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Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

Icahn School of Medicine at Mount Sinai

To investigate the efficacy of EP regimen combined with split-course hyperfractionated accelerated irradiation for locally advanced non-small cell lung cancer.

[Combination of EP regimen and split-course hyperfractionated accelerated irradiation in locally advanced non-small cell lung cancer].

The narrow species tropism of hepatitis C virus (HCV) limits animal studies. We found that pigtail macaque (Macaca nemestrina) hepatic cells derived from induced pluripotent stem cells support the entire HCV life cycle, although infection efficiency was limited by defects in the HCV cell entry process. This block was overcome by either increasing occludin expression, complementing the cells with human CD81, or infecting them with a strain of HCV with less restricted requirements for CD81. Using this system, we can modify viral and host cell genetics to make pigtail macaques a suitable, clinically relevant model for the study of HCV infection.

Hepatic cells derived from induced pluripotent stem cells of pigtail macaques support hepatitis C virus infection.

To find new biomarkers and establish histopathology protein fingerprint models for early detection of colorectal cancer (CRC), laser capture microdissection (LCM) was utilized to obtain 3 groups of cells of interest--CRC tissues, their adjacent normal colorectal tissues, and their adjacent adenomatous polyps tissues--from the same 12 CRC patients. Each sample was then detected by surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS) technology and CM10 protein chip as well as bioinformatics tools. Model 1 formed by 15 protein peaks could be used to distinguish CRC tissues from normal tissues. The diagnostic pattern constructed using support vector machine (SVM) including the 15 proteins showed maximum Youden index (YI). Model 2 formed by 14 protein peaks could be used to distinguish CRC tissues from adenomatous polyps tissues. The two patterns were validated and the results showed that the sensitivity and specificity were both 100.0%. Model 3 formed by 15 protein peaks could distinguish adenomatous polyps tissues from normal tissues with a sensitivity of 92.3% and specificity of 100.0%. The protein peaks m/z 3570 and 5224 were identified in screening for changes during cancer progression. Peak 5224 was significantly upregulated in CRC. However, peak 3570 was significantly downregulated in CRC. LCM technology coupled with SELDI protein chip and bioinformatics approaches could effectively screen the differentially expressed protein profiles and establish molecular diagnosis models with high sensitivity and specificity for CRC.

Protein fingerprint of colorectal cancer, adenomatous polyps, and normal mucosa using ProteinChip analysis on laser capture microdissected cells.

Current influenza virus vaccines rely upon the accurate prediction of circulating virus strains months in advance of the actual influenza season in order to allow time for vaccine manufacture. Unfortunately, mismatches occur frequently, and even when perfect matches are achieved, suboptimal vaccine efficacy leaves several high-risk populations vulnerable to infection. However, the recent discovery of broadly neutralizing antibodies that target the hemagglutinin (HA) stalk domain has renewed hope that the development of "universal" influenza virus vaccines may be within reach. Here, we examine the functions of influenza A virus hemagglutinin stalk-binding antibodies in an endogenous setting, i.e., as polyclonal preparations isolated from human sera. Relative to monoclonal antibodies that bind to the HA head domain, the neutralization potency of monoclonal stalk-binding antibodies was vastly inferior in vitro but was enhanced by several orders of magnitude in the polyclonal context. Furthermore, we demonstrated a surprising enhancement in IgA-mediated HA stalk neutralization relative to that achieved by antibodies of IgG isotypes. Mechanistically, this could be explained in two ways. Identical variable regions consistently neutralized virus more potently when in an IgA backbone compared to an IgG backbone. In addition, HA-specific memory B cells isolated from human peripheral blood were more likely to be stalk specific when secreting antibodies of IgA isotypes compared to those secreting IgG. Taken together, our data provide strong evidence that HA stalk-binding antibodies perform optimally when in a polyclonal context and that the targeted elicitation of HA stalk-specific IgA should be an important consideration during "universal" influenza virus vaccine design.

Broadly neutralizing anti-influenza virus antibodies: enhancement of neutralizing potency in polyclonal mixtures and IgA backbones.

The discovery of broadly-neutralizing antibodies that bind to the hemagglutinin stalk/stem domain has opened exciting new avenues for the development of "universal" influenza virus vaccines and therapeutics. Unlike strain-specific antibodies which bind to the hemagglutinin head domain and inhibit receptor binding, antibodies that bind to the stalk domain function to inhibit later stages of infection. The hemagglutination inhibition assay has long been the standard for evaluating titers of neutralizing hemagglutinin-specific antibodies in serum. The assay has the beneficial properties of being relatively rapid, easy-to-perform, and requires very little specialized equipment. Historically, hemagglutination inhibition titers of 40 or above against a given strain of influenza has been considered a correlate of protection on a population level. Unfortunately, this assay cannot be used to measure titers of hemagglutinin stalk-specific antibodies due to their lack of hemagglutination inhibiting activity. This has necessitated the development of novel reagents and assays capable of sensitive and specific detection of broadly-neutralizing HA stalk-binding antibodies in polyclonal mixtures. Here, we describe a novel microneutralization-based assay that utilizes recombinant influenza A viruses expressing chimeric hemagglutinin molecules and 'exotic' neuraminidase to measure titers of broadly-neutralizing antibodies in polyclonal preparations.

Measuring the neutralization potency of influenza A virus hemagglutinin stalk/stem-binding antibodies in polyclonal preparations by microneutralization assay.

Host response to RNA virus infection is sensed by RNA sensors such as RIG-I, which induces MAVS-mediated NF-κB and IRF3 activation to promote inflammatory and antiviral responses, respectively. Here, we have found that CARMA3, a scaffold protein previously shown to mediate NF-κB activation induced by GPCR and EGFR, positively regulates MAVS-induced NF-κB activation. However, our data suggest that CARMA3 sequesters MAVS from forming high-molecular-weight aggregates, thereby suppressing TBK1/IRF3 activation. Interestingly, following NF-κB activation upon virus infection, CARMA3 is targeted for proteasome-dependent degradation, which releases MAVS to activate IRF3. When challenged with vesicular stomatitis virus or influenza A virus, CARMA3-deficient mice showed reduced disease symptoms compared to those of wild-type mice as a result of less inflammation and a stronger ability to clear infected virus. Altogether, our results reveal the role of CARMA3 in regulating the balance of host antiviral and pro-inflammatory responses against RNA virus infection.

CARMA3 Is a Host Factor Regulating the Balance of Inflammatory and Antiviral Responses against Viral Infection.

High-affinity-antibody production, T-cell activation, and interferon upregulation all contribute to protective immunity that occurs in humans following influenza immunization. Hematopoietic cell-specific PTPN22 encodes lymphoid phosphatase (Lyp), which regulates lymphocyte antigen receptor and pattern recognition receptor (PRR) signaling. A PTPN22 variant, R620W (LypW), predisposes to autoimmune and infectious diseases and confers altered signaling through antigen receptors and PRRs. We tested the hypothesis that LypW-bearing humans would have diminished immune response to trivalent influenza vaccine (TIV). LypW carriers exhibited decreased induction of influenza virus-specific CD4(+) T cells expressing effector cytokines and failed to increase antibody affinity following TIV receipt. No differences between LypW carriers and noncarriers were observed in virus-specific CD8(+) T-cell responses, early interferon transcriptional responses, or myeloid antigen-presenting cell costimulatory molecule upregulation. The association of LypW with defects in TIV-induced CD4(+) T-cell expansion and antibody affinity maturation suggests that LypW may predispose individuals to have a diminished capacity to generate protective immunity against influenza virus.

Autoimmune Variant PTPN22 C1858T Is Associated With Impaired Responses to Influenza Vaccination.

Influenza virus strain-specific monoclonal antibodies (mAbs) provide protection independent of Fc gamma receptor (FcγR) engagement. In contrast, optimal in vivo protection achieved by broadly reactive mAbs requires Fc-FcγR engagement. Most strain-specific mAbs target the head domain of the viral hemagglutinin (HA), whereas broadly reactive mAbs typically recognize epitopes within the HA stalk. This observation has led to questions regarding the mechanism regulating the activation of Fc-dependent effector functions by broadly reactive antibodies. To dissect the molecular mechanism responsible for this dichotomy, we inserted the FLAG epitope into discrete locations on HAs. By characterizing the interactions of several FLAG-tagged HAs with a FLAG-specific antibody, we show that in addition to Fc-FcγR engagement mediated by the FLAG-specific antibody, a second intermolecular bridge between the receptor-binding region of the HA and sialic acid on effector cells is required for optimal activation. Inhibition of this second molecular bridge, through the use of an F(ab')2 or the mutation of the sialic acid-binding site, renders the Fc-FcγR interaction unable to optimally activate effector cells. Our findings indicate that broadly reactive mAbs require two molecular contacts to possibly stabilize the immunologic synapse and potently induce antibody-dependent cell-mediated antiviral responses: (i) the interaction between the Fc of a mAb bound to HA with the FcγR of the effector cell and (ii) the interaction between the HA and its sialic acid receptor on the effector cell. This concept might be broadly applicable for protective antibody responses to viral pathogens that have suitable receptors on effector cells.

Optimal activation of Fc-mediated effector functions by influenza virus hemagglutinin antibodies requires two points of contact.

The generation of strain-specific neutralizing antibodies against influenza A virus is known to confer potent protection against homologous infections. The majority of these antibodies bind to the hemagglutinin (HA) head domain and function by blocking the receptor binding site, preventing infection of host cells. Recently, elicitation of broadly neutralizing antibodies which target the conserved HA stalk domain has become a promising "universal" influenza virus vaccine strategy. The ability of these antibodies to elicit Fc-dependent effector functions has emerged as an important mechanism through which protection is achieved in vivo. However, the way in which Fc-dependent effector functions are regulated by polyclonal influenza virus-binding antibody mixtures in vivo has never been defined. Here, we demonstrate that interactions among viral glycoprotein-binding antibodies of varying specificities regulate the magnitude of antibody-dependent cell-mediated cytotoxicity induction. We show that the mechanism responsible for this phenotype relies upon competition for binding to HA on the surface of infected cells and virus particles. Nonneutralizing antibodies were poor inducers and did not inhibit antibody-dependent cell-mediated cytotoxicity. Interestingly, anti-neuraminidase antibodies weakly induced antibody-dependent cell-mediated cytotoxicity and enhanced induction in the presence of HA stalk-binding antibodies in an additive manner. Our data demonstrate that antibody specificity plays an important role in the regulation of ADCC, and that cross-talk among antibodies of varying specificities determines the magnitude of Fc receptor-mediated effector functions.

Epitope specificity plays a critical role in regulating antibody-dependent cell-mediated cytotoxicity against influenza A virus.

Broadly neutralizing antibodies that recognize the conserved hemagglutinin (HA) stalk have emerged as exciting new biotherapeutic tools to combat seasonal and pandemic influenza viruses. Our general understanding of the mechanisms by which stalk-specific antibodies achieve protection is rapidly evolving. It has recently been demonstrated that broadly neutralizing HA stalk-specific IgG antibodies require Fc-Fcγ receptor (FcγR) interactions for optimal protection in vivo Here we examine the neutrophil effector functions induced by stalk-specific antibodies. As the most abundant subset of blood leukocytes, neutrophils represent a critical innate effector cell population and serve an instrumental role in orchestrating downstream adaptive responses to influenza virus infection. Yet, the interplay of HA stalk-specific IgG, Fc-FcγR engagement, and neutrophils has remained largely uncharacterized. Using an in vitro assay to detect the production of reactive oxygen species (ROS), we show that human and mouse monoclonal HA stalk-specific IgG antibodies are able to induce the production of ROS by neutrophils, while HA head-specific antibodies do not. Furthermore, our results indicate that the production of ROS is dependent on Fc receptor (FcR) engagement and phagocytosis. We went on to assess the ability of monoclonal HA stalk-specific IgA antibodies to induce ROS. Consistent with our findings for monoclonal IgGs, only HA stalk-specific IgA antibodies elicited ROS production by neutrophils. This induction is dependent on the engagement of FcαR1. Taken together, our findings describe a novel FcR-dependent effector function induced by HA stalk-specific IgG and IgA antibodies, and importantly, our studies shed light on the mechanisms by which HA stalk-specific antibodies achieve protection.

Broadly Neutralizing Hemagglutinin Stalk-Specific Antibodies Induce Potent Phagocytosis of Immune Complexes by Neutrophils in an Fc-Dependent Manner.

We have previously demonstrated that the combination of synthetic small molecule Toll-like receptor 4 (TLR4) and TLR7 ligands is a potent adjuvant for recombinant influenza virus hemagglutinin, inducing rapid and sustained immunity protective against influenza viruses in homologous, heterologous and heterosubtypic murine challenge models. Combining TLR4 and TLR7 ligands balances Th1/Th2-type immune responses for long-lived cellular and neutralizing humoral immunity against the viral hemagglutinin. Here, we demonstrate that the protective response induced in mice by this combined adjuvant is dependent upon TLR4 and TLR7 signaling via myeloid differentiation primary response gene 88 (MyD88), indicating that the adjuvants function in vivo via their known receptors with negligible off-target effects to induce protective immunity. The combined adjuvant acts via MyD88 in both bone marrow and non-bone marrow derived, radioresistant cells to induce hemagglutinin-specific antibodies and protect mice against influenza virus challenge. The protective efficacy generated by immunizing with this adjuvant and recombinant hemagglutinin antigen could be transferred with serum from immunized mice to recipient mice in a homologous, but not a heterologous, H1N1 viral challenge model. Depletion of CD4(+) cells after an established humoral response in immunized mice did not impair protection from a homologous challenge; however, it did significantly impair recovery from a heterologous challenge virus, highlighting an important role for vaccine-induced CD4(+) cells in cross-protective vaccine efficacy. The combination of the two TLR agonists allowed for significant dose reduction of each component to achieve a level of protection equivalent to that afforded by either single agent at full dose.Importance Development of novel adjuvants is needed to enhance immunogenicity to provide better protection from seasonal influenza virus infection and improve pandemic preparedness. We show here that several dose combinations of synthetic TLR4 and TLR7 ligands are potent adjuvants for recombinant influenza virus hemagglutinin antigen to induce humoral and cellular immunity against viral challenges. The components of the combined adjuvant work additively to enable both antigen and adjuvant dose sparing while retaining efficacy. Understanding an adjuvant's mechanism of action is a critical component for preclinical safety evaluation, and we demonstrate here that a combined TLR4 and TLR7 adjuvant signals via the appropriate receptors and the MyD88 adaptor protein. This novel adjuvant combination contributes to a more broadly protective vaccine while demonstrating an attractive safety profile.

Wenqian He

Department of Microbiology,
Graduate School of Biomedical Sciences

Icahn School of Medicine at Mount Sinai

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Wenqian He

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Microbiology,Graduate School of Biomedical Sciences

Icahn School of Medicine at Mount Sinai

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Department of Microbiology,
Graduate School of Biomedical Sciences