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

Non-sterilizing, Infection-Permissive Vaccination With Inactivated Influenza Virus Vaccine Reshapes Subsequent Virus Infection-Induced Protective Heterosubtypic Immunity From Cellular to Humoral Cross-Reactive Immune Responses

Conventional influenza vaccines aim at the induction of virus-neutralizing antibodies that provide with sterilizing immunity. However, influenza vaccination often confers protection from disease but not from infection. The impact of infection-permissive vaccination on the immune response elicited by subsequent influenza virus infection is not well-understood. Here, we investigated to what extent infection-permissive immunity, in contrast to virus-neutralizing immunity, provided by a trivalent inactivated virus vaccine (TIV) modulates disease and virus-induced host immune responses after sublethal vaccine-matching H1N1 infection in a mouse model. More than one TIV vaccination was needed to induce a serum HI titer and provide sterilizing immunity upon homologous virus infection. However, single TIV administration provided infection-permissive immunity, characterized by lower viral lung titers and faster recovery. Despite the presence of replicating virus, single TIV vaccination prevented induction of pro-inflammatory cyto- and chemokines, alveolar macrophage depletion as well as the establishment of lung-resident B and T cells after infection. To investigate virus infection-induced cross-protective heterosubtypic immune responses in vaccinated and unvaccinated animals, mice were re-infected with a lethal dose of H3N2 virus 4 weeks after H1N1 infection. Single TIV vaccination did not prevent H1N1 virus infection-induced heterosubtypic cross-protection, but shifted the mechanism of cross-protection from the cellular to the humoral branch of the immune system. These results suggest that suboptimal vaccination with conventional influenza vaccines may still positively modulate disease outcome after influenza virus infection, while promoting humoral heterosubtypic immunity after virus infection.Fil: Choi, Angela. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Ibañez, Lorena Itatí. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología "Dr. César Milstein"; ArgentinaFil: Strohmeier, Shirin. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Krammer, Florian. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: García Sastre, Adolfo. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Schotsaert, Michael. Icahn School of Medicine at Mount Sinai; Estados Unido

Generation of an international standard serum to measure influenza virus hemagglutinin stalk-reactive antibodies

Current efforts towards the development of a universal influenza virus vaccine rely on induction of effective long-term antibody responses against conserved regions of the influenza virus glycoproteins. The stalk domain of the hemagglutinin has been targeted for this purpose due to its high degree of conservation among numerous influenza subtypes and strains. Several vaccine candidates targeting this domain are in late pre-clinical or early clinical stage of development. Currently assays to measure stalk-based immunity are not standardized and no international standard is available. As various vaccine developers are generating data from pre-clinical and clinical studies, harmonization of assay read-outs will help in the comparison of experiments conducted in different laboratories and increase confidence in results. Moreover, researchers will aim to define correlates of protection for new vaccines targeting the hemagglutinin stalk domain. Joint efforts between the Krammer Laboratory at the Icahn School of Medicine at Mount Sinai and the National Institute for Biological Standards and Control (NIBSC) led to a collaborative project to generate an international standard human serum to measure hemagglutinin stalk-reactive antibodies. The WHO Expert Committee on Biological Standardization (ECBS) is currently evaluating this project for its endorsement to generate the first international standard for antibodies to the influenza virus hemagglutinin stalk domain. Please click Download on the upper right corner to see the full abstract

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 universal Influenza B vaccine using mosaic-hemagglutinin vaccine candidates

Influenza viruses undergo antigenic changes in the immuno-dominant hemagglutinin (HA) head domain, necessitating annual re-formulation of and re-vaccination with seasonal influenza virus vaccines for maintaining the protection. We previously synthesized mosaic HA (mHA) proteins of influenza B viruses which redirect the immune response towards the immuno-subdominant conserved epitopes of the B virus HA via sequential immunization. As ~90% of current influenza virus vaccines are manufactured using the inactivated virus platform, we generated and sequentially vaccinated mice with inactivated influenza B viruses displaying either the homologous (same B HA backbones) or the heterologous (different B HA backbones) mosaic HAs. Both approaches induced long-lasting and cross-protective antibody responses showing strong antibody-dependent cellular cytotoxicity (ADCC) activity. Thereafter, we tested different inactivation methods and adjuvants to increase the cross-protection against phylogenetically distant influenza B viruses from both lineages. The use of CpG 1018 or AddaVax boosted the humoral immune response and protection when combined with any inactivation method. Beta-propiolactone (BPL) inactivation was the best method, with high serum HA antibodies levels that correlated with optimal protection in BALB/C mice challenge studies. We believe that these B virus mHA vaccine candidates represent a major step towards a universal influenza B virus vaccine. Please click Download on the upper right corner to see the full abstract

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

Pre-existing Hemagglutinin Stalk Antibodies Correlate with Protection of Lower Respiratory Symptoms in Flu-Infected Transplant Patients

Hemagglutination-inhibitory antibodies are usually highly strain specific with little effect on infection with drifted or shifted strains. The significance of broadly cross-reactive non-HAI anti-influenza antibodies against conserved domains of virus glycoproteins, such as the hemagglutinin (HA) stalk, is of great interest. We characterize a cohort of 40 H1N1pmd09 influenza-infected patients and identify lower respiratory symptoms (LRSs) as a predictor for development of pneumonia. A binomial logistic regression of log10 pre-existing antibody values shows that the probability of LRS occurrence decreased with increased anti-HA full-length and stalk antibody ELISA titers. However, a multilevel logistic regression model adjusted by other potential serocorrelates demonstrates that only antibodies directed against the stalk of HA correlate with protection from lower respiratory infection, limiting disease progression. Our predictive model indicates that a threshold of protective immunity based on broadly cross-reactive HA stalk antibodies could be feasible

A serological assay to detect SARS-CoV-2 seroconversion in humans

Development of an enzyme-linked immunosorbent assay to detect antibodies to the SARS-CoV-2 spike protein in human sera and plasma. Here, we describe a serological enzyme-linked immunosorbent assay for the screening and identification of human SARS-CoV-2 seroconverters. This assay does not require the handling of infectious virus, can be adjusted to detect different antibody types in serum and plasma and is amenable to scaling. Serological assays are of critical importance to help define previous exposure to SARS-CoV-2 in populations, identify highly reactive human donors for convalescent plasma therapy and investigate correlates of protection.Peer reviewe

Sterilizing immunity against SARS-CoV-2 infection in mice by a single-shot and modified imidazoquinoline TLR7/8 agonist-adjuvanted recombinant spike protein vaccine

The search for vaccines that protect from severe morbidity and mortality as a result of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019 (COVID-19) is a race against the clock and the virus. Several vaccine candidates are currently being tested in the clinic. Inactivated virus and recombinant protein vaccines can be safe options but may require adjuvants to induce robust immune responses efficiently. In this work we describe the use of a novel amphiphilic imidazoquinoline (IMDQ-PEG-CHOL) TLR7/8 adjuvant, consisting of an imidazoquinoline conjugated to the chain end of a cholesterol-poly(ethylene glycol) macromolecular amphiphile). This amphiphile is water soluble and exhibits massive translocation to lymph nodes upon local administration, likely through binding to albumin. IMDQ-PEG-CHOL is used to induce a protective immune response against SARS-CoV-2 after single vaccination with trimeric recombinant SARS-CoV-2 spike protein in the BALB/c mouse model. Inclusion of amphiphilic IMDQ-PEG-CHOL in the SARS-CoV-2 spike vaccine formulation resulted in enhanced immune cell recruitment and activation in the draining lymph node. IMDQ-PEG-CHOL has a better safety profile compared to native soluble IMDQ as the former induces a more localized immune response upon local injection, preventing systemic inflammation. Moreover, IMDQ-PEG-CHOL adjuvanted vaccine induced enhanced ELISA and in vitro microneutralization titers, and a more balanced IgG2a/IgG1 response. To correlate vaccine responses with control of virus replication in vivo, vaccinated mice were challenged with SARS-CoV-2 virus after being sensitized by intranasal adenovirus-mediated expression of the human angiotensin converting enzyme 2 (ACE2) gene. Animals vaccinated with trimeric recombinant spike protein vaccine without adjuvant had lung virus titers comparable to non-vaccinated control mice, whereas animals vaccinated with IMDQ-PEG-CHOL-adjuvanted vaccine controlled viral replication and infectious viruses could not be recovered from their lungs at day 4 post infection. In order to test whether IMDQ-PEG-CHOL could also be used to adjuvant vaccines currently licensed for use in humans, proof of concept was also provided by using the same IMDQ-PEG-CHOL to adjuvant human quadrivalent inactivated influenza virus split vaccine, which resulted in enhanced hemagglutination inhibition titers and a more balanced IgG2a/IgG1 antibody response. Enhanced influenza vaccine responses correlated with better virus control when mice were given a lethal influenza virus challenge. Our results underscore the potential use of IMDQ-PEG-CHOL as an adjuvant to achieve protection after single immunization with recombinant protein and inactivated virus vaccines against respiratory viruses, such as SARS-CoV-2 and influenza viruses

Development and Assessment of a Pooled Serum as Candidate Standard to Measure Influenza A Virus Group 1 Hemagglutinin Stalk-Reactive Antibodies

The stalk domain of the hemagglutinin has been identified as a target for induction of protective antibody responses due to its high degree of conservation among numerous influenza subtypes and strains. However, current assays to measure stalk-based immunity are not standardized. Hence, harmonization of assay readouts would help to compare experiments conducted in different laboratories and increase confidence in results. Here, serum samples from healthy individuals (n = 110) were screened using a chimeric cH6/1 hemagglutinin enzyme-linked immunosorbent assay (ELISA) that measures stalk-reactive antibodies. We identified samples with moderate to high IgG anti-stalk antibody levels. Likewise, screening of the samples using the mini-hemagglutinin (HA) headless construct #4900 and analysis of the correlation between the two assays confirmed the presence and specificity of anti-stalk antibodies. Additionally, samples were characterized by a cH6/1N5 virus-based neutralization assay, an antibody-dependent cell-mediated cytotoxicity (ADCC) assay, and competition ELISAs, using the stalk-reactive monoclonal antibodies KB2 (mouse) and CR9114 (human). A “pooled serum” (PS) consisting of a mixture of selected serum samples was generated. The PS exhibited high levels of stalk-reactive antibodies, had a cH6/1N5-based neutralization titer of 320, and contained high levels of stalk-specific antibodies with ADCC activity. The PS, along with blinded samples of varying anti-stalk antibody titers, was distributed to multiple collaborators worldwide in a pilot collaborative study. The samples were subjected to different assays available in the different laboratories, to measure either binding or functional properties of the stalk-reactive antibodies contained in the serum. Results from binding and neutralization assays were analyzed to determine whether use of the PS as a standard could lead to better agreement between laboratories. The work presented here points the way towards the development of a serum standard for antibodies to the HA stalk domain of phylogenetic group 1