Virus-Like Particles of the Murine Leukemia Virus

A Novel Modular Vaccine-Platform?

  • Fig. 1: Schematic illustration of VLP. MLV-derived modular VLP generated within this study present the model antigen OVA and the adjuvant GM-CSF on their surface. “Naked” particles, which display none of these proteins, were used as control for all experiments. Non-modular VLP display either OVA or GM-CSF on their surface. Modular VLP display OVA and GM-CSF on their surface, simultaneously. Fig. 1: Schematic illustration of VLP. MLV-derived modular VLP generated within this study present the model antigen OVA and the adjuvant GM-CSF on their surface. “Naked” particles, which display none of these proteins, were used as control for all experiments. Non-modular VLP display either OVA or GM-CSF on their surface. Modular VLP display OVA and GM-CSF on their surface, simultaneously.
  • Fig. 1: Schematic illustration of VLP. MLV-derived modular VLP generated within this study present the model antigen OVA and the adjuvant GM-CSF on their surface. “Naked” particles, which display none of these proteins, were used as control for all experiments. Non-modular VLP display either OVA or GM-CSF on their surface. Modular VLP display OVA and GM-CSF on their surface, simultaneously.
  • Fig. 2: Effects of modular VLP on primary immune cells. Incubation of primary immune cells with GM-CSF-displaying VLP results in the expansion of CD11b+ cells no matter whether VLP display GM-CSF alone or along with OVA. Proliferation of antigen-specific T cells is significantly enhanced when primary spleen cells were incubated with modular VLP. Proliferation was less pronounced if cell cultures were incubated with a mixture of non-modular-VLP presenting either OVA or GM-CSF.

The fight against infectious diseases remains a challenge. Some pathogens, for which few therapeutics are available and which can cause severe diseases, can still not be prevented by vaccinations. At the same time, some established vaccination strategies would profit from optimization of their safety and efficacy-profile (i.e. the induction of long-lasting protection while being safe) as well as of their production processes. Hence, the development of novel vaccination strategies remains an important scientific task.

Modular vaccines are defined by the physical conjugation of the two main vaccine-components, the antigen and the adjuvant. It has been shown that such conjugation can remarkably enhance the immunogenicity of the vaccine. Apparently, the pivotal mechanism of action is provided by the simultaneous targeting of a given antigen presenting cell (APC) by these two components. Thereby, the adjuvant will induce APC-activation, while the co-delivered antigen is taken up. Antigen-uptake, -processing, and -presentation can thus be enhanced by the adjuvant-mediated activation. Finally, this can result in enhanced adaptive immune responses [1].

Virus-Like Particles of the Murine Leukemia Virus

In this project, virus-like particles (VLP) of the Murine leukemia virus (MLV) have been investigated as novel platform for modular vaccination. These VLP do not contain any genomic material or viral surface proteins. Hence, they are not infectious and cannot replicate. VLP produced for this project present antigen or adjuvant as fusion proteins with a transmembrane domain. Thereby, both are incorporated into the VLP envelope and presented on the particle surface (model in fig. 1). These particles combine two features which are known to contribute to enhanced immunogenicity: On the one hand, the simultaneous presentation of antigen and adjuvant and, on the other hand, the particulate structure of the VLP. Additionally, the production process of these particles enables a rather simple exchange of antigen and adjuvant by replacement of the respective modular components. Thereby, specificity and mode of APC-activation by the platform can be adjusted quickly.

In this study, we used ovalbumin (OVA) as well-established model-antigen and granulocyte-macrophage colony-stimulating factor (GM-CSF) as “non-classical” adjuvant to be displayed on VLP.

GM-CSF is an endogenous cytokine. Its receptor is expressed i.a. by cells of the myeloid lineage, which include dendritic cells (DC). DC present antigens on their major histocompatibility complexes (MHC) and can induce activation of antigen-specific T cells. Thus, DC are at the interface between the innate and adaptive immune system. Consequently, targeting vaccines to DC can increase the efficacy of the former significantly [2,3]. In addition, our adjuvant of choice, GMCSF, induces differentiation and proliferation of DC, which can also contribute to an enhanced immune response.

Effects of Modular VLP on Primary Immune Cells

To confirm the biological activity of GM-CSF presented on the VLP, undifferentiated murine bone marrow cells as source of APC-precursors were incubated with GM-CSF-displaying VLP. Regardless of whether GM-CSF was displayed alone or along with OVA, differentiation and expansion of cells expressing the myeloid surface marker CD11b could be observed. This biological activity of GM-CSF was verified within total spleen cell cultures. The spleen is a secondary lymphoid organ harboring numerous different immune cells including DC, DC-precursors, and T cells. Therefore, total spleen cell cultures were used for further experiments.

Next, we investigated whether VLP-mediated expansion of APC translates into an adaptive immune response. However, the endogenous frequency for a given subset of antigen-specific T cells within the naïve murine system is 1:10,000 - 1:100,000 of the total T cell repertoire [4], which makes antigen-specific T cell responses hardly detectable. Hence, we used spleen cell cultures of transgenic mice (OT-I and OT-II mice), whose T cells express an OVA-specific T cell receptor (TCR) on their killer-T cells (TK) or helper-T cells (TH). Stimulation of mixed OT-I/OT-II cell cultures with VLP, co-displaying GM-CSF and OVA, induced strong OVA-specific T cell activation and proliferation. The effects of an equimolar mixture of non-modular VLP displaying either GM-CSF or OVA, were significantly lower (fig. 2). These experiments confirm that such VLP could provide a potential platform for modular vaccination.

Since VLP are not infectious, we expected phagocytic up-take of these particles by APCs, followed by processing in the endosome, and presentation on MHC class II that stimulate antigen-specific TH cells. Indeed, OVA-specific TH strongly proliferated upon incubation of splenocyte cultures with modular VLP, while TH cell activation was significantly decreased when the cells were incubated with the equimolar, non-modular VLP-mixture. In contrast, we could not show activation of OVA-specific TK by modular VLP, if no OVA-specific TH cells were present during stimulation. Thus, we exclude that these particles are cross-presented in vitro.

These results draw the following picture: Modular VLP are taken up as extracellular antigen by APCs, which were probably targeted, but definitively differentiated and stimulated for proliferation by GM-CSF. The co-delivered antigen OVA is processed endosomally to be presented on MHC class II. Thereby, mainly OVA-specific TH cells are activated, which in turn help to activate TK of the same specificity.

Future immunization experiments with mice will indicate the efficacy and the protective potential of the modular VLP when used as vaccine. If this is successful, the VLP generated here indeed provide an attractive platform for novel modular vaccination strategies. The modular conception potentially enables a rapid exchange of the antigen/adjuvant-composition on the used VLP-backbone which could give rise to tailored vaccines against numerous pathogens.

Affiliations
1 Paul-Ehrlich-Institut, Bundesinstitut für Impfstoffe und biomedizinische Arzneimittel, Langen, Germany
2 Now: University Medical Center of the Johannes Gutenberg University Mainz, Institute for Virology, Mainz, Germany

Authors
Patricia Gogesch1, Michael D. Mühlebach1, Zoe Waibler1

Kontakt
Prof. Dr. Zoe Waibler

Paul-Ehrlich-Institut
Bundesinstitut für Impfstoffe und biomedizinische Arzneimittel
Fachgebiet: Produktprüfung immunologischer Arzneimittel
Langen, Germany
zoe.waibler@pei.de
www.pei.de/zoe-waibler
 

References

[1] Schülke, Stefan; Wolfheimer, Sonja; Gadermaier, Gabriele; Wangorsch, Andrea; Siebeneicher, Susanne; Briza, Peter et al. (2014): Prevention of intestinal allergy in mice by rflaA:Ova is associated with enforced antigen processing and TLR5-dependent IL-10 secretion by mDC. In: PloS one 9 (2), e87822. DOI: 10.1371/journal.pone.0087822.
[2] Gardner, Thomas A.; Elzey, Bennett D.; Hahn, Noah M. (2012): Sipuleucel-T (Provenge) autologous vaccine approved for treatment of men with asymptomatic or minimally symptomatic castrate-resistant metastatic prostate cancer. In: Human vaccines & immunotherapeutics 8 (4), S. 534–539. DOI: 10.4161/hv.19795.
[3] Yin, Wenjie; Gorvel, Laurent; Zurawski, Sandra; Li, Dapeng; Ni, Ling; Duluc, Dorothée et al. (2016): Functional Specialty of CD40 and Dendritic Cell Surface Lectins for Exogenous Antigen Presentation to CD8(+) and CD4(+) T Cells. In: EBioMedicine 5, S. 46–58. DOI: 10.1016/j.ebiom.2016.01.02
[4] Jenkins, Marc K.; Moon, James J. (2012): The role of naive T cell precursor frequency and recruitment in dictating immune response magnitude. In: Journal of immunology (Baltimore, Md. : 1950) 188 (9), S. 4135–4140. DOI: 10.4049/jimmunol.1102661.

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