Publications by authors named "Veronika Chromikova"

20 Publications

  • Page 1 of 1

Influenza hemagglutinin-specific IgA Fc-effector functionality is restricted to stalk epitopes.

Proc Natl Acad Sci U S A 2021 Feb;118(8)

Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029;

In this study, we utilized a panel of human immunoglobulin (Ig) IgA monoclonal antibodies isolated from the plasmablasts of eight donors after 2014/2015 influenza virus vaccination (Fluarix) to study the binding and functional specificities of this isotype. In this cohort, isolated IgA monoclonal antibodies were primarily elicited against the hemagglutinin protein of the H1N1 component of the vaccine. To compare effector functionalities, an H1-specific subset of antibodies targeting distinct epitopes were expressed as monomeric, dimeric, or secretory IgA, as well as in an IgG1 backbone. When expressed with an IgG Fc domain, all antibodies elicited Fc-effector activity in a primary polymorphonuclear cell-based assay which differs from previous observations that found only stalk-specific antibodies activate the low-affinity FcγRIIIa. However, when expressed with IgA Fc domains, only antibodies targeting the stalk domain showed Fc-effector activity in line with these previous findings. To identify the cause of this discrepancy, we then confirmed that IgG signaling through the high-affinity FcγI receptor was not restricted to stalk epitopes. Since no corresponding high-affinity Fcα receptor exists, the IgA repertoire may therefore be limited to stalk-specific epitopes in the context of Fc receptor signaling.
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http://dx.doi.org/10.1073/pnas.2018102118DOI Listing
February 2021

A chimeric hemagglutinin-based universal influenza virus vaccine approach induces broad and long-lasting immunity in a randomized, placebo-controlled phase I trial.

Nat Med 2021 01 7;27(1):106-114. Epub 2020 Dec 7.

Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

Seasonal influenza viruses constantly change through antigenic drift and the emergence of pandemic influenza viruses through antigenic shift is unpredictable. Conventional influenza virus vaccines induce strain-specific neutralizing antibodies against the variable immunodominant globular head domain of the viral hemagglutinin protein. This necessitates frequent re-formulation of vaccines and handicaps pandemic preparedness. In this completed, observer-blind, randomized, placebo-controlled phase I trial (NCT03300050), safety and immunogenicity of chimeric hemagglutinin-based vaccines were tested in healthy, 18-39-year-old US adults. The study aimed to test the safety and ability of the vaccines to elicit broadly cross-reactive antibodies against the hemagglutinin stalk domain. Participants were enrolled into five groups to receive vaccinations with live-attenuated followed by AS03-adjuvanted inactivated vaccine (n = 20), live-attenuated followed by inactivated vaccine (n = 15), twice AS03-adjuvanted inactivated vaccine (n = 16) or placebo (n = 5, intranasal followed by intramuscular; n = 10, twice intramuscular) 3 months apart. Vaccination was found to be safe and induced a broad, strong, durable and functional immune response targeting the conserved, immunosubdominant stalk of the hemagglutinin. The results suggest that chimeric hemagglutinins have the potential to be developed as universal vaccines that protect broadly against influenza viruses.
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http://dx.doi.org/10.1038/s41591-020-1118-7DOI Listing
January 2021

Adjuvanted H5N1 influenza vaccine enhances both cross-reactive memory B cell and strain-specific naive B cell responses in humans.

Proc Natl Acad Sci U S A 2020 07 13;117(30):17957-17964. Epub 2020 Jul 13.

Emory Vaccine Center, School of Medicine, Emory University, Atlanta, GA 30322;

There is a need for improved influenza vaccines. In this study we compared the antibody responses in humans after vaccination with an AS03-adjuvanted versus nonadjuvanted H5N1 avian influenza virus inactivated vaccine. Healthy young adults received two doses of either formulation 3 wk apart. We found that AS03 significantly enhanced H5 hemagglutinin (HA)-specific plasmablast and antibody responses compared to the nonadjuvanted vaccine. Plasmablast response after the first immunization was exclusively directed to the conserved HA stem region and came from memory B cells. Monoclonal antibodies (mAbs) derived from these plasmablasts had high levels of somatic hypermutation (SHM) and recognized the HA stem region of multiple influenza virus subtypes. Second immunization induced a plasmablast response to the highly variable HA head region. mAbs derived from these plasmablasts exhibited minimal SHM (naive B cell origin) and largely recognized the HA head region of the immunizing H5N1 strain. Interestingly, the antibody response to H5 HA stem region was much lower after the second immunization, and this suppression was most likely due to blocking of these epitopes by stem-specific antibodies induced by the first immunization. Taken together, these findings show that an adjuvanted influenza vaccine can substantially increase antibody responses in humans by effectively recruiting preexisting memory B cells as well as naive B cells into the response. In addition, we show that high levels of preexisting antibody can have a negative effect on boosting. These findings have implications toward the development of a universal influenza vaccine.
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http://dx.doi.org/10.1073/pnas.1906613117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7395544PMC
July 2020

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

medRxiv 2020 Apr 16. Epub 2020 Apr 16.

Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

SARS-Cov-2 (severe acute respiratory disease coronavirus 2), which causes Coronavirus Disease 2019 (COVID19) was first detected in China in late 2019 and has since then caused a global pandemic. While molecular assays to directly detect the viral genetic material are available for the diagnosis of acute infection, we currently lack serological assays suitable to specifically detect SARS-CoV-2 antibodies. Here we describe serological enzyme-linked immunosorbent assays (ELISA) that we developed using recombinant antigens derived from the spike protein of SARS-CoV-2. Using negative control samples representing pre-COVID 19 background immunity in the general adult population as well as samples from COVID19 patients, we demonstrate that these assays are sensitive and specific, allowing for screening and identification of COVID19 seroconverters using human plasma/serum as early as two days post COVID19 symptoms onset. Importantly, these assays do not require handling of infectious virus, can be adjusted to detect different antibody types and are amendable to scaling. Such serological assays are of critical importance to determine seroprevalence in a given population, define previous exposure and identify highly reactive human donors for the generation of convalescent serum as therapeutic. Sensitive and specific identification of coronavirus SARS-Cov-2 antibody titers may, in the future, also support screening of health care workers to identify those who are already immune and can be deployed to care for infected patients minimizing the risk of viral spread to colleagues and other patients.
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http://dx.doi.org/10.1101/2020.03.17.20037713DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7239062PMC
April 2020

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

Nat Med 2020 07 12;26(7):1033-1036. Epub 2020 May 12.

Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

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.
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http://dx.doi.org/10.1038/s41591-020-0913-5DOI Listing
July 2020

SARS-CoV-2 Seroconversion in Humans: A Detailed Protocol for a Serological Assay, Antigen Production, and Test Setup.

Curr Protoc Microbiol 2020 06;57(1):e100

Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York.

In late 2019, cases of atypical pneumonia were detected in China. The etiological agent was quickly identified as a betacoronavirus (named SARS-CoV-2), which has since caused a pandemic. Several methods allowing for the specific detection of viral nucleic acids have been established, but these only allow detection of the virus during a short period of time, generally during acute infection. Serological assays are urgently needed to conduct serosurveys, to understand the antibody responses mounted in response to the virus, and to identify individuals who are potentially immune to re-infection. Here we describe a detailed protocol for expression of antigens derived from the spike protein of SARS-CoV-2 that can serve as a substrate for immunological assays, as well as a two-stage serological enzyme-linked immunosorbent assay (ELISA). These assays can be used for research studies and for testing in clinical laboratories. © 2020 The Authors. Basic Protocol 1: Mammalian cell transfection and protein purification Basic Protocol 2: A two-stage ELISA for high-throughput screening of human serum samples for antibodies binding to the spike protein of SARS-CoV-2.
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http://dx.doi.org/10.1002/cpmc.100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7235504PMC
June 2020

Activity of human serum antibodies in an influenza virus hemagglutinin stalk-based ADCC reporter assay correlates with activity in a CD107a degranulation assay.

Vaccine 2020 02 17;38(8):1953-1961. Epub 2020 Jan 17.

Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA. Electronic address:

The stalk of the influenza virus hemagglutinin (HA) is an attractive target for antibody-based universal influenza virus vaccine development. While antibodies that target this part of the virus can be neutralizing, it has been shown in recent years that Fc receptor-mediated effector functions are of significant importance for the protective effect of anti-stalk antibodies. Several assays to measure Fc-Fc receptor interaction-based effector functions like antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis exist, but they suffer from limitations such as low throughput and high run-to-run variability. Reporter assays for antibody-dependent cellular cytotoxicity based on reporter cells that express luciferase upon engagement of human FcγRIIIa with the Fc of antigen-bound antibodies have been developed as well. These reporter assays can be used in a higher throughput setting with limited run-to-run assay variability but since they express only one Fc receptor, their biological relevance is unclear. Here we optimized an antibody-dependent cellular cytotoxicity reporter assay to measure the activity of antibodies to the conserved stalk domain of H1 hemagglutinin. The assay was then correlated to a CD107a-based degranulation assay, and a strong and significant correlation could be observed. This data suggests that the FcγRIIIa-based reporter assay is a good substitute for functional assays, especially in settings where larger sample numbers need to be analyzed.
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http://dx.doi.org/10.1016/j.vaccine.2020.01.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7117648PMC
February 2020

Prolonged evolution of the memory B cell response induced by a replicating adenovirus-influenza H5 vaccine.

Sci Immunol 2019 04;4(34)

HIV-Specific Immunity Section of the Laboratory of Immunoregulation, National Institutes of Health (NIH), Bethesda, MD 20892, USA.

Induction of an antibody response capable of recognizing highly diverse strains is a major obstacle to the development of vaccines for viruses such as HIV and influenza. Here, we report the dynamics of B cell expansion and evolution at the single-cell level after vaccination with a replication-competent adenovirus type 4 recombinant virus expressing influenza H5 hemagglutinin. Fluorescent H1 or H5 probes were used to quantitate and isolate peripheral blood B cells and their antigen receptors. We observed increases in H5-specific antibody somatic hypermutation and potency for several months beyond the period of active viral replication that was not detectable at the serum level. Individual broad and potent antibodies could be isolated, including one stem-specific antibody that is part of a new multidonor class. These results demonstrate prolonged evolution of the B cell response for months after vaccination and should be considered in efforts to evaluate or boost vaccine-induced immunity.
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http://dx.doi.org/10.1126/sciimmunol.aau2710DOI Listing
April 2019

Human immunodeficiency virus type-1 (HIV-1) evades antibody-dependent phagocytosis.

PLoS Pathog 2017 12 27;13(12):e1006793. Epub 2017 Dec 27.

Department of Medicine, Division of Infectious Diseases, University of California, Irvine School of Medicine, Irvine, California, United States of America.

Fc gamma receptor (FcyR)-mediated antibody functions play a crucial role in preventing HIV infection. One such function, antibody-dependent phagocytosis (ADP), is thought to be involved in controlling other viral infections, but its role in HIV infection is unknown. We measured the ability of HIV-specific polyclonal and monoclonal antibodies (mAbs) to mediate the internalization of HIV-1 virions and HIV-1-decorated cells by phagocytes. To measure ADP of virions, we primarily used a green-fluorescent protein-expressing molecular clone of HIV-1JRFL, an R5, clinical isolate, in combination with polyclonal HIVIG or mAbs known to capture and/or neutralize HIV-1. THP-1 and U937 cells, as well as freshly isolated primary monocytes from healthy individuals, were used as phagocytic effector cells, and uptake of virions was measured by cytometry. We surprisingly found minimal or no ADP of virions with any of the antibodies. However, after coating virions with gp41 or with gp41-derived peptides, gp41- (but not gp120-) specific mAbs efficiently mediated phagocytosis. We estimated that a minimum of a few hundred gp41 molecules were needed for successful phagocytosis, which is similar to the number of envelope spikes on viruses that are readily phagocytosed (e.g. influenza virus). Furthermore, by employing fluorescence correlation spectroscopy, a well-established technique to measure particle sizes and aggregation phenomena, we found a clear association between virus aggregation and ADP. In contrast to virions themselves, virion-decorated cells were targets for ADP or trogocytosis in the presence of HIV-specific antibodies. Our findings indicate that ADP of virions may not play a role in preventing HIV infection, likely due to the paucity of trimers and the consequent inability of virion-bound antibody to cross-link FcyRs on phagocytes. However, ADP or trogocytosis could play a role in clearing HIV-infected cells and cells on the verge of infection.
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http://dx.doi.org/10.1371/journal.ppat.1006793DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5760106PMC
December 2017

Alveolar macrophages are critical for broadly-reactive antibody-mediated protection against influenza A virus in mice.

Nat Commun 2017 10 10;8(1):846. Epub 2017 Oct 10.

Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA.

The aim of candidate universal influenza vaccines is to provide broad protection against influenza A and B viruses. Studies have demonstrated that broadly reactive antibodies require Fc-Fc gamma receptor interactions for optimal protection; however, the innate effector cells responsible for mediating this protection remain largely unknown. Here, we examine the roles of alveolar macrophages, natural killer cells, and neutrophils in antibody-mediated protection. We demonstrate that alveolar macrophages play a dominant role in conferring protection provided by both broadly neutralizing and non-neutralizing antibodies in mice. Our data also reveal the potential mechanisms by which alveolar macrophages mediate protection in vivo, namely antibody-induced inflammation and antibody-dependent cellular phagocytosis. This study highlights the importance of innate effector cells in establishing a broad-spectrum antiviral state, as well as providing a better understanding of how multiple arms of the immune system cooperate to achieve an optimal antiviral response following influenza virus infection or immunization.Broadly reactive antibodies that recognize influenza A virus HA can be protective, but the mechanism is not completely understood. Here, He et al. show that the inflammatory response and phagocytosis mediated by the interaction between protective antibodies and macrophages are essential for protection.
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http://dx.doi.org/10.1038/s41467-017-00928-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5635038PMC
October 2017

Generation of a serum free CHO DG44 cell line stably producing a broadly protective anti-influenza virus monoclonal antibody.

PLoS One 2017 14;12(9):e0183315. Epub 2017 Sep 14.

Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, United States of America.

Because of the broad neutralization and in vivo protection across influenza A and influenza B virus strains, monoclonal antibody CR9114 is widely used in influenza virus research as a positive control in many experiments. To produce amounts sufficient for the demand requires regular transient transfections, resulting in varying yield as well as differing batch to batch quality. Here, we report the development of a serum-free CHO DG44 cell line, stably producing a CR9114-like antibody with a potential to become a useful influenza virus research tool.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0183315PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5598927PMC
October 2017

Broadly protective murine monoclonal antibodies against influenza B virus target highly conserved neuraminidase epitopes.

Nat Microbiol 2017 Oct 21;2(10):1415-1424. Epub 2017 Aug 21.

Department of Microbiology, Icahn School of Medicine at Mount Sinai, 1 Gustave L. Levy Place, New York, NY, 10029, USA.

A substantial proportion of influenza-related childhood deaths are due to infection with influenza B viruses, which co-circulate in the human population as two antigenically distinct lineages defined by the immunodominant receptor binding protein, haemagglutinin. While broadly cross-reactive, protective monoclonal antibodies against the haemagglutinin of influenza B viruses have been described, none targeting the neuraminidase, the second most abundant viral glycoprotein, have been reported. Here, we analyse a panel of five murine anti-neuraminidase monoclonal antibodies that demonstrate broad binding, neuraminidase inhibition, in vitro antibody-dependent cell-mediated cytotoxicity and in vivo protection against influenza B viruses belonging to both haemagglutinin lineages and spanning over 70 years of antigenic drift. Electron microscopic analysis of two neuraminidase-antibody complexes shows that the conserved neuraminidase epitopes are located on the head of the molecule and that they are distinct from the enzymatic active site. In the mouse model, one therapeutic dose of antibody 1F2 was more protective than the current standard of treatment, oseltamivir, given twice daily for six days.
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http://dx.doi.org/10.1038/s41564-017-0011-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5819343PMC
October 2017

Novel Cross-Reactive Monoclonal Antibodies against Ebolavirus Glycoproteins Show Protection in a Murine Challenge Model.

J Virol 2017 08 27;91(16). Epub 2017 Jul 27.

Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA

Out of an estimated 31,100 cases since their discovery in 1976, ebolaviruses have caused approximately 13,000 deaths. The vast majority (∼11,000) of these occurred during the 2013-2016 West African epidemic. Three out of five species in the genus are known to cause Ebola Virus Disease in humans. Several monoclonal antibodies against the ebolavirus glycoprotein are currently in development as therapeutics. However, there is still a paucity of monoclonal antibodies that can cross-react between the glycoproteins of different ebolavirus species, and the mechanism of these monoclonal antibody therapeutics is still not understood in detail. Here, we generated a panel of eight murine monoclonal antibodies (MAbs) utilizing a prime-boost vaccination regimen with a glycoprotein expression plasmid followed by infection with a vesicular stomatitis virus expressing the glycoprotein. We tested the binding breadth of the resulting monoclonal antibodies using a set of recombinant surface glycoproteins from Reston, Taï Forest, Bundibugyo, Zaire, Sudan, and Marburg viruses and found two antibodies that showed pan-ebolavirus binding. An mouse model was utilized to test the ability of these MAbs to protect from infection with a vesicular stomatitis virus expressing the glycoprotein. Several of our antibodies, including the broadly binding ones, protected mice from mortality despite lacking neutralization capability , suggesting their protection may be mediated by Fc-FcR interactions. Indeed, three antibodies displayed cellular phagocytosis and/or antibody-dependent cell-mediated cytotoxicity Our antibodies, specifically the two identified cross-reactive monoclonal antibodies (KL-2E5 and KL-2H7), might add to the understanding of anti-ebolavirus humoral immunity. This study describes the generation of a panel of novel anti-ebolavirus glycoprotein monoclonal antibodies, including two antibodies with broad cross-reactivity to all known ebolavirus species. The antibodies were raised using a heterologous DNA-viral vector prime-boost regimen, resulting in a high proportion of cross-reactive antibodies (25%). Similar vaccination regimens have been used successfully to induce broad protection against influenza viruses in humans, and our limited data indicate that this might be a useful strategy for filovirus vaccines as well. Several of our antibodies showed protective efficacy when tested in a novel murine challenge model and may be developed into future therapeutics.
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http://dx.doi.org/10.1128/JVI.00652-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5533894PMC
August 2017

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

J Virol 2017 06 26;91(12). Epub 2017 May 26.

Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA

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. 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.
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http://dx.doi.org/10.1128/JVI.00286-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5446656PMC
June 2017

Molecular-level analysis of the serum antibody repertoire in young adults before and after seasonal influenza vaccination.

Nat Med 2016 12 7;22(12):1456-1464. Epub 2016 Nov 7.

Department of Chemical Engineering, University of Texas at Austin, Austin, Texas, USA.

Molecular understanding of serological immunity to influenza has been confounded by the complexity of the polyclonal antibody response in humans. Here we used high-resolution proteomics analysis of immunoglobulin (referred to as Ig-seq) coupled with high-throughput sequencing of transcripts encoding B cell receptors (BCR-seq) to quantitatively determine the antibody repertoire at the individual clonotype level in the sera of young adults before and after vaccination with trivalent seasonal influenza vaccine. The serum repertoire comprised between 40 and 147 clonotypes that were specific to each of the three monovalent components of the trivalent influenza vaccine, with boosted pre-existing clonotypes accounting for ∼60% of the response. An unexpectedly high fraction of serum antibodies recognized both the H1 and H3 monovalent vaccines. Recombinant versions of these H1 + H3 cross-reactive antibodies showed broad binding to hemagglutinins (HAs) from previously circulating virus strains; several of these antibodies, which were prevalent in the serum of multiple donors, recognized the same conserved epitope in the HA head domain. Although the HA-head-specific H1 + H3 antibodies did not show neutralization activity in vitro, they protected mice against infection with the H1N1 and H3N2 virus strains when administered before or after challenge. Collectively, our data reveal unanticipated insights regarding the serological response to influenza vaccination and raise questions about the added benefits of using a quadrivalent vaccine instead of a trivalent vaccine.
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http://dx.doi.org/10.1038/nm.4224DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5301914PMC
December 2016

Both Neutralizing and Non-Neutralizing Human H7N9 Influenza Vaccine-Induced Monoclonal Antibodies Confer Protection.

Cell Host Microbe 2016 Jun;19(6):800-13

The Department of Medicine, Section of Rheumatology, The Knapp Center for Lupus and Immunology Research, The University of Chicago, Chicago, IL 60637, USA. Electronic address:

Pathogenic H7N9 avian influenza viruses continue to represent a public health concern, and several candidate vaccines are currently being developed. It is vital to assess if protective antibodies are induced following vaccination and to characterize the diversity of epitopes targeted. Here we characterized the binding and functional properties of twelve H7-reactive human antibodies induced by a candidate A/Anhui/1/2013 (H7N9) vaccine. Both neutralizing and non-neutralizing antibodies protected mice in vivo during passive transfer challenge experiments. Mapping the H7 hemagglutinin antigenic sites by generating escape mutant variants against the neutralizing antibodies identified unique epitopes on the head and stalk domains. Further, the broadly cross-reactive non-neutralizing antibodies generated in this study were protective through Fc-mediated effector cell recruitment. These findings reveal important properties of vaccine-induced antibodies and provide a better understanding of the human monoclonal antibody response to influenza in the context of vaccines.
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http://dx.doi.org/10.1016/j.chom.2016.05.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4901526PMC
June 2016

Hemagglutinin Stalk- and Neuraminidase-Specific Monoclonal Antibodies Protect against Lethal H10N8 Influenza Virus Infection in Mice.

J Virol 2016 01 28;90(2):851-61. Epub 2015 Oct 28.

Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA

Unlabelled: Between November 2013 and February 2014, China reported three human cases of H10N8 influenza virus infection in the Jiangxi province, two of which were fatal. Using hybridoma technology, we isolated a panel of H10- and N8-directed monoclonal antibodies (MAbs) and further characterized the binding reactivity of these antibodies (via enzyme-linked immunosorbent assay) to a range of purified virus and recombinant protein substrates. The H10-directed MAbs displayed functional hemagglutination inhibition (HI) and neutralization activity, and the N8-directed antibodies displayed functional neuraminidase inhibition (NI) activity against H10N8. Surprisingly, the HI-reactive H10 antibodies, as well as a previously generated, group 2 hemagglutinin (HA) stalk-reactive antibody, demonstrated NI activity against H10N8 and an H10N7 strain; this phenomenon was absent when virus was treated with detergent, suggesting the anti-HA antibodies inhibited neuraminidase enzymatic activity through steric hindrance. We tested the prophylactic efficacy of one representative H10-reactive, N8-reactive, and group 2 HA stalk-reactive antibody in vivo using a BALB/c challenge model. All three antibodies were protective at a high dose (5 mg/kg). At a low dose (0.5 mg/kg), only the anti-N8 antibody prevented weight loss. Together, these data suggest that antibody targets other than the globular head domain of the HA may be efficacious in preventing influenza virus-induced morbidity and mortality.

Importance: Avian H10N8 and H10N7 viruses have recently crossed the species barrier, causing morbidity and mortality in humans and other mammals. Although these reports are likely isolated incidents, it is possible that more cases may emerge in future winter seasons, similar to H7N9. Furthermore, regular transmission of avian influenza viruses to humans increases the risk of adaptive mutations and reassortment events, which may result in a novel virus with pandemic potential. Currently, no specific therapeutics or vaccines are available against the H10N8 influenza virus subtype. We generated a panel of H10- and N8-reactive MAbs. Although these antibodies may practically be developed into therapeutic agents, characterizing the protective potential of MAbs that have targets other than the HA globular head domain will provide insight into novel antibody-mediated mechanisms of protection and help to better understand correlates of protection for influenza A virus infection.
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http://dx.doi.org/10.1128/JVI.02275-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4702667PMC
January 2016

Introduction of germline residues improves the stability of anti-HIV mAb 2G12-IgM.

Biochim Biophys Acta 2015 Oct 5;1854(10 Pt A):1536-44. Epub 2015 Mar 5.

Department of Biotechnology, Vienna Institute of BioTechnology at BOKU, University of Natural Resources and Life Sciences, Vienna, Austria. Electronic address:

Immunoglobulins M (IgMs) are gaining increasing attention as biopharmaceuticals since their multivalent mode of binding can give rise to high avidity. Furthermore, IgMs are potent activators of the complement system. However, they are frequently difficult to express recombinantly and can suffer from low conformational stability. Here, the broadly neutralizing anti-HIV-1 antibody 2G12 was class-switched to IgM and then further engineered by introduction of 17 germline residues. The impact of these changes on the structure and conformational stability of the antibody was then assessed using a range of biophysical techniques. We also investigated the effects of the class switch and germline substitutions on the ligand-binding properties of 2G12 and its capacity for HIV-1 neutralization. Our results demonstrate that the introduced germline residues improve the conformational and thermal stability of 2G12-IgM without altering its overall shape and ligand-binding properties. Interestingly, the engineered protein was found to exhibit much lower neutralization potency than its wild-type counterpart, indicating that potent antigen recognition is not solely responsible for IgM-mediated HIV-1 inactivation.
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http://dx.doi.org/10.1016/j.bbapap.2015.02.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4582045PMC
October 2015

Evaluating the bottlenecks of recombinant IgM production in mammalian cells.

Cytotechnology 2015 Mar 11;67(2):343-56. Epub 2014 Mar 11.

Department of Biotechnology, Vienna Institute of BioTechnology (BOKU - VIBT), University of Natural Resources and Life Sciences, Muthgasse 11, 1190, Vienna, Austria.

Despite the fact, that monoclonal antibodies are the fastest growing group of biopharmaceuticals in development, this is not true for the IgM class, which remains as enigmatic as ever. While more examples of usefulness of IgMs for medical applications are emerging, their recombinant production is still not common. In our study, stable monoclonal IgM producing CHO DG44 and HEK 293 cell lines, expressing two model IgM molecules (IgM-617 and IgM-012) were established. Recombinant cell lines were compared in regard of specific productivity, specific growth rate, maximal achieved antibody titer, gene copy numbers and transcription levels of transgene. IgM-617 cell lines were identified as high while IgM-012 clones were low producers. Although differences in gene copy numbers as well as in transcription levels were observed, they did not seem to be a limitation. Levels of relevant endoplasmic reticulum-stress related proteins were analyzed and no indications of unfolded protein response were detected. This could indicate that the difference in the intrinsic protein stability of our model proteins (as was previously observed on purified samples) might cause lower yields of IgM-012. Transcriptomics and/or proteomics follow up studies might be necessary for identification of potential bottlenecks in IgM producing cell lines.
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http://dx.doi.org/10.1007/s10616-014-9693-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4329305PMC
March 2015

HIV-1 specific antibody titers and neutralization among chronically infected patients on long-term suppressive antiretroviral therapy (ART): a cross-sectional study.

PLoS One 2014 15;9(1):e85371. Epub 2014 Jan 15.

Division of Infectious Diseases and Center for Global Health, Northwestern University, Chicago, Illinois, United States of America.

The majority of potent and broadly neutralizing antibodies against HIV-1 have been isolated from untreated patients with acute or chronic infection. To assess the extent of HIV-1 specific antibody response and neutralization after many years of virologic suppression from potent combination ART, we examined antibody binding titers and neutralization of 51 patients with chronic HIV-1 infection on suppressive ART for at least three years. In this cross-sectional analysis, we found high antibody titers against gp120, gp41, and the membrane proximal external region (MPER) in 59%, 43%, and 27% of patients, respectively. We observed significantly higher endpoint binding titers for gp120 and gp41 for patients with >10 compared to ≤ 10 years of detectable HIV RNA. Additionally, we observed higher median gp120 and gp41 antibody titers in patients with HIV RNA <50 copies/mL for ≤ 5 years. 22% of patients neutralized a HIV-1 primary isolate (HIV-1(JR-FL)) and 8% neutralized a HIV-2/HIV-1 MPER chimera. Significantly greater HIV-1(JR-FL) neutralization was found among patients with >10 years of detectable HIV RNA (8/20 [40.0%] versus 3/31 [9.7%] for ≤ 10 years, p = 0.02) and a trend toward greater neutralization in patients with ≤ 5 years of HIV RNA <50 copies/mL (7/20 [35.0%] versus 4/31 [12.9%] for >5 years, p = 0.08). All patients with neutralizing activity mediated successful phagocytosis of VLPs by THP-1 cells after antibody opsonization. Our findings of highly specific antibodies to several structural epitopes of HIV-1 with antibody effector functions and neutralizing activity after long-term suppressive ART, suggest continuous antigenic stimulation and evolution of HIV-specific antibody response occurs before and after suppression with ART. These patients, particularly those with slower HIV progression and more time with detectable viremia prior to initiation of suppressive ART, are a promising population to identify and further study functional antibodies against HIV-1.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0085371PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3893210PMC
December 2014