Monoclonal antibodies targeting the influenza virus N6 neuraminidase

Influenza A viruses are a diverse species that include 16 true hemagglutinin (HA) subtypes and 9 true neuraminidase (NA) subtypes. While the antigenicity of many HA subtypes is reasonably well studied, less is known about NA antigenicity, especially when it comes to non-human subtypes that only circulate in animal reservoirs. The N6 subtype NAs are mostly found in viruses infecting birds. However, they have also been identified in viruses that infect mammals, such as swine and seals. More recently, highly pathogenic H5N6 subtype viruses have caused rare infections and mortality in humans. Here, we generated murine mAbs to the N6 NA, characterized their breadth and antiviral properties in vitro and in vivo and mapped their epitopes by generating escape mutant viruses. We found that the antibodies had broad reactivity across the American and Eurasian N6 lineages, but relatively little binding to the H5N6 NA. Several of the antibodies exhibited strong NA inhibition activity and some also showed activity in the antibody dependent cellular cytotoxicity reporter assay and neutralization assay. In addition, we generated escape mutant viruses for six monoclonal antibodies and found mutations on the lateral ridge of the NA. Lastly, we observed variable protection in H4N6 mouse challenge models when the antibodies were given prophylactically

Chimeric Hemagglutinin Constructs Induce Broad Protection against Influenza B Virus Challenge in the Mouse Model.

Seasonal influenza virus epidemics represent a significant public health burden. Approximately 25% of all influenza virus infections are caused by type B viruses, and these infections can be severe, especially in children. Current influenza virus vaccines are an effective prophylaxis against infection but are impacted by rapid antigenic drift, which can lead to mismatches between vaccine strains and circulating strains. Here, we describe a broadly protective vaccine candidate based on chimeric hemagglutinins, consisting of globular head domains from exotic influenza A viruses and stalk domains from influenza B viruses. Sequential vaccination with these constructs in mice leads to the induction of broadly reactive antibodies that bind to the conserved stalk domain of influenza B virus hemagglutinin. Vaccinated mice are protected from lethal challenge with diverse influenza B viruses. Results from serum transfer experiments and antibody-dependent cell-mediated cytotoxicity (ADCC) assays indicate that this protection is antibody mediated and based on Fc effector functions. The present data suggest that chimeric hemagglutinin-based vaccination is a viable strategy to broadly protect against influenza B virus infection.IMPORTANCE While current influenza virus vaccines are effective, they are affected by mismatches between vaccine strains and circulating strains. Furthermore, the antiviral drug oseltamivir is less effective for treating influenza B virus infections than for treating influenza A virus infections. A vaccine that induces broad and long-lasting protection against influenza B viruses is therefore urgently needed

Asymptomatic Seroconversion of Immunoglobulins to SARS-CoV-2 in a Pediatric Dialysis Unit

This article is made available for unrestricted research re-use and secondary analysis in any form or be any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.Dialysis units are at especially high risk of infectious disease transmission, and concern exists about spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Dialysis units in Wuhan, China, have reported high coronavirus disease 2019 (COVID-19) prevalence, due in part to unique exposure challenges that limit social distancing efforts, including open bay formats and rotating/multiple nursing assignments. This study describes SARS-CoV-2 seroconversion in patients and health care workers in a pediatric dialysis unit.The study was supported by the Lilly Endowment Inc. Physician Scientist Initiative to Drs. Hains and Schwaderer. Dr. Krammer was supported by institutional seed funding

Vaccination with recombinant neuraminidase protects against influenza virus infection in mice

While the efficacy of most influenza virus vaccines is measured by the ability to induce antibodies against the hemagglutinin (HA), antibodies against the viral neuraminidase (NA) are also correlated with less severe disease in humans and animal models. Yet, neither the amount nor the enzymatic activity of NA is standardized in current seasonal vaccines, and the breadth of NA-based protection is unknown. In the present study, different subtypes of recombinant NA were expressed in a baculovirus system and used to vaccinate mice prior to homologous, heterologous, or heterosubtypic virus challenge. Additionally, pre- and post-vaccination human serum samples from vaccinees that received TIV were studied to compare induction of antibodies against the HA and NA. Finally, the amounts of NA in 4 different vaccine formulations from 2013-2014 were quantified using ELISA. Mice immunized with N2 were 100% protected from morbidity and mortality in a homologous challenge and displayed significantly reduced viral lung titers. Heterologous challenge with a drifted strain resulted in morbidity but no mortality. Mice immunized with B/Yamagata/16/88 NA were 100% protected from morbidity and mortality when lethally challenged with a recent Victoria lineage strain. In our human cohorts, the increase in endpoint titers against N1 NA post-vaccination was less robust than that against HA and, as our quantification data suggests, the N1 NA amounts in seasonal vaccine formulations is quite variable. To confirm the broad protective effects of anti-influenza B NA antibodies on a monoclonal level, a panel of mouse monoclonal antibodies was generated against influenza B virus NA; several of these displayed broad reactivity in ELISA to whole virus and recombinant NA and protected against lethal influenza B virus challenge in mice when delivered at a dose of 5 mg/kg prophylactically, or therapeutically, 48 hours post-infection. Analysis of the protective epitopes is currently in progress. The demonstrated protective capacity of anti-NA antibodies suggests that targeting the NA through vaccination may offer increased protection against influenza virus infection

A 10-Minute “Mix and Read” Antibody Assay for SARS-CoV-2

Accurate and rapid diagnostic tools are needed for management of the ongoing coronavirus disease 2019 (COVID-19) pandemic. Antibody tests enable detection of individuals past the initial phase of infection and help examine vaccine responses. The major targets of human antibody response in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are the spike glycoprotein (SP) and nucleocapsid protein (NP). We have developed a rapid homogenous approach for antibody detection termed LFRET (protein L-based time-resolved Förster resonance energy transfer immunoassay). In LFRET, fluorophore-labeled protein L and antigen are brought to close proximity by antigen-specific patient immunoglobulins of any isotype, resulting in TR-FRET signal. We set up LFRET assays for antibodies against SP and NP and evaluated their diagnostic performance using a panel of 77 serum/plasma samples from 44 individuals with COVID-19 and 52 negative controls. Moreover, using a previously described SP and a novel NP construct, we set up enzyme linked immunosorbent assays (ELISAs) for antibodies against SARS-CoV-2 SP and NP. We then compared the LFRET assays with these ELISAs and with a SARS-CoV-2 microneutralization test (MNT). We found the LFRET assays to parallel ELISAs in sensitivity (90–95% vs. 90–100%) and specificity (100% vs. 94–100%). In identifying individuals with or without a detectable neutralizing antibody response, LFRET outperformed ELISA in specificity (91–96% vs. 82–87%), while demonstrating an equal sensitivity (98%). In conclusion, this study demonstrates the applicability of LFRET, a 10-min “mix and read” assay, to detection of SARS-CoV-2 antibodies

Vaccination with SARS-CoV-2 variants of concern protects mice from challenge with wild-type virus.

Funder: open philanthropy projectFunder: jpb foundationVaccines against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) have been highly efficient in protecting against Coronavirus Disease 2019 (COVID-19). However, the emergence of viral variants that are more transmissible and, in some cases, escape from neutralizing antibody responses has raised concerns. Here, we evaluated recombinant protein spike antigens derived from wild-type SARS-CoV-2 and from variants B.1.1.7, B.1.351, and P.1 for their immunogenicity and protective effect in vivo against challenge with wild-type SARS-CoV-2 in the mouse model. All proteins induced high neutralizing antibodies against the respective viruses but also induced high cross-neutralizing antibody responses. The decline in neutralizing titers between variants was moderate, with B.1.1.7-vaccinated animals having a maximum fold reduction of 4.8 against B.1.351 virus. P.1 induced the most cross-reactive antibody responses but was also the least immunogenic in terms of homologous neutralization titers. However, all antigens protected from challenge with wild-type SARS-CoV-2 in a mouse model

A 10-Minute “Mix and Read” Antibody Assay for SARS-CoV-2

Accurate and rapid diagnostic tools are needed for management of the ongoing coronavirus disease 2019 (COVID-19) pandemic. Antibody tests enable detection of individuals past the initial phase of infection and help examine vaccine responses. The major targets of human antibody response in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are the spike glycoprotein (SP) and nucleocapsid protein (NP). We have developed a rapid homogenous approach for antibody detection termed LFRET (protein L-based time-resolved Förster resonance energy transfer immunoassay). In LFRET, fluorophore-labeled protein L and antigen are brought to close proximity by antigen-specific patient immunoglobulins of any isotype, resulting in TR-FRET signal. We set up LFRET assays for antibodies against SP and NP and evaluated their diagnostic performance using a panel of 77 serum/plasma samples from 44 individuals with COVID-19 and 52 negative controls. Moreover, using a previously described SP and a novel NP construct, we set up enzyme linked immunosorbent assays (ELISAs) for antibodies against SARS-CoV-2 SP and NP. We then compared the LFRET assays with these ELISAs and with a SARS-CoV-2 microneutralization test (MNT). We found the LFRET assays to parallel ELISAs in sensitivity (90–95% vs. 90–100%) and specificity (100% vs. 94–100%). In identifying individuals with or without a detectable neutralizing antibody response, LFRET outperformed ELISA in specificity (91–96% vs. 82–87%), while demonstrating an equal sensitivity (98%). In conclusion, this study demonstrates the applicability of LFRET, a 10-min “mix and read” assay, to detection of SARS-CoV-2 antibodies

Ectomycorrhizal Fungi as Biofertilizers in Forestry

Ectomycorrhiza (ECM) is an association of fungi with the roots of higher plants in which both the species are equally benefited and appears to be important for the survival of both parties, and no doubt this association offered extensive benefits in the restoration of forest and ecosystem soil aggregation and stabilization. The most important and vital role of ECM fungi, which is analyzed globally, is that they are best and environment friendly biofertilizers. ECM fungi considered as a biotechnological tool in forest management because their role in reforestation, bioremediation, control of soil pathogen, and restoration of ecosystem is reviewed extensively. ECM fungi not only increase the biomass of edible fruiting bodies but also improve soil structure, nutrient cycle, and also produce phytohormones, which increase the growth and survival of seedlings and enhance the photosynthetic rate of plants and also maintain their tolerance level against environmental stresses in forest nursery. Ectomycorrhizas also reduce fertilization expenses in an environment friendly manner. The importance of ECM fungi and mycorrhizal helper bacteria for the growth enhancement of the economically important trees and significantly important role in restoration of sites degraded by forestry operation

Immunological imprinting of the antibody response in COVID-19 patients

In addition to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), humans are also susceptible to six other coronaviruses, for which consecutive exposures to antigenically related and divergent seasonal coronaviruses are frequent. Despite the prevalence of COVID-19 pandemic and ongoing research, the nature of the antibody response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is unclear. Here we longitudinally profile the early humoral immune response against SARS-CoV-2 in hospitalized coronavirus disease 2019 (COVID-19) patients and quantify levels of pre-existing immunity to OC43, HKU1 and 229E seasonal coronaviruses, and find a strong back-boosting effect to conserved but not variable regions of OC43 and HKU1 betacoronaviruses spike protein. However, such antibody memory boost to human coronaviruses negatively correlates with the induction of IgG and IgM against SARS-CoV-2 spike and nucleocapsid protein. Our findings thus provide evidence of immunological imprinting by previous seasonal coronavirus infections that can potentially modulate the antibody profile to SARS-CoV-2 infection

Functionality of the putative surface glycoproteins of the Wuhan spiny eel influenza virus

A panel of influenza virus-like sequences were recently documented in fish and amphibians. Of these, the Wuhan spiny eel influenza virus (WSEIV) was found to phylogenetically cluster with influenza B viruses as a sister clade. Influenza B viruses have been documented to circulate only in humans, with certain virus isolates found in harbor seals. It is therefore interesting that a similar virus was potentially found in fish. Here we characterize the putative hemagglutinin (HA) and neuraminidase (NA) surface glycoproteins of the WSEIV. Functionally, we show that the WSEIV NA-like protein has sialidase activity comparable to B/Malaysia/2506/2004 influenza B virus NA, making it a bona fide neuraminidase that is sensitive to NA inhibitors. We tested the functionality of the HA by addressing the receptor specificity, stability, preferential airway protease cleavage, and fusogenicity. We show highly specific binding to monosialic ganglioside 2 (GM2) and fusogenicity at a range of different pH conditions. In addition, we found limited antigenic conservation of the WSEIV HA and NA relative to the B/Malaysia/2506/2004 virus HA and NA. In summary, we perform a functional and antigenic characterization of the glycoproteins of WSEIV to assess if it is indeed a bona fide influenza virus potentially circulating in ray-finned fish