RESUMO
Neutralization by antibodies and complement limits the effective dose and thus the therapeutic efficacy of oncolytic viruses after systemic application. We and others previously showed that pseudotyping of oncolytic rhabdoviruses such as maraba virus and vesicular stomatitis virus (VSV) with the lymphocytic choriomeningitis virus glycoprotein (LCMV-GP) results in only a weak induction of neutralizing antibodies. Moreover, LCMV-GP-pseudotyped VSV (VSV-GP) was significantly more stable in normal human serum (NHS) than VSV. Here, we demonstrate that depending on the cell line used for virus production, VSV-GP showed different complement sensitivities in nonimmune NHS. The NHS-mediated titer reduction of VSV-GP was dependent on activation of the classical complement pathway, mainly by natural IgM antibodies against xenoantigens such as galactose-α-(1,3)-galactose (α-Gal) or N-glycolylneuraminic acid (Neu5Gc) expressed on nonhuman production cell lines. VSV-GP produced on human cell lines was stable in NHS. However, VSV-GP generated in transduced human cells expressing α-Gal became sensitive to NHS. Furthermore, GP-specific antibodies induced complement-mediated neutralization of VSV-GP independently of the producer cell line, suggesting that complement regulatory proteins potentially acquired by the virus during the budding process are not sufficient to rescue the virus from antibody-dependent complement-mediated lysis. Thus, our study points to the importance of a careful selection of cell lines for viral vector production for clinical use.IMPORTANCE Systemic application aims to deliver oncolytic viruses to tumors as well as to metastatic lesions. However, we found that xenoantigens incorporated onto the viral surface from nonhuman production cell lines are recognized by natural antibodies in human serum and that the virus is thereby inactivated by complement lysis. Hence, to maximize the effective dose, careful selection of cell lines for virus production is crucial.
Assuntos
Vírus da Coriomeningite Linfocítica/imunologia , Estomatite Vesicular/imunologia , Vírus da Estomatite Vesicular Indiana/imunologia , Células A549 , Animais , Anticorpos Neutralizantes/imunologia , Antígenos Heterófilos/imunologia , Linhagem Celular , Chlorocebus aethiops , Proteínas do Sistema Complemento/imunologia , Cricetinae , Vetores Genéticos , Glicoproteínas/genética , Humanos , Camundongos , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/metabolismo , Vírus da Estomatite Vesicular Indiana/genética , Vírus da Estomatite Vesicular Indiana/fisiologia , Vesiculovirus/genéticaRESUMO
Mucosal-associated invariant T (MAIT) cells are an abundant human T-cell subset with antimicrobial properties. They can respond to bacteria presented via antigen-presenting cells (APCs) such as macrophages, which present bacterially derived ligands from the riboflavin synthesis pathway on MR1. Moreover, MAIT cells are also highly responsive to cytokines which enhance and even substitute for T-cell receptor-mediated signaling. The mechanisms leading to an efficient presentation of bacteria to MAIT cells by APCs have not been fully elucidated. Here, we showed that the monocytic cell line THP-1 and B cells activated MAIT cells differentially in response to Escherichia coli. THP-1 cells were generally more potent in inducing IFNγ and IFNγ/TNF production by MAIT cells. Furthermore, THP-1, but not B, cells produced TNF upon bacterial stimulation, which in turn supported IFNγ production by MAIT cells. Finally, we addressed the role of antibody-dependent opsonization of bacteria in the activation of MAIT cells using in vitro models. We found that opsonization had a substantial impact on downstream MAIT cell activation by monocytes. This was associated with enhanced activation of monocytes and increased TNF release. Importantly, this TNF acted in concert with other cytokines to drive MAIT cell activation. These data indicate both a significant interaction between adaptive and innate immunity in the response to bacteria, and an important role for TNF in MAIT cell triggering.
Assuntos
Linfócitos B/imunologia , Infecções por Escherichia coli/imunologia , Escherichia coli/fisiologia , Monócitos/imunologia , Células T Invariantes Associadas à Mucosa/imunologia , Imunidade Adaptativa , Anticorpos Antibacterianos/metabolismo , Apresentação de Antígeno , Humanos , Imunidade Inata , Interferon gama/metabolismo , Ativação Linfocitária , Proteínas Opsonizantes/metabolismo , Fagocitose , Transdução de Sinais , Células THP-1 , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The antitumor activity of monoclonal antibodies in the treatment of chronic lymphocytic leukemia is mediated mainly by antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. Unfortunately, the efficacy of complement-dependent cytotoxicity is strongly restricted due to the expression and acquisition of regulators of complement activation by lymphocytic leukemia cells. Whereas the role of membrane regulators of complement activation, such as CD55 and CD59, has been investigated in detail in chronic lymphocytic leukemia, the involvement of soluble regulators of complement activation, such as complement factor H, has not yet been reported. Propidium iodide staining was performed to investigate the efficacy of ofatumumab and factor H-derived short-consensus-repeat 18-20 in the induction of complement-dependent cytotoxicity on primary chronic lymphocytic leukemia cells from 20 patients. Deposition of complement C3 fragments was monitored by western blot analysis. Expression of CD20, CD55 or CD59 was determined by FACS analysis. Replacement of factor H with short consensus repeat 18-20 significantly increased the susceptibility of primary chronic lymphocytic leukemia cells to ofatumumab-induced complement-dependent cytotoxicity. More importantly, addition of short-consensus-repeat 18-20 was able to overcome complement- resistance occurring during treatment with ofatumumab alone. Use of short consensus repeat 18-20 is likely to prolong the turnover time of active C3b fragments generated on the target cells following ofatumumab-induced complement activation, thereby improving specific killing of chronic lymphocytic leukemia cells by complement-dependent cytotoxicity. The relative contribution of factor H to the protection of chronic lymphocytic leukemia cells against complement-dependent cytotoxicity was comparable to that of CD55. Our data suggest that, by abrogating factor H function, short consensus repeat 18-20 may provide a novel approach that improves the complement-dependent efficacy of therapeutic monoclonal antibodies.
Assuntos
Anticorpos Monoclonais/farmacologia , Fator H do Complemento/farmacologia , Sequência Consenso/efeitos dos fármacos , Citotoxinas/farmacologia , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados , Células Cultivadas , Fator H do Complemento/genética , Sequência Consenso/genética , Citotoxinas/uso terapêutico , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Humanos , Leucemia Linfocítica Crônica de Células B/patologiaRESUMO
N-chlorotaurine (NCT) a long-lived oxidant generated by leukocytes, can be synthesized chemically and applied topically as an anti-infective to different body sites, including the lung via inhalation. Here, we demonstrate the activity of NCT against viruses causing acute respiratory tract infections, namely severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza viruses, and respiratory syncytial virus (RSV). Virucidal activity of NCT was tested in plaque assays, confirmed by RT-qPCR assays. Attack on virus proteins was investigated by mass spectrometry. NCT revealed broad virucidal activity against all viruses tested at 37°C and pH 7. A significant reduction in infectious particles of SARS-CoV-2 isolates from early 2020 by 1 log10 was detected after 15â min of incubation in 1% NCT. Proteinaceous material simulating body fluids enhanced this activity by transchlorination mechanisms (1 -2 log10 reduction within 1-10â min). Tested SARS-CoV-2 variants B.1.1.7 (Alpha) und B.1.351 (Beta) showed a similar susceptibility. Influenza virus infectious particles were reduced by 3 log10 (H3N2) to 5 log10 (H1N1pdm), RSV by 4 log10 within a few min. Mass spectrometry of NCT-treated SARS-CoV-2 spike protein and 3C-like protease, influenza virus haemagglutinin and neuraminidase, and RSV fusion glycoprotein disclosed multiple sites of chlorination and oxidation as the molecular mechanism of action. Application of 1.0% NCT as a prophylactic and therapeutic strategy against acute viral respiratory tract infections deserves comprehensive clinical investigation.
Assuntos
Tratamento Farmacológico da COVID-19 , Infecções Respiratórias , Humanos , Vírus da Influenza A Subtipo H3N2 , Vírus Sinciciais Respiratórios , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Taurina/análogos & derivadosRESUMO
Dendritic cells (DCs) express Fcγ receptors (FcγRs) for the binding immune complexes (ICs) consisting of IgG and antigens (Ags). ICâ»FcγR interactions have been demonstrated to enhance activation and antigen-presenting functions of DCs. Utilizing Friend virus (FV), an oncogenic mouse retrovirus, we investigated the effect of IgG-opsonization of retroviral particles on the infection of DCs and the subsequent presentation of viral antigens by DCs to virus-specific CD8 T cells. We found that opsonization by virus-specific non-neutralizing IgG abrogated DC infection and as a consequence significantly reduced the capacity of DCs to activate virus-specific CD8 T cells. Effects of IgG-opsonization were mediated by the high-affinity FcγR type I, CD64, expressed on DCs. Our results suggest that different opsonization patterns on the retroviral surface modulate infection and antigen-presenting functions of DCs, whereby, in contrast to complement, IgG reduces the capacity of DCs to activate cytotoxic T cell (CTL) responses.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Vírus da Leucemia Murina de Friend/imunologia , Ativação Linfocitária , Receptores de IgG/imunologia , Animais , Apresentação de Antígeno , Complexo Antígeno-Anticorpo/imunologia , Células Dendríticas/virologia , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Receptores de IgG/genéticaRESUMO
During (nearly) all steps in retroviral pathogenesis, viruses are confronted with complement and complement receptor (CR)-positive cells. As all of the retroviruses tested so far activate the complement system, members of this virus family have adapted different protection mechanisms to keep complement activation under the threshold necessary to avoid complement-mediated lysis. As a consequence of complement activation, retroviruses are covered with complement proteins and thus provide additional ligands to interact with CR-expressing cells. This review discusses the complex complement-retroviral interactions and follows the fate of the virus on its way to the lymphatic tissue.
Assuntos
Ativação do Complemento , Tecido Linfoide/virologia , Mucosa/virologia , Infecções por Retroviridae/imunologia , Infecções por Retroviridae/virologia , Retroviridae/patogenicidade , Animais , Proteínas do Sistema Complemento/metabolismo , Humanos , Tecido Linfoide/imunologia , Mucosa/imunologia , Receptores de Complemento/metabolismoRESUMO
Our study demonstrates that binding of complement-opsonized HIV to complement receptor type 1 on human erythrocytes (E) via C3b fragments is followed by a rapid normal human serum-mediated detachment of HIV from E. The release was dependent on the presence of factor I indicating a conversion of C3b fragments to iC3b and C3d on the viral surface. This in turn resulted in an efficient binding of opsonized HIV to CR2-expressing B cells, thus facilitating B cell-mediated transmission of HIV to T cells. These data provide a new dynamic view of complement opsonization of HIV, suggesting that association of virus with E might be a transient phenomenon and the factor I-mediated processing of C3b to iC3b and C3d on HIV targets the virus to complement receptor type 2-expressing cells. Thus, factor I in concert with CR1 on E and factor H in serum due to their cofactor activity are likely to be important contributors for the generation of C3d-opsonized infectious HIV reservoirs on follicular dendritic cells and/or B cells in HIV-infected individuals.
Assuntos
Linfócitos B/virologia , Proteínas do Sistema Complemento/imunologia , Fibrinogênio/metabolismo , Infecções por HIV/imunologia , HIV/imunologia , Receptores de Complemento 3d/imunologia , Linfócitos T/virologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Proteínas do Sistema Complemento/química , Proteínas do Sistema Complemento/metabolismo , Infecções por HIV/virologia , Humanos , Cinética , Soro , Linfócitos T/imunologiaRESUMO
From the site of transmission at mucosal surfaces, HIV is thought to be transported by DCs to lymphoid tissues. To initiate migration, HIV needs to activate DCs. This activation, reflected by intra- and extracellular changes in cell phenotype, is investigated in the present study. In two-thirds of the donors, R5- and X4-tropic HIV-1 strains induced partial up-regulation of DC activation markers such as CD83 and CD86. In addition, CCR7 expression was increased. HIV-1 initiated a transient phosphorylation of p44/p42 ERK1/2 in iDCs, whereas p38 MAPK was activated in both iDCs and mDCs. Up-regulation of CD83 and CD86 on DCs was blocked when cells were incubated with specific p38 MAPK inhibitors before HIV-1-addition. CCR7 expression induced by HIV-1 was sufficient to initiate migration of DCs in the presence of secondary lymphoid tissue chemokine (CCL21) and MIP-3beta (CCL19). Preincubation of DCs with a p38 MAPK inhibitor blocked CCR7-dependent DC migration. Migrating DCs were able to induce infection of autologous unstimulated PBLs in the Transwell system. These data indicate that HIV-1 triggers a cell-specific signaling machinery, thereby manipulating DCs to migrate along a chemokine gradient, which results in productive infection of nonstimulated CD4(+) cells.
Assuntos
Movimento Celular/imunologia , Células Dendríticas/enzimologia , Células Dendríticas/virologia , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , HIV-1/imunologia , Sistema de Sinalização das MAP Quinases/imunologia , Monócitos/citologia , Antígenos de Diferenciação Mielomonocítica/biossíntese , Linfócitos T CD4-Positivos/virologia , Diferenciação Celular/imunologia , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/citologia , Células Dendríticas/imunologia , Ativação Enzimática/imunologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , Fosforilação , Receptores CCR7 , Receptores de Quimiocinas/biossíntese , Regulação para Cima/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologiaRESUMO
Dendritic cells (DC) represent a class of professional antigen-presenting cells whose primary function is to alert the immune system, not to clear invading microorganisms. The objective of our study was to compare the abilities of polymorphonuclear neutrophilic leukocytes (PMN), monocytes, monocyte-derived macrophages (MDM), monocyte-derived immature DC (imDC), and mature DC (maDC) to ingest and destroy Staphylococcus aureus and Escherichia coli. Acridine orange staining and fluorescence microscopy demonstrated that MDM, followed by monocytes, imDC, and PMN, internalized bacteria well but that maDC exhibited less pronounced phagocytic activity. PMN, monocytes, and MDM exhibited a much higher capacity to kill ingested bacteria than both imDC and maDC. In summary, these data are in agreement with the generally accepted idea that different types of leukocytes fulfill specialized tasks in antigen presentation and killing of pathogens.
Assuntos
Atividade Bactericida do Sangue , Células Dendríticas/imunologia , Fagócitos/imunologia , Fagocitose , Humanos , Monócitos/imunologia , Neutrófilos/imunologiaRESUMO
Dendritic cells (DC) represent a unique set of APCs that initiate immune responses through priming of naive T cells. Maturation of DC is a crucial step during Ag presentation and can be induced by triggering a broad spectrum of DC surface receptors. Although human DC express several receptors for the Fc portion of IgG which were described to play an important role in Ag internalization, little is known about the effects of IgG or immune complexes on DC maturation. In this study, we show that cross-linking of FcgammaR-type II (CD32) with immobilized IgG (imIgG) can induce maturation of human monocyte-derived DC via the NF-kappaB signaling pathway. IgG-mediated maturation was accompanied by a moderate increase of IL-10 secretion, whereas no IL-12 production was observed. Involvement of CD32 was further supported by experiments with the anti-CD32 mAb, which blocked IgG-triggered DC maturation and cytokine secretion significantly. Furthermore, DC cultivated in the presence of imIgG induced allogeneic T cell proliferation. Because this imIgG-induced maturation was considerably impaired in monocyte-derived DC from systemic lupus erythematosus patients, we suggest that DC, which matured in the presence of immune complexes, may contribute to prevention of pathological immune responses.