemagglutinin (HA) induces strongly neutralizing and broadly protective antibodies in naïve mice and ferrets with pre-existing influenza immunity. Following a lethal viral challenge, the rAAV-COBRA vaccine allowed for significantly reduced viral loads in the upper and lower respiratory tracts and complete protection from morbidity and mortality that lasted for at least 5 months post-vaccination. We observed no signs of antibody waning during this study. CpG motif enrichment of the antigen can act as an internal adjuvant to further enhance the immune responses to allow for lower vaccine dosages with the induction of unique interferon-producing CD4+ and CD8+ T cells specific to HA head and stem peptide sequences. Our studies highlight the utility of rAAV as an effective platform to improve seasonal influenza vaccines. IMPORTANCE: Developing an improved seasonal influenza vaccine remains an ambitious goal of researchers and clinicians alike. With influenza routinely causing severe epidemics with the potential to rise to pandemic levels, it is critical to create an effective, broadly protective, and durable vaccine to improve public health worldwide. As a potential solution, we created a rAAV viral vector expressing a COBRA-optimized influenza hemagglutinin antigen with modestly enriched CpG motifs to evoke a robust and long-lasting immune response after a single intramuscular dose without needing boosts or adjuvants. Importantly, the rAAV vaccine boosted antibody breadth to future strains in ferrets with pre-existing influenza immunity. Together, our data support further investigation into the utility of viral vectors as a potential avenue to improve our seasonal influenza vaccines.

Assessment of Humoral Immune Responses to Repeated Influenza Vaccination in a Multiyear Cohort: A 5-Year Follow-up.

The long-term effects of host factors on vaccine-elicited immune responses have not been well studied, and the interactions of host factors with annual influenza vaccinations are yet to be explored. We analyzed data from a cohort of 386 individuals who received the standard-dose influenza vaccine and enrolled in ≥2 seasons from 2016 to 2020. Our analyses indicated disparate vaccine-elicited immune responses between males and females in adults when they were repeatedly vaccinated for at least 2 seasons. Notably, we found interactive effects between age and body mass index (BMI) on overall immune responses, and between sex at birth and BMI in adults.

The Pre-Existing Human Antibody Repertoire to Computationally Optimized Influenza H1 Hemagglutinin Vaccines.

Computationally optimized broadly reactive Ag (COBRA) hemagglutinin (HA) immunogens have previously been generated for several influenza subtypes to improve vaccine-elicited Ab breadth. As nearly all individuals have pre-existing immunity to influenza viruses, influenza-specific memory B cells will likely be recalled upon COBRA HA vaccination. We determined the epitope specificity and repertoire characteristics of pre-existing human B cells to H1 COBRA HA Ags. Cross-reactivity between wild-type HA and H1 COBRA HA proteins P1, X6, and Y2 were observed for isolated mAbs. The mAbs bound five distinct epitopes on the pandemic A/California/04/2009 HA head and stem domains, and most mAbs had hemagglutination inhibition and neutralizing activity against 2009 pandemic H1 strains. Two head-directed mAbs, CA09-26 and CA09-45, had hemagglutination inhibition and neutralizing activity against a prepandemic H1 strain. One mAb, P1-05, targeted the stem region of H1 HA, but did not compete with a known stem-targeting H1 mAb. We determined that mAb P1-05 recognizes a recently discovered HA epitope, the anchor epitope, and we identified similar mAbs using B cell repertoire sequencing. In addition, the trimerization domain distance from HA was critical to recognition of this epitope by mAb P1-05, suggesting the importance of protein design for vaccine formulations. Overall, these data indicate that seasonally vaccinated individuals possess a population of functional H1 COBRA HA-reactive B cells that target head, central stalk, and anchor epitopes, and they demonstrate the importance of structure-based assessment of subunit protein vaccine candidates to ensure accessibility of optimal protein epitopes.

Dual oxidase 1 promotes antiviral innate immunity.

Dual oxidase 1 (DUOX1) is an NADPH oxidase that is highly expre-ssed in respiratory epithelial cells and produces H2O2 in the airway lumen. While a line of prior in vitro observations suggested that DUOX1 works in partnership with an airway peroxidase, lactoperoxidase (LPO), to produce antimicrobial hypothiocyanite (OSCN-) in the airways, the in vivo role of DUOX1 in mammalian organisms has remained unproven to date. Here, we show that Duox1 promotes antiviral innate immunity in vivo. Upon influenza airway challenge, Duox1-/- mice have enhanced mortality, morbidity, and impaired lung viral clearance. Duox1 increases the airway levels of several cytokines (IL-1ß, IL-2, CCL1, CCL3, CCL11, CCL19, CCL20, CCL27, CXCL5, and CXCL11), contributes to innate immune cell recruitment, and affects epithelial apoptosis in the airways. In primary human tracheobronchial epithelial cells, OSCN- is generated by LPO using DUOX1-derived H2O2 and inactivates several influenza strains in vitro. We also show that OSCN- diminishes influenza replication and viral RNA synthesis in infected host cells that is inhibited by the H2O2 scavenger catalase. Binding of the influenza virus to host cells and viral entry are both reduced by OSCN- in an H2O2-dependent manner in vitro. OSCN- does not affect the neuraminidase activity or morphology of the influenza virus. Overall, this antiviral function of Duox1 identifies an in vivo role of this gene, defines the steps in the infection cycle targeted by OSCN-, and proposes that boosting this mechanism in vivo can have therapeutic potential in treating viral infections.

Bivalent H1 and H3 COBRA Recombinant Hemagglutinin Vaccines Elicit Seroprotective Antibodies against H1N1 and H3N2 Influenza Viruses from 2009 to 2019.

Commercial influenza virus vaccines often elicit strain-specific immune responses and have difficulties preventing illness caused by antigenically drifted viral variants. In the last 20 years, the H3N2 component of the annual vaccine has been updated nearly twice as often as the H1N1 component, and in 2019, a mismatch between the wild-type (WT) H3N2 vaccine strain and circulating H3N2 influenza strains led to a vaccine efficacy of ∼9%. Modern methods of developing computationally optimized broadly reactive antigens (COBRAs) for H3N2 influenza viruses utilize current viral surveillance information to design more broadly reactive vaccine antigens. Here, 7 new recombinant hemagglutinin (rHA) H3 COBRA hemagglutinin (HA) antigens were evaluated in mice. Subsequently, two candidates, J4 and NG2, were selected for further testing in influenza-preimmune animals based on their ability to elicit broadly reactive antibodies against antigenically drifted H3N2 viral isolates. In the preimmune model, monovalent formulations of J4 and NG2 elicited broadly reactive antibodies against recently circulating H3N2 influenza viruses from 2019. Bivalent mixtures of COBRA H1 and H3 rHA, Y2 + J4, and Y2 + NG2 outperformed multiple WT H1+H3 bivalent rHA mixtures by eliciting seroprotective antibodies against H1N1 and H3N2 isolates from 2009 to 2019. Overall, the newly generated COBRA HA antigens, namely, Y2, J4, and NG2, had the ability to induce broadly reactive antibodies in influenza-naive and preimmune animals in both monovalent and bivalent formulations, and these antigens outperformed H1 and H3 WT rHA vaccine antigens by eliciting seroprotective antibodies against panels of antigenically drifted historical H1N1 and H3N2 vaccine strains from 2009 to 2019. IMPORTANCE Standard-of-care influenza virus vaccines are composed of a mixture of antigens from different influenza viral subtypes. For the first time, lead COBRA H1 and H3 HA antigens, formulated as a bivalent vaccine, have been investigated in animals with preexisting immunity to influenza viruses. The cocktail of COBRA HA antigens elicited more broadly reactive anti-HA antibodies than those elicited by a comparator bivalent wild-type HA vaccine against H1 and H3 influenza viruses isolated between 2009 and 2019.

Differential Recognition of Computationally Optimized H3 Hemagglutinin Influenza Vaccine Candidates by Human Antibodies.

Among circulating influenza viruses in humans, H3N2 viruses typically evolve faster than other subtypes and have caused disease in millions of people since emerging in 1968. Computationally optimized broadly reactive antigen (COBRA) technology is one strategy to broaden vaccine-elicited antibody responses among influenza subtypes. In this study, we determined the structural integrity of an H3N2 COBRA hemagglutinin (HA), TJ5, and we probed the antigenic profile of several H3N2 COBRA HAs by assessing recognition of these immunogens by human B cells from seasonally vaccinated human subjects. Of three recently described COBRA H3 HA antigens (TJ5, NG2, and J4), we determined that TJ5 and J4 HA proteins recognize pre-existing B cells more effectively than NG2 HA and a wild-type Hong Kong/4801/2014 protein. We also isolated a panel of 12 H3 HA-specific human monoclonal antibodies (MAbs) and identified that most MAbs recognize both wild-type and COBRA HA proteins and have functional activity against a broad panel of H3N2 viruses. Most MAbs target the receptor-binding site, and one MAb targets the HA stem. MAb TJ5-5 recognizes TJ5 and J4 COBRA HA proteins but has poor recognition of NG2 HA, similar to the global B-cell analysis. We determined a 3.4 Å structure via cryo-electron microscopy of Fab TJ5-5 complexed with the H3 COBRA TJ5, which revealed residues important to the differential binding. Overall, these studies determined that COBRA H3 HA proteins have correct antigenic and structural features, and the proteins are recognized by B cells and MAbs isolated from seasonally vaccinated humans. IMPORTANCE Vaccine development for circulating influenza viruses, particularly for the H3N2 subtype, remains challenging due to consistent antigenic drift. Computationally optimized broadly reactive antigen (COBRA) technology has proven effective for broadening influenza hemagglutinin (HA)-elicited antibody responses compared to wild-type immunogens. Here, we determined the structural features and antigenic profiles of H3 COBRA HA proteins. Two H3 COBRA HA proteins, TJ5 and J4, are better recognized by pre-existing B cells and monoclonal antibodies from the 2017 to 2018 vaccine season compared to COBRA NG2 and a wild-type A/Hong Kong/2014 HA protein. We determined a cryo-electron microscopy (cryo-EM) structure of one MAb that poorly recognizes NG2, MAb TJ5-5, in complex with the TJ5 COBRA HA protein and identified residues critical to MAb recognition. As NG2 is more effective than TJ5 for the recent Hong Kong/2019 virus, these data provide insights into the diminished effectiveness of influenza vaccines across vaccine seasons.

SARS-CoV-2 and Influenza A Virus Coinfections in Ferrets.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and seasonal influenza viruses are cocirculating in the human population. However, only a few cases of viral coinfection with these two viruses have been documented in humans with some people having severe disease and others mild disease. To examine this phenomenon, ferrets were coinfected with SARS-CoV-2 and human seasonal influenza A viruses (IAVs; H1N1 or H3N2) and were compared to animals that received each virus alone. Ferrets were either immunologically naive to both viruses or vaccinated with the 2019 to 2020 split-inactivated influenza virus vaccine. Coinfected naive ferrets lost significantly more body weight than ferrets infected with each virus alone and had more severe inflammation in both the nose and lungs compared to that of ferrets that were single infected with each virus. Coinfected, naive animals had predominantly higher IAV titers than SARS-CoV-2 titers, and IAVs were efficiently transmitted by direct contact to the cohoused ferrets. Comparatively, SARS-CoV-2 failed to transmit to the ferrets that cohoused with coinfected ferrets by direct contact. Moreover, vaccination significantly reduced IAV titers and shortened the viral shedding but did not completely block direct contact transmission of the influenza virus. Notably, vaccination significantly ameliorated influenza-associated disease by protecting vaccinated animals from severe morbidity after IAV single infection or IAV and SARS-CoV-2 coinfection, suggesting that seasonal influenza virus vaccination is pivotal to prevent severe disease induced by IAV and SARS-CoV-2 coinfection during the COVID-19 pandemic. IMPORTANCE Influenza A viruses cause severe morbidity and mortality during each influenza virus season. The emergence of SARS-CoV-2 infection in the human population offers the opportunity to potential coinfections of both viruses. The development of useful animal models to assess the pathogenesis, transmission, and viral evolution of these viruses as they coinfect a host is of critical importance for the development of vaccines and therapeutics. The ability to prevent the most severe effects of viral coinfections can be studied using effect coinfection ferret models described in this report.

Evaluation of determinants of the serological response to the quadrivalent split-inactivated influenza vaccine.

The seasonal influenza vaccine is only effective in half of the vaccinated population. To identify determinants of vaccine efficacy, we used data from > 1,300 vaccination events to predict the response to vaccination measured as seroconversion as well as hemagglutination inhibition (HAI) titer levels one year after. We evaluated the predictive capabilities of age, body mass index (BMI), sex, race, comorbidities, vaccination history, and baseline HAI titers, as well as vaccination month and vaccine dose in multiple linear regression models. The models predicted the categorical response for > 75% of the cases in all subsets with one exception. Prior vaccination, baseline titer level, and age were the major determinants of seroconversion, all of which had negative effects. Further, we identified a gender effect in older participants and an effect of vaccination month. BMI had a surprisingly small effect, likely due to its correlation with age. Comorbidities, vaccine dose, and race had negligible effects. Our models can generate a new seroconversion score that is corrected for the impact of these factors which can facilitate future biomarker identification.

Zinc Carnosine Metal-Organic Coordination Polymer as a Potent Broadly Active Influenza Vaccine Platform with In Vitro Shelf-Stability.

Current seasonal influenza vaccines are limited in that they need to be reformulated every year in order to account for the constant mutation of the virus. Hemagglutinin (HA) immunogens have been developed using a computationally optimized broadly reactive antigen (COBRA) methodology, which are able to elicit an antibody response that neutralizes antigenically distinct influenza strains; however, subunit proteins are not immunogenic enough on their own to generate a substantial immune response. Due to this, different delivery strategies and adjuvants can be used to improve immunogenicity. Recently, we reported a new coordination polymer composed of the dipeptide carnosine and zinc (ZnCar) that is able to deliver protein antigens along with CpG to generate a potent immune response. In the present work, ZnCar was used to deliver the COBRA HA immunogen Y2 and the adjuvant CpG. We incorporated Y2 into ZnCar using two different methods to assess which would be the most immunogenic. Mice vaccinated with Y2 and CpG complexed with ZnCar showed an improved humoral and cellular response when compared to mice vaccinated with soluble Y2 and CpG. Further, we demonstrate in vitro that when Y2 and CpG are coordinated with ZnCar, they are protected from degradation at 40 °C for 3 months or 24 °C for 6 months. Overall, ZnCar shows promise as a delivery vehicle for subunit vaccines, given its superior immunogenicity and in vitro storage stability.

New Proteomic Signatures to Distinguish Between Zika and Dengue Infections.

Distinguishing between Zika and dengue virus infections is critical for accurate treatment, but we still lack detailed understanding of their impact on their host. To identify new protein signatures of the two infections, we used next-generation proteomics to profile 122 serum samples from 62 Zika and dengue patients. We quantified >500 proteins and identified 13 proteins that were significantly differentially expressed (adjusted p-value < 0.05). These proteins typically function in infection and wound healing, with several also linked to pregnancy and brain function. We successfully validated expression differences with Carbonic Anhydrase 2 in both the original and an independent sample set. Three of the differentially expressed proteins, i.e., Fibrinogen Alpha, Platelet Factor 4 Variant 1, and Pro-Platelet Basic Protein, predicted Zika virus infection at a ∼70% true-positive and 6% false-positive rate. Further, we showed that intraindividual temporal changes in protein signatures can disambiguate diagnoses and serve as indicators for past infections. Taken together, we demonstrate that serum proteomics can provide new resources that serve to distinguish between different viral infections.

Glycomic analysis of host response reveals high mannose as a key mediator of influenza severity.

Influenza virus infections cause a wide variety of outcomes, from mild disease to 3 to 5 million cases of severe illness and ∼290,000 to 645,000 deaths annually worldwide. The molecular mechanisms underlying these disparate outcomes are currently unknown. Glycosylation within the human host plays a critical role in influenza virus biology. However, the impact these modifications have on the severity of influenza disease has not been examined. Herein, we profile the glycomic host responses to influenza virus infection as a function of disease severity using a ferret model and our lectin microarray technology. We identify the glycan epitope high mannose as a marker of influenza virus-induced pathogenesis and severity of disease outcome. Induction of high mannose is dependent upon the unfolded protein response (UPR) pathway, a pathway previously shown to associate with lung damage and severity of influenza virus infection. Also, the mannan-binding lectin (MBL2), an innate immune lectin that negatively impacts influenza outcomes, recognizes influenza virus-infected cells in a high mannose-dependent manner. Together, our data argue that the high mannose motif is an infection-associated molecular pattern on host cells that may guide immune responses leading to the concomitant damage associated with severity.

Prevaccination Glycan Markers of Response to an Influenza Vaccine Implicate the Complement Pathway.

A key to improving vaccine design and vaccination strategy is to understand the mechanism behind the variation of vaccine response with host factors. Glycosylation, a critical modulator of immunity, has no clear role in determining vaccine responses. To gain insight into the association between glycosylation and vaccine-induced antibody levels, we profiled the pre- and postvaccination serum protein glycomes of 160 Caucasian adults receiving the FLUZONE influenza vaccine during the 2019-2020 influenza season using lectin microarray technology. We found that prevaccination levels of Lewis A antigen (Lea) are significantly higher in nonresponders than responders. Glycoproteomic analysis showed that Lea-bearing proteins are enriched in complement activation pathways, suggesting a potential role of glycosylation in tuning the activities of complement proteins, which may be implicated in mounting vaccine responses. In addition, we observed a postvaccination increase in sialyl Lewis X antigen (sLex) and a decrease in high mannose glycans among high responders, which were not observed in nonresponders. These data suggest that the immune system may actively modulate glycosylation as part of its effort to establish effective protection postvaccination.

Template-Assisted De Novo Sequencing of SARS-CoV-2 and Influenza Monoclonal Antibodies by Mass Spectrometry.

In this study, we used multiple enzyme digestions, coupled with higher-energy collisional dissociation (HCD) and electron-transfer/higher-energy collision dissociation (EThcD) fragmentation to develop a mass-spectrometric (MS) method for determining the complete protein sequence of monoclonal antibodies (mAbs). The method was refined on an mAb of a known sequence, a SARS-CoV-1 antireceptor binding domain (RBD) spike monoclonal antibody. The data were searched using Supernovo to generate a complete template-assisted de novo sequence for this and two SARS-CoV-2 mAbs of known sequences resulting in correct sequences for the variable regions and correct distinction of Ile and Leu residues. We then used the method on a set of 25 antihemagglutinin (HA) influenza antibodies of unknown sequences and determined high confidence sequences for >99% of the complementarity determining regions (CDRs). The heavy-chain and light-chain genes were cloned and transfected into cells for recombinant expression followed by affinity purification. The recombinant mAbs displayed binding curves matching the original mAbs with specificity to the HA influenza antigen. Our findings indicate that this methodology results in almost complete antibody sequence coverage with high confidence results for CDR regions on diverse mAb sequences.

Universal Dengue Vaccine Elicits Neutralizing Antibodies against Strains from All Four Dengue Virus Serotypes.

Any potential dengue virus (DENV) vaccine needs to elicit protective immunity against strains from all four serotypes to avoid potential antibody-dependent enhancement (ADE). In this study, four independent DENV envelope (E) glycoproteins were generated using wild-type E sequences from viruses isolated between 1943 and 2006 using computationally optimized broadly reactive antigen (COBRA) methodology. COBRA and wild-type E antigens were expressed on the surface of subvirion viral particles (SVPs). Four separate wild-type E antigens were used for each serotype. Mice vaccinated with wild-type DENV SVPs had anti-E IgG antibodies that neutralized serotype-specific viruses. COBRA DENV SVPs elicited a broader breadth of antibodies that neutralized strains across all four serotypes. Two COBRA DENV vaccine candidates that elicited the broadest breadth of neutralizing antibodies in mice were used to vaccinate rhesus macaques (Macaca mulatta) that either were immunologically naive to any DENV serotype or had preexisting antibodies to DENV. Antibodies elicited by COBRA DENV E immunogens neutralized all 12 strains of DENV in vitro, which was comparable to antibodies elicited by a tetravalent wild-type E SVP vaccination mixture. Therefore, using a single DENV COBRA E protein can elicit neutralizing antibodies against strains representing all four serotypes of DENV in both naive and dengue virus-preimmune populations.IMPORTANCE Dengue virus infects millions of people living in tropical areas of the world. Dengue virus-induced diseases can range from mild to severe with death. An effective vaccine will need to neutralize viruses from all four serotypes of dengue virus without inducing enhanced disease. A dengue virus E vaccine candidate generated by computationally optimized broadly reactive antigen algorithms elicits broadly neutralizing protection for currently circulating strains from all four serotypes regardless of immune status. Most dengue vaccines in development formulate four separate components based on prM-E from a wild-type strain representing each serotype. Designing a monovalent vaccine that elicits protective immunity against all four serotypes is an effective and economical strategy.