Agrarstruktur in Ghandruk und Umgebung : Schwerpunkt Ackerbau und Hausgärten

Immunity to human influenza A virus (IAV) infection is only partially understood. Broadly non-neutralizing antibodies may assist in reducing disease but have not been well characterized.We measured internalization of opsonized, influenza protein-coated fluorescent beads and live IAV into a monocytic cell line to study antibody-dependent phagocytosis (ADP) against multiple influenza hemagglutinin (HA) subtypes. We analyzed influenza HA-specific ADP in healthy human donors, in preparations of intravenous immunoglobulin (IVIG), and following IAV infection of humans and macaques.We found that both sera from healthy adults and IVIG preparations had broad ADP to multiple seasonal HA proteins and weak cross-reactive ADP to non-circulating HA proteins. The ADP in experimentally influenza-infected macaque plasma and naturally influenza-infected human sera mediated phagocytosis of both homologous and heterologous IAVs. Further, the IAV phagocytosed in an antibody-mediated manner had reduced infectivity in vitro.We conclude that IAV infections in humans and macaques leads to the development of influenza-specific ADP that can clear IAV infection in vitro. Repeated exposure of humans to multiple IAV infections likely leads to the development of ADP that is cross-reactive to strains not previously encountered. Further analyses of the protective capacity of broadly reactive influenza-specific ADP is warranted

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

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

Comparison of Influenza and SIV Specific CD8 T Cell Responses in Macaques

Macaques are a potentially useful non-human primate model to compare memory T-cell immunity to acute virus pathogens such as influenza virus and effector T-cell responses to chronic viral pathogens such as SIV. However, immunological reagents to study influenza CD8+ T-cell responses in the macaque model are limited. We recently developed an influenza-SIV vaccination model of pigtail macaques (Macaca nemestrina) and used this to study both influenza-specific and SIV-specific CD8+ T-cells in 39 pigtail macaques expressing the common Mane-A*10+ (Mane-A01*084) MHC-I allele. To perform comparative studies between influenza and SIV responses a common influenza nucleoprotein-specific CD8+ T-cell response was mapped to a minimal epitope (termed RA9), MHC-restricted to Mane-A*10 and an MHC tetramer developed to study this response. Influenza-specific memory CD8+ T-cell response maintained a highly functional profile in terms of multitude of effector molecule expression (CD107a, IFN-γ, TNF-α, MIP-1β and IL-2) and showed high avidity even in the setting of SIV infection. In contrast, within weeks following active SIV infection, SIV-specific CD8+ effector T-cells expressed fewer cytokines/degranulation markers and had a lower avidity compared to influenza specific CD8+ T-cells. Further, the influenza specific memory CD8 T-cell response retained stable expression of the exhaustion marker programmed death-marker-1 (PD-1) and co-stimulatory molecule CD28 following infection with SIV. This contrasted with the effector SIV-specific CD8+ T-cells following SIV infection which expressed significantly higher amounts of PD-1 and lower amounts of CD28. Our results suggest that strategies to maintain a more functional CD8+ T-cell response, profile may assist in controlling HIV disease

The Potential Role of Fc-Receptor Functions in the Development of a Universal Influenza Vaccine

Despite global vaccination efforts, influenza virus continues to cause yearly epidemics and periodic pandemics throughout most of the world. Many of us consider the generation of broader, potent and long-lasting immunity against influenza viruses as critical in curtailing the global health and economic impact that influenza currently plays. To date, classical vaccinology has relied on the generation of neutralizing antibodies as the benchmark to measure vaccine effectiveness. However, recent developments in numerous related fields of biomedical research including, HIV, HSV and DENV have emphasized the importance of Fc-mediate effector functions in pathogenesis and immunity. The concept of Fc effector functions in contributing to protection from illness is not a new concept and has been investigated in the field for over four decades. However, in recent years the application and study of Fc effector functions has become revitalized with new knowledge and technologies to characterize their potential importance in immunity. In this perspective, we describe the current state of the field of Influenza Fc effector functions and discuss its potential utility in universal vaccine design in the future

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

© 2013 Dr. Sinthujan JegaskandaPublications included in thesis:Jegaskanda, S., Job, E. R., Kramski, M., Laurie, K., Isitman, G., De Rose, R., et al. (2012). Cross-reactive influenza-specific antibody-dependent cellular cytotoxicity antibodies in the absence of neutralizing antibodies. Journal of Immunology, 190(3), 1837-1848. DOI: 10.4049/jimmunol.1201574Jegaskanda, S., Weinfurter, J. T., Friedrich, T. C., & Kent, S. J. (2013). Antibody-dependent cellular cytotoxicity is associated with control of pandemic H1N1 influenza virus infection of macaques. Journal of Virology, 87(10), 5512-5522. DOI: 10.1128/JVI.03030-12Jegaskanda, S., Laurie, K., Amarasena, T. H., Winnall, W., Kramski, M., De Rose, R., et al. (in press 2013). Age-associated cross-reactive antibody-dependent cellular cytotoxicity toward 2009 pandemic influenza A virus subtype H1N1. Journal of Infectious Diseases. DOI: 10.1093/infdis/jit294Jegaskanda, S., Vandenberg, K., Rockman, S., & Kent, S. J. Cross-reactive influenza-specific antibody-dependent cellular cytotoxicity present in intravenous immunoglobulin. (Submitted to Journal of Immunology, August 2013)Jegaskanda, S., Amarasena, T. H., Laurie, K. L., Tan, H-X., Butler, J., Parsons, M. S., et al. (2013). Standard trivalent influenza protein vaccination does not prime antibody-dependent cellular cytotoxicity (ADCC) in macaques. Journal of Virology, 87(24), 13706-13718. DOI: 10.1128/JVI.01666-13Influenza virus is a major cause of morbidity and mortality worldwide. Yearly trivalent influenza vaccination partially reduces the severity of influenza infection. Seasonal influenza vaccines mediate their protective activity through the induction of neutralising antibodies towards the influenza virus surface hemagglutinin (HA) and neuraminidase (NA) glycoproteins. HA- and NA-specific antibodies act by either inhibiting virus entry or preventing the release of new virions from host cells, respectively. However, influenza-specific neutralising antibodies are susceptible to loss of recognition by accumulation of point mutations in the HA and NA over time (antigenic drift), or by the introduction of new viral HA and NA glycoproteins (antigenic shift). Fortunately, antibodies have a number of non-neutralising effector functions including antibody-dependent cellular cytotoxicity (ADCC). Non-neutralising antibody effector functions rely on binding of the antibody fragment antigen-binding (Fab) region to surface antigens, but mediate their effector activity through the fragment crystallisable (Fc) region of the antibody. ADCC in particular may play an important role in protection from influenza infection, but few studies have been undertaken in this area for over 3 decades. Previous studies on influenza-specific ADCC were performed using either chromium-release assays or in in vivo using FcR-knockout mice. Until recently no flow-cytometry based assay was available to measure and characterise influenza-specific ADCC-mediating antibodies in human and non-human primate sera samples. Using newly developed assays we showed that healthy individuals aged between 18-43 years of age did not have neutralising antibodies to a 1968 H3N2 influenza virus, but they did possess binding antibodies. Furthermore, a portion of these non-neutralising antibodies mediated ADCC effector functions including activating NK cells and eliminating virus-infected respiratory epithelial cells (Chapter 2). This highlighted that ADCC-mediating antibodies have broad cross-reactive capacity that may play an important role in protection against influenza virus infections. In April 2009, a swine-origin H1N1 influenza (A(H1N1)pdm09) virus caused a pandemic in the human population. The pandemic was estimated to have resulted in 4 million cases worldwide, but only lead to approximately 106,700-395,699 deaths. This was a comparatively milder influenza pandemic, as seasonal influenza epidemics generally lead to approximately 350,000-500,000 deaths worldwide. Moreover, yearly influenza epidemics generally cause deaths in younger (65 years old), particularly targeting individuals with major risk factors such as cardiovascular disease. In contrast, the 2009 H1N1 pandemic had a lower mortality in older individuals (>65 years old), with younger healthy individuals experiencing greater morbidity. These two separate observations suggested that there were two levels of protection detected in the population prior to the 2009 H1N1 pandemic: (1) the reduced severity of the 2009 H1N1 pandemic on the population and (2) the lower incidence of mortality in older individuals in the population. The potential role of cross-reactive ADCC-mediating antibodies in providing protection during the 2009 H1N1 pandemic was investigated. To determine if there was ADCC-mediated protection against A(H1N1)pdm09 virus in older individuals, we obtained human sera samples collected from separate individuals (aged between 1-72 years) prior to or following the 2009 H1N1 pandemic. We showed that individuals greater than 45 years of age had a moderate level of cross-reactive A(H1N1)pdm09-specific ADCC-mediating antibodies prior to the 2009 H1N1 pandemic (Chapter 3). To understand the role that ADCC-mediating antibodies may have played in reducing the severity of A(H1N1)pdm09 virus on the whole population, we obtained intravenous immunoglobulin (IVIG) manufactured either prior to the 2009 or post 2009 pandemic. We showed that there was a level of cross-reactive A(H1N1)pdm09-specific ADCC-mediating antibodies in the whole population prior to the 2009 H1N1 pandemic (Chapter 4). Lastly, to examine the mechanism by which cross-reactive A(H1N1)pdm09-specific ADCC-mediating antibodies are induced, we measured ADCC-mediating antibodies in naïve rhesus macaques (Macaca mulatta) infected with seasonal H1N1 influenza virus and subsequently challenged with the A(H1N1)pdm09 virus (Chapter 5). We showed that seasonal H1N1 infection of macaques induced cross-reactive ADCC-mediating antibodies towards the A(H1N1)pdm09 virus. Collectively, of these studies implicate ADCC in the protection observed during the 2009 H1N1 pandemic. The induction of influenza-specific ADCC-mediating antibodies by trivalent vaccines remains unknown. To investigate whether trivalent influenza vaccines can prime or induce ADCC-mediating antibodies we vaccinated influenza naïve pigtail macaques (Macaca nemestrina) with two doses of trivalent influenza vaccine (2012, A/California/07/2009 (H1N1), A/Perth/16/2009 (H3N2) and B/Brisbane/60/2008 (Type B), Sanofi Pasteur) and subsequently serially infected them with either H1N1 or H3N2 influenza strains. Our results showed that ADCC-mediating antibodies and cytotoxic T lymphocytes (CTL) responses were not primed or induced by vaccinations with seasonal trivalent influenza vaccine (Chapter 6). These studies suggested that the induction of ADCC-mediating antibodies by trivalent vaccination most likely relied on a background of previous influenza exposures. The design of new vaccines that induce broad ADCC-mediating antibodies may provide greater protection from divergent influenza strains and should be an important direction for future studies. Together, these studies serve as a basis for future research into influenza-specific ADCC immunity. The association of cross-reactive ADCC mediating antibodies with the protection observed during the 2009 H1N1 pandemic has important implications for the control of potential future influenza pandemics. The induction of broadly cross-reactivity ADCC mediating antibodies in nearly all individuals could be utilised for universal influenza vaccine design

Antibody-dependent cellular cytotoxicity and influenza virus

Antibodies are a key defence against influenza infection and disease, but neutralizing antibodies are often strain-specific and of limited utility against divergent or pandemic viruses. There is now considerable evidence that influenza-specific antibodies with Fc-mediated effector functions, such as antibody-dependent cellular cytotoxicity (ADCC), can assist in the clearance of influenza infection in vitro and in animal models. Further, ADCC-mediating antibodies that recognize a broad array of influenza strains are common in humans, likely as a result of being regularly exposed to influenza infections. The concept that influenza-specific ADCC can assist in the partial control of influenza infections in humans is gaining momentum. This review examines the utility of influenza specific ADCC antibodies

Poor protective potential of influenza nucleoprotein antibodies despite wide prevalence

Humans are exposed to influenza virus through periodic infections. Due to these repeated exposures, human populations commonly have elevated antibody titers targeting the conserved internal influenza virus nucleoprotein (NP). Despite the presence of anti-NP antibodies, humans are acutely susceptible to drifted influenza viruses with antigenically different surface proteins and the protective potential of human NP antibodies is unclear. In this study, high levels of anti-NP antibody and NP-specific B cells were detected in both adult humans and influenza-infected mice, confirming that NP is a major target of humoral immunity. Through sorting single B cells from influenza-exposed human adults, we generated a panel of 11 anti-NP monoclonal antibodies (mAbs). The majority of anti-NP human mAbs generated were capable of engaging cellular Fc receptors and bound NP on the surface of influenza-infected cell lines in vitro, suggesting that anti-NP mAbs have the potential to mediate downstream Fc effector functions such as antibody-dependent cellular cytotoxicity and antibody-dependent phagocytosis. However, human anti-NP mAbs were not protective in vivo when passively transferred into a murine influenza challenge model. Future in vivo studies examining the synergistic effect of anti-NP mAbs infused with other influenza-specific mAbs are warranted

Antibody-dependent cellular cytotoxicity responses to seasonal influenza vaccination in older adults

Background. Older adults are at high risk of influenza disease, but generally respond poorly to vaccination. Antibody-dependent cellular cytotoxicity (ADCC) may be an important component of protection against influenza infection. An improved understanding of the ADCC response to influenza vaccination in older adults is required. Methods. We studied sera samples from 3 groups of subjects aged >= 65 years (n = 16-17/group) receiving the 2008/2009 seasonal trivalent influenza vaccine (TIV). Subjects had minimal pre-existing hemagglutination inhibiting (HAI) antibodies and TIV induced either no, low, or high HAI responses. Serum ADCC activity was analyzed using Fc receptor cross-linking, NK cell activation, and influenza-infected cell killing. Results. Most subjects from TIV nonresponder, low responder, and high responder groups had detectable ADCC antibodies prevaccination, but baseline ADCC was not predictive of HAI vaccine responsiveness. Interestingly, ADCC and HAI responses tracked closely across all groups, against all 3 TIV hemagglutinins, and in all ADCC assays tested. Conclusions. Older adults commonly have pre-existing ADCC antibodies in the absence of high HAI titers to circulating influenza strains. In older vaccinees, ADCC response mirrored HAI antibodies and was readily detectable despite high postvaccination HAI titers. Alternate measures of vaccine responsiveness and improved vaccinations in this at-risk group are needed