Coexpression of GM-CSF and antigen in DNA prime-adenoviral vector boost immunization enhances polyfunctional CD8+ T cell responses, whereas expression of GM-CSF antigen fusion protein induces autoimmunity

<p>Abstract</p> <p>Background</p> <p>Granulocyte-macrophage colony-stimulating factor (GM-CSF) has shown promising results as a cytokine adjuvant for antiviral vaccines and in various models of tumor gene therapy. To explore whether the targeting of antigens to GM-CSF receptors on antigen-presenting cells enhances antigen-specific CD8 T-cell responses, fusion proteins of GM-CSF and ovalbumin (OVA) were expressed by DNA and adenoviral vector vaccines. In addition, bicistronic vectors allowing independent expression of the antigen and the cytokine were tested in parallel.</p> <p>Results</p> <p><it>In vitro</it>, the GM-CSF ovalbumin fusion protein (GM-OVA) led to the better stimulation of OVA-specific CD8+ T cells by antigen-presenting cells than OVA and GM-CSF given as two separate proteins. However, prime-boost immunizations of mice with DNA and adenoviral vector vaccines encoding GM-OVA suppressed CD8+ T-cell responses to OVA. OVA-specific IgG2a antibody levels were also reduced, while the IgG1 antibody response was enhanced. Suppression of CD8+ T cell responses by GM-OVA vaccines was associated with the induction of neutralizing antibodies to GM-CSF. In contrast, the coexpression of GM-CSF and antigens in DNA prime adenoviral boost immunizations led to a striking expansion of polyfunctional OVA-specific CD8+ T cells without the induction of autoantibodies.</p> <p>Conclusion</p> <p>The induction of autoantibodies suggests a general note of caution regarding the use of highly immunogenic viral vector vaccines encoding fusion proteins between antigens and host proteins. In contrast, the expansion of polyfunctional OVA-specific CD8+ T cells after immunizations with bicistronic vectors further support a potential application of GM-CSF as an adjuvant for heterologous prime-boost regimens with genetic vaccines. Since DNA prime adenoviral vector boost regimenes are presently considered as one of the most efficient ways to induce CD8+ T cell responses in mice, non-human primates and humans, further enhancement of this response by GM-CSF is a striking observation.</p

Distinct roles of CD4+ T cell subpopulations in retroviral immunity: lessons from the Friend virus mouse model

It is well established that CD4+ T cells play an important role in immunity to infections with retroviruses such as HIV. However, in recent years CD4+ T cells have been subdivided into several distinct populations that are differentially regulated and perform widely varying functions. Thus, it is important to delineate the separate roles of these subsets, which range from direct antiviral activities to potent immunosuppression. In this review, we discuss contributions from the major CD4+ T cell subpopulations to retroviral immunity. Fundamental concepts obtained from studies on numerous viral infections are presented along with a more detailed analysis of studies on murine Friend virus. The relevance of these studies to HIV immunology and immunotherapy is reviewed

The Cytotoxic Activity of Natural Killer Cells Is Suppressed by IL-10+ Regulatory T Cells During Acute Retroviral Infection

Natural killer (NK) cells play a key role in host defense against cancer and viral infections. It was shown that NK cells are important for the control of acute retroviral infections, but their antiviral activity depends on multiple parameters such as viral inoculation dose, interactions with myeloid cell types and the cytokine milieu. In addition, during an ongoing retroviral infection regulatory T cells (Tregs) can suppress NK cell functions. However, the precise role of Tregs on the initial NK cell response and their immediate antiviral activity after an acute retroviral infection is still unknown. Here we show that thymus-derived Tregs suppress the proliferation, effector functions and cytotoxicity of NK cells very early during acute Friend Retrovirus (FV) infection. Tregs exhibited an activated phenotype and increased the production of the immunosuppressive cytokines IL-10 and TGF-β after FV infection of mice. Neutralization of the immunosuppressive cytokine IL-10 resulted in a significant augmentation of NK cell functions. Although the activation of dendritic cells (DCs) and macrophages as well as the IL-15 cytokine levels were increased after Treg depletion, Tregs mainly affect the NK cell activity in an IL-10-regulated pathway. In this study we demonstrate an IL-10-dependent suppression of NK cells by activated Tregs during the first days of a retroviral infection

The PD-1/PD-L1 Pathway Affects the Expansion and Function of Cytotoxic CD8+ T Cells During an Acute Retroviral Infection

Cytotoxic CD8+ T lymphocytes (CTL) efficiently control acute virus infections but can become exhausted when a chronic infection develops. The checkpoint receptor PD-1 suppresses the functionality of virus-specific CD8+ T cells during chronic infection. However, the role of the PD-L1/PD-1 pathway during the acute phase of infections has not been well characterized. In the current study the effects of PD-1 or PD-L1 deficiency on the CD8+ T cell response against Friend retroviral (FV) infection of knockout mice was analyzed during acute infection. We observed an enhanced proliferation, functional maturation, and reduced apoptosis of effector CD8+ T cells in the absence of PD-1 or PD-L1. The knockout of PD-L1 had a stronger effect on the functionality of CD8+ T cells than that of PD-1. Augmented CTL responses were associated with an improved control of FV replication. The strong phenotype of FV-infected PD-L1 knockout mice was independent of the interaction with CD80 as an additional receptor for PD-L1. Furthermore, we performed a detailed analysis of the production of different granzymes in virus-specific CD8+ T cells and observed that especially the simultaneous production of multiple granzymes in individual T cells (multifunctionality) was under the control of the PD-1/PD-L1 pathway. The findings from this study allow for a better understanding of the development of antiviral cytotoxic immunity during acute viral infections

Identification of herbal teas and their compounds eliciting antiviral activity against SARS-CoV-2 in vitro

Background: The SARS-CoV-2/COVID-19 pandemic has inflicted medical and socioeconomic havoc, and despite the current availability of vaccines and broad implementation of vaccination programs, more easily accessible and cost-effective acute treatment options preventing morbidity and mortality are urgently needed. Herbal teas have historically and recurrently been applied as self-medication for prophylaxis, therapy, and symptom alleviation in diverse diseases, including those caused by respiratory viruses, and have provided sources of natural products as basis for the development of therapeutic agents. To identify affordable, ubiquitously available, and effective treatments, we tested herbs consumed worldwide as herbal teas regarding their antiviral activity against SARS-CoV-2. Results: Aqueous infusions prepared by boiling leaves of the Lamiaceae perilla and sage elicit potent and sustained antiviral activity against SARS-CoV-2 when applied after infection as well as prior to infection of cells. The herbal infusions exerted in vitro antiviral effects comparable to interferon-β and remdesivir but outperformed convalescent sera and interferon-α2 upon short-term treatment early after infection. Based on protein fractionation analyses, we identified caffeic acid, perilla aldehyde, and perillyl alcohol as antiviral compounds. Global mass spectrometry (MS) analyses performed comparatively in two different cell culture infection models revealed changes of the proteome upon treatment with herbal infusions and provided insights into the mode of action. As inferred by the MS data, induction of heme oxygenase 1 (HMOX-1) was confirmed as effector mechanism by the antiviral activity of the HMOX-1-inducing compounds sulforaphane and fraxetin. Conclusions: In conclusion, herbal teas based on perilla and sage exhibit antiviral activity against SARS-CoV-2 including variants of concern such as Alpha, Beta, Delta, and Omicron, and we identified HMOX-1 as potential therapeutic target. Given that perilla and sage have been suggested as treatment options for various diseases, our dataset may constitute a valuable resource also for future research beyond virology

Immune Response in Moderate to Critical Breakthrough COVID-19 Infection After mRNA Vaccination

SARS-CoV-2 variants of concern (VOCs) can trigger severe endemic waves and vaccine breakthrough infections (VBI). We analyzed the cellular and humoral immune response in 8 patients infected with the alpha variant, resulting in moderate to fatal COVID-19 disease manifestation, after double mRNA-based anti-SARS-CoV-2 vaccination. In contrast to the uninfected vaccinated control cohort, the diseased individuals had no detectable high-avidity spike (S)-reactive CD4+ and CD8+ T cells against the alpha variant and wild type (WT) at disease onset, whereas a robust CD4+ T-cell response against the N- and M-proteins was generated. Furthermore, a delayed alpha S-reactive high-avidity CD4+ T-cell response was mounted during disease progression. Compared to the vaccinated control donors, these patients also had lower neutralizing antibody titers against the alpha variant at disease onset. The delayed development of alpha S-specific cellular and humoral immunity upon VBI indicates reduced immunogenicity against the S-protein of the alpha VOC, while there was a higher and earlier N- and M-reactive T-cell response. Our findings do not undermine the current vaccination strategies but underline a potential need for the inclusion of VBI patients in alternative vaccination strategies and additional antigenic targets in next-generation SARS-CoV-2 vaccines

Fatty acids are crucial to fuel NK cells upon acute retrovirus infection

Natural killer (NK) cells are cytotoxic innate immune cells, able to recognize and eliminate virus-infected as well as cancer cells. Metabolic reprogramming is crucial for their activity as they have enhanced energy and nutritional demands for their functions during an infection. Fatty acids (FAs) represent an important source of cellular energy and are essential for proliferation of immune cells. However, the precise role of FAs for NK cells activity in retrovirus infection was unknown. Here we show that activated NK cells increase the expression of the FA uptake receptor CD36 and subsequently the uptake of FAs upon acute virus infection. We found an enhanced flexibility of NK cells to utilize FAs as source of energy compare to naïve NK cells. NK cells that were able to generate energy from FAs showed an augmented target cell killing and increased expression of cytotoxic parameters. However, NK cells that were unable to generate energy from FAs exhibited a severely decreased migratory capacity. Our results demonstrate that NK cells require FAs in order to fight acute virus infection. Susceptibility to severe virus infections as it is shown for people with malnutrition may be augmented by defects in the FA processing machinery, which might be a target to therapeutically boost NK cell functions in the future

Woodchuck hepatitis virus core antigen-based DNA and protein vaccines induce qualitatively different immune responses that affect T cell recall responses and antiviral effects

AbstractT helper type 1 (Th1) immunity was considered to play a dominant role in viral clearance of hepadnaviral infection. However, pre-primed Th2 type responses were able to efficiently control hepadnaviral infection in animal models. We investigated how pre-primed Th1/2 responses control hepadnaviral replication using the newly established mouse models. DNA (pWHcIm, pCTLA-4-C) and protein vaccines based on the nucleocapsid protein (WHcAg) of woodchuck hepatitis virus (WHV) primed specific immune responses with distinct features. The pre-primed responses determined the characteristics of recall responses if challenged with a WHcAg-expressing adenoviral vector. Vaccination with pWHcIm and pCTLA4-C facilitated viral control in the hydrodynamic injection model and reduced WHV loads by about 3 and 2 logs in WHV-transgenic mice, respectively, despite of different kinetics of specific CD8+ T cell responses. Thus, pre-primed Th2-biased responses facilitate the development of CD8+ T cell responses in mice compared with naïve controls and thereby confer better viral control