Sinthujan Jegaskanda

Cross-Reactive Influenza-Specific Antibody-Dependent Cellular Cytotoxicity Antibodies in the Absence of Neutralizing Antibodies

A better understanding of immunity to influenza virus is needed to generate cross-protective vaccines. Engagement of Ab-dependent cellular cytotoxicity (ADCC) Abs by NK cells leads to killing of virus-infected cells and secretion of antiviral cytokines and chemokines. ADCC Abs may target more conserved influenza virus Ags compared with neutralizing Abs. There has been minimal interest in influenza-specific ADCC in recent decades. In this study, we developed novel assays to assess the specificity and function of influenza-specific ADCC Abs. We found that healthy influenza-seropositive young adults without detectable neutralizing Abs to the hemagglutinin of the 1968 H3N2 influenza strain (A/Aichi/2/1968) almost always had ADCC Abs that triggered NK cell activation and in vitro elimination of influenza-infected human blood and respiratory epithelial cells. Furthermore, we detected ADCC in the absence of neutralization to both the recent H1N1 pandemic strain (A/California/04/2009) as well as the avian H5N1 influenza hemagglutinin (A/Anhui/01/2005). We conclude that there is a remarkable degree of cross-reactivity of influenza-specific ADCC Abs in seropositive humans. Targeting cross-reactive influenza-specific ADCC epitopes by vaccination could lead to improved influenza vaccines.

Evaluation of influenza-specific antibody-dependent cellular cytotoxicity (ADCC) in human and non-human primates

ABSTRACT Emerging influenza viruses pose a serious risk to global human health. Recent studies in ferrets, macaques, and humans suggest that seasonal H1N1 (sH1N1) infection provides some cross-protection against 2009 pandemic influenza viruses (H1N1pdm), but the correlates of cross-protection are poorly understood. Here we show that seasonal infection of influenza-naïve Indian rhesus macaques (Macaca mulatta) with A/Kawasaki/173/2001 (sH1N1) virus induces antibodies capable of binding the hemagglutinin (HA) of both the homologous seasonal virus and the antigenically divergent A/California/04/2009 (H1N1pdm) strain in the absence of detectable H1N1pdm-specific neutralizing antibodies. These influenza virus-specific antibodies activated macaque NK cells to express both CD107a and gamma interferon (IFN-γ) in the presence of HA proteins from either sH1N1 or H1N1pdm viruses. Although influenza virus-specific antibody-dependent cellular cytotoxicity (ADCC)-mediated NK cell activation diminished in titer over time following sH1N1 infection, these cells expanded rapidly within 7 days following H1N1pdm exposure. Furthermore, we found that influenza virus-specific ADCC was present in bronchoalveolar lavage fluid and was able to activate lung NK cells. We concluded that infection with a seasonal influenza virus can induce antibodies that mediate ADCC capable of recognizing divergent influenza virus strains. Cross-reactive ADCC may provide a mechanism for reducing the severity of divergent influenza virus infections.

Antibody-Dependent Cellular Cytotoxicity Is Associated with Control of Pandemic H1N1 Influenza Virus Infection of Macaques

ABSTRACT Emerging influenza viruses pose a serious risk to global human health. Recent studies in ferrets, macaques, and humans suggest that seasonal H1N1 (sH1N1) infection provides some cross-protection against 2009 pandemic influenza viruses (H1N1pdm), but the correlates of cross-protection are poorly understood. Here we show that seasonal infection of influenza-naïve Indian rhesus macaques (Macaca mulatta) with A/Kawasaki/173/2001 (sH1N1) virus induces antibodies capable of binding the hemagglutinin (HA) of both the homologous seasonal virus and the antigenically divergent A/California/04/2009 (H1N1pdm) strain in the absence of detectable H1N1pdm-specific neutralizing antibodies. These influenza virus-specific antibodies activated macaque NK cells to express both CD107a and gamma interferon (IFN-γ) in the presence of HA proteins from either sH1N1 or H1N1pdm viruses. Although influenza virus-specific antibody-dependent cellular cytotoxicity (ADCC)-mediated NK cell activation diminished in titer over time following sH1N1 infection, these cells expanded rapidly within 7 days following H1N1pdm exposure. Furthermore, we found that influenza virus-specific ADCC was present in bronchoalveolar lavage fluid and was able to activate lung NK cells. We concluded that infection with a seasonal influenza virus can induce antibodies that mediate ADCC capable of recognizing divergent influenza virus strains. Cross-reactive ADCC may provide a mechanism for reducing the severity of divergent influenza virus infections.

Human mucosal-associated invariant T cells contribute to antiviral influenza immunity via IL-18–dependent activation

Significance Mucosal-associated invariant T (MAIT) cells are innate-like T lymphocytes with potent antibacterial reactivity. In this study, we investigated whether MAIT cells also contribute to immunity against influenza A viruses. Compared with those who succumbed, hospitalized patients who recovered from severe avian H7N9 influenza infection had higher numbers of MAIT cells. Subsequent in vitro analysis established that MAIT cells from healthy donors are indirectly activated by influenza infection via an IL-18–dependent (but not IL-12–dependent) mechanism requiring the involvement of CD14+ monocytes. Our findings highlight the potential for MAIT cells to promote protective immunity in human influenza. Mucosal-associated invariant T (MAIT) cells are innate-like T lymphocytes known to elicit potent immunity to a broad range of bacteria, mainly via the rapid production of inflammatory cytokines. Whether MAIT cells contribute to antiviral immunity is less clear. Here we asked whether MAIT cells produce cytokines/chemokines during severe human influenza virus infection. Our analysis in patients hospitalized with avian H7N9 influenza pneumonia showed that individuals who recovered had higher numbers of CD161+Vα7.2+ MAIT cells in peripheral blood compared with those who succumbed, suggesting a possible protective role for this lymphocyte population. To understand the mechanism underlying MAIT cell activation during influenza, we cocultured influenza A virus (IAV)-infected human lung epithelial cells (A549) and human peripheral blood mononuclear cells in vitro, then assayed them by intracellular cytokine staining. Comparison of influenza-induced MAIT cell activation with the profile for natural killer cells (CD56+CD3−) showed robust up-regulation of IFNγ for both cell populations and granzyme B in MAIT cells, although the individual responses varied among healthy donors. However, in contrast to the requirement for cell-associated factors to promote NK cell activation, the induction of MAIT cell cytokine production was dependent on IL-18 (but not IL-12) production by IAV-exposed CD14+ monocytes. Overall, this evidence for IAV activation via an indirect, IL-18–dependent mechanism indicates that MAIT cells are protective in influenza, and also possibly in any human disease process in which inflammation and IL-18 production occur.

Influenza-Specific Antibody-Dependent Cellular Cytotoxicity: Toward a Universal Influenza Vaccine

There is an urgent need for universal influenza vaccines that can control emerging pandemic influenza virus threats without the need to generate new vaccines for each strain. Neutralizing Abs to the influenza virus hemagglutinin glycoprotein are effective at controlling influenza infection but generally target highly variable regions. Abs that can mediate other functions, such as killing influenza-infected cells and activating innate immune responses (termed “Ab-dependent cellular cytotoxicity [ADCC]-mediating Abs”), may assist in protective immunity to influenza. ADCC-mediating Abs can target more conserved regions of influenza virus proteins and recognize a broader array of influenza strains. We review recent research on influenza-specific ADCC Abs and their potential role in improved influenza-vaccination strategies.

Cross-Reactive Influenza-Specific Antibody-Dependent Cellular Cytotoxicity in Intravenous Immunoglobulin as a Potential Therapeutic Against Emerging Influenza Viruses

BACKGROUND Intravenous immunoglobulin (IVIG) is a purified pool of human antibodies from thousands of donors that is used to prevent or treat primary immune deficiency, several infectious diseases, and autoimmune diseases. The antibodies that mediate antibody-dependent cellular cytotoxicity (ADCC) against heterologous influenza strains may be present in IVIG preparations. METHODS We tested 8 IVIG preparations prior to the 2009 H1N1 swine-origin influenza pandemic and 10 IVIG preparations made after 2010 for their ability to mediate influenza-specific ADCC. RESULTS ADCC mediating antibodies to A(H1N1)pdm09 hemagglutinin (HA) and neuraminidase (NA) were detected in IVIG preparations prior to the 2009-H1N1 pandemic. The HA-specific ADCC targeted both the HA1 and HA2 regions of A(H1N1)pdm09 HA and was capable of recognizing a broad range of HA proteins including those from recent avian influenza strains A(H5N1) and A(H7N9). The low but detectable ADCC recognition of A(H7N9) was likely due to rare individuals in the population contributing cross-reactive antibodies to IVIG. CONCLUSIONS IVIG preparations contain broadly cross-reactive ADCC mediating antibodies. IVIG may provide at least some level of protection for individuals at high risk of severe influenza disease, especially during influenza pandemics prior to the development of effective vaccines.

Age-associated cross-reactive antibody-dependent cellular cytotoxicity toward 2009 pandemic influenza A virus subtype H1N1.

BACKGROUND During the 2009 pandemic of influenza A virus subtype H1N1 (A[H1N1]pdm09) infection, older individuals were partially protected from severe disease. It is not known whether preexisting antibodies with effector functions such as antibody-dependent cellular cytotoxicity (ADCC) contributed to the immunity observed. METHODS We tested serum specimens obtained from 182 individuals aged 1-72 years that were collected either immediately before or after the A(H1N1)pdm09 pandemic for ADCC antibodies to the A(H1N1)pdm09 hemagglutinin (HA) protein. RESULTS A(H1N1)pdm09 HA-specific ADCC antibodies were detected in almost all individuals aged >45 years (28/31 subjects) before the 2009 A(H1N1) pandemic. Conversely, only approximately half of the individuals aged 1-14 years (11/31) and 15-45 years (17/31) had cross-reactive ADCC antibodies before the 2009 A(H1N1) pandemic. The A(H1N1)pdm09-specific ADCC antibodies were able to efficiently mediate the killing of influenza virus-infected respiratory epithelial cells. Further, subjects >45 years of age had higher ADCC titers to a range of seasonal H1N1 HA proteins, including from the 1918 virus, compared with younger individuals. CONCLUSIONS ADCC antibodies may have contributed to the protection exhibited in older individuals during the 2009 A(H1N1) pandemic. This work has significant implications for improved vaccination strategies for future influenza pandemics.

Role of monocytes in mediating HIV-specific antibody-dependent cellular cytotoxicity

Antibodies (Abs) that mediate antibody-dependent cellular cytotoxicity (ADCC) activity against HIV-1 are of major interest. A widely used method to measure ADCC Abs is the rapid and fluorometric antibody-dependent cellular cytotoxicity (RFADCC) assay. Antibody-dependent killing of a labelled target cell line by PBMC is assessed by loss of intracellular CFSE but retention of membrane dye PKH26 (CFSE-PKH26+). Cells of this phenotype are assumed to be derived from CFSE+PKH26+ target cells killed by NK cells. We assessed the effector cells that mediate ADCC in this assay. Backgating analysis and phenotyping of CFSE-PKH26+ revealed that the RFADCC assays readout mainly represents CD3-CD14+ monocytes taking up the PKH26 dye. This was confirmed for 53 HIV+plasma-purified IgG samples when co-cultured with PBMC from three separate healthy donors. Emergence of the CFSE-PKH26+ monocyte population was observed upon co-culture of targets with purified monocytes but not with purified NK cells. Image flow cytometry and microscopy showed a monocyte-specific interaction with target cells without typical morphological changes associated with phagocytosis, suggesting a monocyte-mediated ADCC process. We conclude that the RFADCC assay primarily reflects Ab-mediated monocyte function. Further studies on the immunological importance of HIV-specific monocyte-mediated ADCC are warranted.

Generation and Protective Ability of Influenza Virus–Specific Antibody-Dependent Cellular Cytotoxicity in Humans Elicited by Vaccination, Natural Infection, and Experimental Challenge

BACKGROUND Nonneutralizing antibodies (Abs) involved in antibody-dependent cellular cytotoxicity (ADCC) may provide some protection from influenza virus infection. The ability of influenza vaccines to induce ADCC-mediating Abs (ADCC-Abs) in adults and children is unclear. METHODS We quantified ADCC-Abs in serum samples from adults who received a dose of inactivated subunit vaccine (ISV) targeting monovalent 2009 pandemic influenza A(H1N1) virus or live-attenuated influenza vaccine (LAIV) or who had laboratory-confirmed influenza A(H1N1) virus infection. We also measured ADCC-Abs in children who either received a dose of trivalent seasonal ISV followed by trivalent seasonal LAIV or 2 doses of LAIV. Finally, we assessed the ability of low and high ADCC-Ab titers to protect adults from experimental challenge with influenza A/Wisconsin/67/131/2005(H3N2) virus. RESULTS Adults and children who received a dose of ISV had a robust increase in ADCC-Ab titers to both recombinant hemagglutinin (rHA) protein and homologous virus-infected cells. There was no detectable increase in titers of ADCC-Abs to rHA or virus-infected cells in adults and children who received LAIV. Higher titers (≥320) of preexisting ADCC-Abs were associated with lower virus replication and a significant reduction in total symptom scores in experimentally infected adults. CONCLUSIONS ADCC-Ab titers increased following experimental influenza virus infection in adults and after ISV administration in both children and adults.

Standard Trivalent Influenza Virus Protein Vaccination Does Not Prime Antibody-Dependent Cellular Cytotoxicity in Macaques

ABSTRACT Yearly vaccination with the trivalent inactivated influenza vaccine (TIV) is recommended, since current vaccines induce little cross neutralization to divergent influenza strains. Whether the TIV can induce antibody-dependent cellular cytotoxicity (ADCC) responses that can cross-recognize divergent influenza virus strains is unknown. We immunized 6 influenza-naive pigtail macaques twice with the 2011–2012 season TIV and then challenged the macaques, along with 12 control macaques, serially with H1N1 and H3N2 viruses. We measured ADCC responses in plasma to a panel of H1 and H3 hemagglutinin (HA) proteins and influenza virus-specific CD8 T cell (CTL) responses using a sensitive major histocompatibility complex (MHC) tetramer reagent. The TIV was weakly immunogenic and, although binding antibodies were detected by enzyme-linked immunosorbent assay (ELISA), did not induce detectable influenza virus-specific ADCC or CTL responses. The H1N1 challenge elicited robust ADCC to both homologous and heterologous H1 HA proteins, but not influenza virus HA proteins from different subtypes (H2 to H7). There was no anamnestic influenza virus-specific ADCC or CTL response in vaccinated animals. The subsequent H3N2 challenge did not induce or boost ADCC either to H1 HA proteins or to divergent H3 proteins but did boost CTL responses. ADCC or CTL responses were not induced by TIV vaccination in influenza-naive macaques. There was a marked difference in the ability of infection compared to that of vaccination to induce cross-reactive ADCC and CTL responses. Improved vaccination strategies are needed to induce broad-based ADCC immunity to influenza.