Establishment and characterization of biodegradable calcium phosphate nanoparticles for the induction of potent anti-viral T cell responses

Die immunologischen Abwehrmechanismen gegen Infektionskrankheiten sind sehr komplex. Dabei sind die verschiedenen Bereiche des Immunsystems gegen die Infektion einzelner Pathogene unterschiedlich effektiv. Während neutralisierende Antikörper schützend gegen einige Virusinfektionen wirken, sind sie gegen andere, wie z.B. HIV Infektionen, nicht ausreichend. Dies ist unter anderem in der Morphologie und Pathologie des jeweiligen Virus begründet. Die Möglichkeit Pathogen-spezifische T-Zellantworten zu induzieren ist in solchen Fällen für neuartige Vakzine von besonderer Wichtigkeit. Nanopartikuläre Ansätze werden dabei als sehr effektive Immunisierungsvehikel angesehen. Die partikuläre Struktur der Nanopartikel, sowie die Funktionalisierung mit immunogenen Strukturen von Bakterien oder Viren, ergeben ein Pathogen-Mimikry, welches eine Pathogen-ähnliche Aktivierung des angeborenen Immunsystems und damit Initiierung einer Immunantwort erlaubt. In dieser Arbeit wurde der Einsatz von biologisch abbaubaren Calciumphosphat (CaP) Nanopartikeln als Immunisierungsvehikel untersucht. CaP Nanopartikel wurden mit Toll-like Rezeptor (TLR)-Liganden CpG (TLR9) oder poly(I:C) (TLR3), sowie viralen Antigenen funktionalisiert und die schnelle Aufnahme dieser Nanopartikel durch Dendritische Zellen (DCs) gezeigt. Die Aufnahme der Nanopartikel in vitro führte zur Reifung der DCs und zur Induktion einer Virus-spezifischen T-Zell-Antwort. Immunisierungsexperimente mit funktionalisierten CaP Nanopartikeln gegen das Influenza Virus und das retrovirale Friend Virus zeigen deutlich, dass es zur Induktion Virus-spezifischer IFN-γ-produzierender CD4+ und CD8+ Effektor T-Zellen kommt. Die anti-virale Funktion dieser Zellen wurde in anschließenden Infektionsexperimenten gezeigt. Sowohl die Influenza-Viruslast als auch die Friend-Viruslast konnte signifikant reduziert werden. Die erzielten Ergebnisse zeigen das Potential des neu entwickelten Nanopartikel-Systems für den Einsatz in der prophylaktischen Vakzinierung sehr deutlich. Einige virale Infektionen resultieren jedoch nicht in der vollständigen Klärung der Pathogene und können zu chronisch persistierenden Infektionen führen. Immunologische Toleranzmechanismen führen dabei oft zu Beeinträchtigung der Immunantwort. Deshalb ist hier die Reaktivierung der Pathogen-spezifischen Immunantwort eines der wichtigen Ziele einer therapeutischen Immunisierung. Im chronischen FV-Mausmodell wurde demonstriert, dass die einmalige therapeutische Immunisierung mit funktionalisierten CaP Nanopartikeln zu einer signifikanten Reduktion der Viruslast führt. Dabei scheint die Reaktivierung von Virus-spezifischen CD8+ T-Zellen und die Reduktion regulatorischer T-Zellen einen wichtigen Beitrag zu leisten. Zusammenfassend machen die Ergebnisse dieser Arbeit sehr deutlich, dass sich CaP Nanopartikel hervorragend als Immunisierungsvehikel eignen. Die umfangreichen Funktionalisierungsmöglichkeiten dieses Systems erlauben zusätzlich einen sehr flexiblen Einsatz dieser Nanopartikel gegen verschiedene akute und chronische Infektionen.The immunological mechanisms against infectious diseases are very complex. The different parts of the immune system thereby show diverse effectivity against several pathogens. Due to morphology and pathology neutralizing antibody responses may protect against some viral infections but fail to prevent infection with others, like HIV. The ability of vaccines to induce T cell responses is crucial for preventing diseases caused by most intracellular pathogens like viruses or bacteria. Nanoparticles are considered as potent vaccination tools in new vaccine designs, since they mimic invading pathogens by their particular structure and can be functionalized with immunogenic parts of bacteria or viruses. The stimulation of certain specialized receptors with these structures therefore leads to an activation of the innate immune system. This study describes a novel vaccination approach with biodegradable calcium phosphate (CaP) nanoparticles functionalized with immunoactive Toll-like receptor 9 ligand (CpG) or Toll-like receptor 3 ligand poly(I:C) co-encapsulating virus derived peptides. Functionalized CaP nanoparticles are rapidly taken up by dendritic cells (DCs) which lead to successful maturation of DCs and the induction of virus-specific T cell responses. Immunization with functionalized CaP nanoparticles against influenza virus or the retroviral Friend virus elicited a potent T cell-mediated immune response with high numbers of anti-viral IFN-γ-producing CD4+ and CD8+ effector T cells. Challenge experiments demonstrated a significant reduction of viral loads. The results of this study demonstrate the high potential of these nanoparticles as novel and flexible tool for prophylactic vaccination. However, many viral infections do not result in pathogen clearance but rather in viral persistence and the development of a chronic state of infection. The immunological mechanisms of tolerance have a great impact on the immune response. Thus, the reactivation of the pathogen-specific immune responses is one of the main goals during therapeutic vaccination. Single shot immunization with functionalized CaP nanoparticles was sufficient to significantly decrease viral load in persistent Friend retroviral infection. Especially the reactivation of virus-specific CD8+ T cells and the reduction of regulatory T cells seem to mediate this effect. Taken together, these results recommend functionalized CaP nanoparticles as a novel immunization tool. The possibility of further modifications implies their use for several prophylactic and therapeutic immunizations

Combination immunotherapy with anti-PD-L1 antibody and depletion of regulatory T cells during acute viral infections results in improved virus control but lethal immunopathology

Combination immunotherapy (CIT) is currently applied as a treatment for different cancers and is proposed as a cure strategy for chronic viral infections. Whether such therapies are efficient during an acute infection remains elusive. To address this, inhibitory receptors were blocked and regulatory T cells depleted in acutely Friend retrovirus-infected mice. CIT resulted in a dramatic expansion of cytotoxic CD4+ and CD8+ T cells and a subsequent reduction in viral loads. Despite limited viral replication, mice developed fatal immunopathology after CIT. The pathology was most severe in the gastrointestinal tract and was mediated by granzyme B producing CD4+ and CD8+ T cells. A similar post-CIT pathology during acute Influenza virus infection of mice was observed, which could be prevented by vaccination. Melanoma patients who developed immune-related adverse events under immune checkpoint CIT also presented with expanded granzyme-expressing CD4+ and CD8+ T cell populations. Our data suggest that acute infections may induce immunopathology in patients treated with CIT, and that effective measures for infection prevention should be applied

CD47 restricts antiviral function of alveolar macrophages during influenza virus infection

CD47 is an ubiquitously expressed surface molecule with significant impact on immune responses. However, its role for antiviral immunity is not fully understood. Here, we revealed that the expression of CD47 on immune cells seemed to disturb the antiviral immune response as CD47-deficient mice (CD47−/−) showed an augmented clearance of influenza A virus (IAV). Specifically, we have shown that enhanced viral clearance is mediated by alveolar macrophages (aMФ). Although aMФ displayed upregulation of CD47 expression during IAV infection in wildtype mice, depletion of aMФ in CD47−/− mice during IAV infection reversed the augmented viral clearance. We have also demonstrated that CD47 restricts hemoglobin (HB) expression in aMФ after IAV and severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection, with HB showing antiviral properties by enhancing the IFN-β response. Our study showed a negative role for CD47 during antiviral immune responses in the lung by confining HB expression in aMФ

Virus-induced type I interferon deteriorates control of systemic pseudomonas aeruginosa infection

BACKGROUND: Type I interferon (IFN-I) predisposes to bacterial superinfections, an important problem during viral infection or treatment with interferon-alpha (IFN-α). IFN-I-induced neutropenia is one reason for the impaired bacterial control; however there is evidence that more frequent bacterial infections during IFN-α-treatment occur independently of neutropenia. METHODS: We analyzed in a mouse model, whether Pseudomonas aeruginosa control is influenced by co-infection with the lymphocytic choriomeningitis virus (LCMV). Bacterial titers, numbers of neutrophils and the gene-expression of liver-lysozyme-2 were determined during a 24 hours systemic infection with P. aeruginosa in wild-type and Ifnar(-/-) mice under the influence of LCMV or poly(I:C). RESULTS: Virus-induced IFN-I impaired the control of Pseudomonas aeruginosa. This was associated with neutropenia and loss of lysozyme-2-expression in the liver, which had captured P. aeruginosa. A lower release of IFN-I by poly(I:C)-injection also impaired the bacterial control in the liver and reduced the expression of liver-lysozyme-2. Low concentration of IFN-I after infection with a virulent strain of P. aeruginosa alone impaired the bacterial control and reduced lysozyme-2-expression in the liver as well. CONCLUSION: We found that during systemic infection with P. aeruginosa Kupffer cells quickly controlled the bacteria in cooperation with neutrophils. Upon LCMV-infection this cooperation was disturbed

Influenza virus infection enhances tumour-specific CD8+ T-cell immunity, facilitating tumour control.

Influenza A virus (IAV) can cause severe respiratory infection leading to significant global morbidity and mortality through seasonal epidemics. Likewise, the constantly increasing number of cancer diseases is a growing problem. Nevertheless, the understanding of the mutual interactions of the immune responses between cancer and infection is still very vague. Therefore, it is important to understand the immunological cross talk between cancer and IAV infection. In several preclinical mouse models of cancer, including melanoma and colorectal cancer, we observed that IAV infection in the lung significantly decreased the tumour burden. Concomitantly, tumour-specific CD8+ T-cells are strongly activated upon infection, both in the tumour tissue and in the lung. CD8+ T-cell depletion during infection reverses the reduced tumour growth. Interestingly, IAV infection orchestrated the migration of tumour-specific CD8+ T-cells from the tumour into the infected lung. Blocking the migration of CD8+ T-cells prevented the anti-tumoural effect. Thus, our findings show that viral respiratory infection has significant impact on the anti-tumour CD8+ T-cell response, which will significantly improve our understanding of the immunological cross talk between cancer and infection

Combination of nanoparticle-based therapeutic vaccination and transient ablation of regulatory T cells enhances anti-viral immunity during chronic retroviral infection.

Regulatory T cells (Tregs) have been shown to limit anti-viral immunity during chronic retroviral infection and to restrict vaccine-induced T cell responses. The objective of the study was to assess whether a combinational therapy of nanoparticle-based therapeutic vaccination and concomitant transient ablation of Tregs augments anti-viral immunity and improves virus control in chronically retrovirus-infected mice. Therefore, chronically Friend retrovirus (FV)-infected mice were immunized with calcium phosphate (CaP) nanoparticles functionalized with TLR9 ligand CpG and CD8(+) or CD4(+) T cell epitope peptides (GagL85-93 or Env gp70123-141) of FV. In addition, Tregs were ablated during the immunization process. Reactivation of CD4(+) and CD8(+) effector T cells was analysed and the viral loads were determined

Induction of Type I Interferons by Therapeutic Nanoparticle-Based Vaccination Is Indispensable to Reinforce Cytotoxic CD8+ T Cell Responses During Chronic Retroviral Infection

T cell dysfunction and immunosuppression are characteristic for chronic viral infections and contribute to viral persistence. Overcoming these burdens is the goal of new therapeutic strategies to cure chronic infectious diseases. We recently described that therapeutic vaccination of chronic retrovirus infected mice with a calcium phosphate (CaP) nanoparticle (NP)-based vaccine carrier, functionalized with CpG and viral peptides is able to efficiently reactivate the CD8+ T cell response and improve the eradication of virus infected cells. However, the mechanisms underlying this effect were largely unclear. While type I interferons (IFNs I) are considered to drive T cell exhaustion by persistent immune activation during chronic viral infection, we here describe an indispensable role of IFN I induced by therapeutic vaccination to efficiently reinforce cytotoxic CD8+ T cells (CTL) and improve control of chronic retroviral infection. The induction of IFN I is CpG dependent and leads to significant IFN signaling indicated by upregulation of IFN stimulated genes. By vaccinating chronically retrovirus-infected mice lacking the IFN I receptor (IFNAR−/−) or by blocking IFN I signaling in vivo during therapeutic vaccination, we demonstrate that IFN I signaling is necessary to drive full reactivation of CTLs. Surprisingly, we also identified an impaired suppressive capability of regulatory T cells in the presence of IFNα, which implicates an important role for vaccine-induced IFNα in the regulation of the T cell response during chronic retroviral infection. Our data suggest that inducing IFN I signaling in conjunction with the presentation of viral antigens can reactivate immune functions and reduce viral loads in chronic infections. Therefore, we propose CaP NPs as potential therapeutic tool to treat chronic infections

CD4⁺ T cells are not altered in the tumour upon infection.

B16 tumours were transplanted and mice were infected as described in Fig 1. Tumours were analysed by flow cytometry 12 days post transplantation. (a) Frequencies of viable CD4+ T cells. (b) Frequencies of CD43 or Granzyme B (GzmB) of CD43 expressing cells of CD4+ T cells. Data from 2 experiments are shown. Error bars represent SEM. (TIF)</p

IAV infection suppresses tumour growth.

(a-d) 1 x 105 B16F1 (B16) tumour cells were transplanted subcutaneously (s.c.) into the right flank per mouse. 4 days after tumour transplantation, mice were infected intranasally with 150 PFU/mL Influenza A/PR/8/34 (IAV). (a) Viral load was measured by M1 expression relative to RPS9 expression in the lungs of infected mice with or without B16 tumour 8 days post infection (dpi). n = 9 for B16+IAV, n = 6 for IAV (b) Viral load as measured from homogenized lung supernatants by counting plaque forming units (PFU) from plaque assay 8 dpi. (c) Body weight change after IAV infection. Data from 2 experiments with 4–6 mice per group per experiment. (d-g) 1 x 105 B16 (d and g), CT26 (e) or 5 x 105 Lewis Lung Carcinoma (LLC) (f) tumour cells were transplanted s.c. and mice were infected as described in a) with (d-f) IAV or (g) inactivated IAV. The tumour volume was measured everyday once palpable upon infection. d&e: Data from 4 experiments with 3–4 mice per group per experiment, f: n = 4, g: Data from 2 experiments with 4 mice per group per experiment. (h) Viral load as measured by M1 expression relative to RPS9 expression in the tumour or lung of infected mice 12 days post tumour transplantation. (i) B16 tumours were transplanted s.c. with 1 x 105 tumour cells and mice were infected intravenously with Friend Virus (FV) 4 days post tumour transplantation. The tumour volume was scored everyday once palpable. n = 4. Error bars represent SEM. Statistical tests on tumour growth development were performed as Two-way-ANOVA, in Sidak’s multiple comparisons test when two groups were compared or Tukey’s multiple comparisons test when more than two groups were compared. Viral titres were compared with non-parametric t tests. * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001, ns = not significant.</p