Prophylactic and therapeutic vaccination with a nanoparticle-based peptide vaccine induces efficient protective immunity during acute and chronic retroviral infection

doi:10.1016/j.nano.2014.06.014

Nanomedicine: Nanotechnology, Biology and Medicine

Volume 10, Issue 8, November 2014, Pages 1787-1798

https://doi.org/10.1016/j.nano.2014.06.014 Get rights and content

Abstract

Retroviral infections e.g. HIV still represent a unique burden in the field of vaccine research. A common challenge in vaccine design is to find formulations that create appropriate immune responses to protect against and/or control the given pathogen. Nanoparticles have been considered to be ideal vaccination vehicles that mimic invading pathogens. In this study, we present biodegradable calcium phosphate (CaP) nanoparticles, functionalized with CpG and retroviral T cell epitopes of Friend virus (FV) as excellent vaccine delivery system. CaP nanoparticles strongly increased antigen delivery to antigen-presenting cells to elicit a highly efficient T cell-mediated immune response against retroviral FV infection. Moreover, single-shot immunization of chronically FV-infected mice with functionalized CaP nanoparticles efficiently reactivated effector T cells which led to a significant decrease in viral loads. Thus, our findings clearly indicate that a nanoparticle-based peptide immunization is a promising approach to improve antiretroviral vaccination.

From the Clinical Editor

In this study, biodegradable calcium phosphate nanoparticles were used as a vaccine delivery system after functionalization with CpG and Friend virus-derived T-cell epitopes. This vaccination strategy resulted in increased T-cell mediated immune response even in chronically infected mice, providing a promising approach to the development of clinically useful antiretroviral vaccination strategies.

Section snippets

Background

The primary goal in vaccine design is to find an immunogenic formulation which sufficiently activates innate and adaptive immunity to generate a protective immune response. Nanoparticles are ideal antigen delivery carriers to target cells or organs for therapeutic purposes. The nanoparticulate character enhances the uptake by antigen-presenting cells (APCs) of the immune system (dendritic cells; DCs) and improves the biodistribution of the antigen.¹ They can be loaded with drugs or biomolecules

Ethics statement

Animal experiments were performed in strict accordance with the German regulations of the Society for Laboratory Animal Science (GV-SOLAS) and the European Health Law of the Federation of Laboratory Animal Science Associations (FELASA). The protocol was approved by the North Rhine-Westphalia State Agency for Nature, Environment and Consumer Protection (LANUV) (Permit number: G G1107/10). All efforts were made to minimize animal suffering.

Mice

CB6F1 hybrid mice (H2^b/d FV2^r/s Rfv3^r/s) were obtained

Characterization of functionalized CaP nanoparticles

Functionalized triple-shell CaP nanoparticles were prepared by subsequent precipitation and functionalization steps as described earlier (Figure 1, A).8, 9 The CaP solid cores were loaded with CpG and CD8⁺ or CD4⁺ epitope peptides (GagL_85–93 or Env gp70_123–141) of FV and finally coated with a second layer of CaP and finally an outer shell of CpG for colloidal stabilization. The goal of this process was to protect bioactive molecules from early degradation (e.g. by nucleases or proteases) within

Discussion

Even after several decades of vaccine research, there is still no protective vaccine in sight against persistently infecting viruses such as HCV or HIV. Protein-based vaccines or adenoviral vectors are commonly used to induce antibody or cytotoxic T cell responses but rarely lead to a sufficient protective effect.30, 31 Therefore, there is a strong need to develop new strategies for prophylactic or therapeutic vaccination. The aim of our study was to determine whether functionalized CaP

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The results demonstrate the high potential of CaP NPs as a vaccine delivery tool for human usage, which could target different subsets of human DCs and lead to a potent virus-specific CD8+ T cell responses [228]. CaP NPs as vaccine delivery nanocarriers are also evaluated in terms of the prevention for chronic retroviral infection and colon cancer control via eliciting a highly efficient T cells-mediated immunity [231,232]. As an important form of innovative drug preparation, several nanomedicines have been approved and marketed at present, such as Doxil, Abraxane and Genexol-PM.
After centuries of development, using vaccination to stimulate immunity has become an effective method for prevention and treatment of a variety of diseases including infective diseases and cancers. However, the tailor-made efficient delivery system for specific antigens is still urgently needed due to the low immunogenicity and stability of antigens, especially for vaccines to induce CD8⁺ T cells-mediated cellular immunity. Unlike B cells-mediated humoral immunity, CD8⁺ T cells-mediated cellular immunity mainly aims at the intracellular antigens from microorganism in virus-infected cells or genetic mutations in tumor cells. Therefore, the vaccines for stimulating CD8⁺ T cells-mediated cellular immunity should deliver the antigens efficiently into the cytoplasm of antigen presenting cells (APCs) to form major histocompatibility complex I (MHCI)-antigen complex through cross-presentation, followed by activating CD8⁺ T cells for immune protection and clearance. Importantly, nanotechnology has been emerged as a powerful tool to facilitate these multiple processes specifically, allowing not only enhanced antigen immunogenicity and stability but also APCs-targeted delivery and elevated cross-presentation. This review summarizes the process of CD8⁺ T cells-mediated cellular immunity induced by vaccines and the technical advantages of nanotechnology implementation in general, then provides an overview of the whole spectrum of nanocarriers studied so far and the recent development of delivery nanotechnology in vaccines against infectious diseases and cancer. Finally, we look forward to the future development of nanotechnology for the next generation of vaccines to induce CD8⁺ T cells-mediated cellular immunity.
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Among the different nanoparticulate delivery systems [7] like magnetite [8], gold [9], chitosan [10], calcium phosphate nanoparticles are very attractive as they combine high biocompatibility with a good biodegradability, have a small size and high affinity to nucleic acids and can be loaded or functionalized on the surface with different biomolecules [11-17]. We have demonstrated earlier that functionalized calcium phosphate nanoparticles are a versatile carrier for small and big molecules (including nucleic acids) both in vitro and in vivo and a promising tool for immunization, e.g. for prophylactic and therapeutic immunization [18-22] and for specific B-cell activation [23-25]. Together with their high biocompatibility and biodegradability, they should be well suited for an in vivo application, also in humans.
Calcium phosphate nanoparticles were loaded with plasmid DNA and toll-like receptor ligands (TLR), i.e. CpG or flagellin, to activate antigen-presenting cells (APCs) like dendritic cells (DCs). The functionalized nanoparticles were studied in vitro on HeLa, C2C12 and BHK-21 cell lines, focusing on the expression of two specific proteins. EGFP-DNA, encoding for enhanced green fluorescent protein (EGFP), was used as a model plasmid to optimize the transfection efficiency in vitro by fluorescence microscopy and flow cytometry. Calcium phosphate nanoparticles loaded with TLR ligands and plasmid DNA encoding for the hepatitis B virus surface antigen (pHBsAg) were evaluated by in vitro and in vivo immunization experiments to identify a possible candidate for a prophylactic hepatitis B virus (HBV) vaccine. The nanoparticles induced a strong expression of HBsAg in the three cell lines. In splenocytes, the expression of the co-stimulatory molecules CD80 and CD86 was enhanced. After intramuscular injection in mice, the nanoparticles induced the expression of HBsAg, the antigen-specific T cell response, and the antigen-specific antibody response (IgG1).
Hepatitis B is one of the most frequent viral infections worldwide. For preventive immunization, nanoparticles can be used which carry both an adjuvant (a stimulatory molecule) and DNA encoding for a viral antigen. After administration of such nanoparticles to cells, they are taken up by cells where the DNA is transcribed into the viral antigen (a protein). This viral antigen is inducing a virus-specific immune response. This was shown both by in vitro cell culture as well as by an extensive in vivo study in mice.
Induction of herpes simplex virus type 1 cell-to-cell spread inhibiting antibodies by a calcium phosphate nanoparticle-based vaccine
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Additionally, Alum and MPL, also known as the GlaxoSmithKline adjuvant system AS04, were used as adjuvants to additionally modulate the adaptive immune response.40 The major advantages of CaP nanoparticles over other nanoparticle-based delivery systems such as virus-like particles are their biodegradability and biocompatibility as CaP is the inorganic component of human hard tissues i.e. bone and teeth.41–43 The calcium phosphate nanoparticles are taken up by the cells via endocytosis and end up in an endolysosome.
Herpes simplex viruses 1 and 2 are among the most ubiquitous human infections and persist lifelong in their host. Upon primary infection or reactivation from ganglia, the viruses spread by direct cell–cell contacts (cell-to-cell spread) and thus escape from the host immune response. We have developed a monoclonal antibody (mAb 2c), which inhibits the HSV cell-to-cell spread, thereby protecting from lethal genital infection and blindness in animal models. In the present study we have designed a nanoparticle-based vaccine to induce protective antibody responses exceeding the cell-to-cell spread inhibiting properties of mAb 2c. We used biodegradable calcium phosphate (CaP) nanoparticles coated with a synthetic peptide that represents the conformational epitope on HSV-1 gB recognized by mAb 2c. The CaP nanoparticles additionally contained a TLR-ligand CpG^m and were formulated with adjuvants to facilitate the humoral immune response. This vaccine effectively protected mice from lethal HSV-1 infection by inducing cell-to-cell spread inhibiting antibodies.
A systematic electron microscopic study on the uptake of barium sulphate nano-, submicro-, microparticles by bone marrow-derived phagocytosing cells
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Nanoparticles can act as transporters for synthetic molecules and biomolecules into cells, also in immunology. Antigen-presenting cells like dendritic cells are important targets for immunotherapy in nanomedicine. Therefore, we have used primary murine bone marrow-derived phagocytosing cells (bmPCs), i.e. dendritic cells and macrophages, to study their interaction with spherical barium sulphate particles of different size (40 nm, 420 nm, and 1 µm) and to follow their uptake pathway. Barium sulphate is chemically and biologically inert (no dissolution, no catalytic effects), i.e. we can separate the particle uptake effect from potential biological reactions. The colloidal stabilization of the nanoparticles was achieved by a layer of carboxymethylcellulose (CMC) which is biologically inert and gives the particles a negative zeta potential (i.e. charge). The particles were made fluorescent by conjugating 6-aminofluoresceine to CMC. Their uptake was visualized by flow cytometry, confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and correlative light and electron microscopy (CLEM). Barium sulphate particles of all sizes were readily taken up by dendritic cells and even more by macrophages, with the uptake increasing with time and particle concentration. They were mainly localized inside phagosomes, heterophagosomes, and in the case of nanoparticles also in the nearby cytosol. No particles were found in the nucleus. In nanomedicine, inorganic nanoparticles from the nanometer to the micrometer size are therefore well suited as transporters of biomolecules, including antigens, into dendritic cells and macrophages. The presented model system may also serve to describe the aseptic loosening of endoprostheses caused by abrasive wear of inert particles and the subsequent cell reaction, a question which relates to the field of nanotoxicology.
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Calcium phosphate nanoparticles are easily taken up by cells and subsequently dissolved in lysosomes [31–33]. We have demonstrated that multi-shell calcium phosphate nanoparticles are very versatile in immunology, e.g. for prophylactic and therapeutic immunization [34–38] and for specific B-cell activation [13,39]. For many viral infections, an effective prophylactic vaccine is not available (e.g. for human immunodeficiency virus [HIV] and C [HCV]) [40–42].
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: Funding Sources: This work was supported by the Deutsche Forschungsgemeinschaft (SFB/Transregio 60 to WB, CJK, UD, ME, JB, and AMW).

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Feature ArticleProphylactic and therapeutic vaccination with a nanoparticle-based peptide vaccine induces efficient protective immunity during acute and chronic retroviral infection

Abstract

From the Clinical Editor

Section snippets

Background

Ethics statement

Mice

Characterization of functionalized CaP nanoparticles

Discussion

Adv Drug Deliv Rev

Int J Pharm

J Control Release

Biomaterials

Virology

J Pharm Sci

Trends Immunol

Curr Opin Immunol

Blood

Immunity

Acta Biomater

Vaccine

J Virol Methods

Blood

Biomaterials

Adv Drug Deliv Rev

Adv Drug Deliv Rev

Biomaterials

Blood

Nanomedicine–challenge and perspectives

Angew Chem Int Ed Engl

Nanoparticles and the immune system

Endocrinology

Virus-like particle (VLP) lymphatic trafficking and immune response generation after immunization by different routes

J Immunother

Endocytosis at the nanoscale

Chem Soc Rev

Immunization with biodegradable nanoparticles efficiently induces cellular immunity and protects against influenza virus infection

J Immunol

Immune responses against persistent viral infections: possible avenues for immunotherapeutic interventions

Crit Rev Immunol

Feature Article
Prophylactic and therapeutic vaccination with a nanoparticle-based peptide vaccine induces efficient protective immunity during acute and chronic retroviral infection