Dominik Damm

Infectious diseases are a predominant problem in human health. While the incidence of many pathogenic infections is controlled by vaccines, some pathogens still pose a challenging task for vaccine researchers. In order to face these challenges, the field of vaccine development has changed tremendously over the last few years. For non-replicating recombinant antigens, novel vaccine delivery systems that attempt to increase the immunogenicity by mimicking structural properties of pathogens are already approved for clinical applications. Lipid-based nanoparticles (LbNPs) of different natures are vesicles made of lipid layers with aqueous cavities, which may carry antigens and other biomolecules either displayed on the surface or encapsulated in the cavity. However, the efficacy profile of recombinant LbNP vaccines is not as high as that of live-attenuated ones. This review gives a compendious picture of two approaches that affect the immunogenicity of recombinant LbNP vaccines: (i) the incorporation of immunostimulatory agents and (ii) the utilization of pre-existing or promiscuous cellular immunity, which might be beneficial for the development of tailored prophylactic and therapeutic LbNP vaccine candidates.

Functionalization of experimental HIV-1 virus-like particle vaccines with heterologous T helper epitopes (T helper VLPs) can modulate the humoral immune response via intrastructural help (ISH). Current advances in the conjugation of native-like HIV-1 envelope trimers (Env) onto liposomes and encapsulation of peptide epitopes into these nanoparticles renders this GMP-scalable liposomal platform a feasible alternative to VLP-based vaccines. In this study, we designed and analyzed customizable Env-conjugated T helper liposomes. First, we passively encapsulated T helper peptides into a well-characterized liposome formulation displaying a dense array of Env trimers on the surface. We confirmed the closed pre-fusion state of the coupled Env trimers by immunogold staining with conformation-specific antibodies. These peptide-loaded Env-liposome conjugates efficiently activated Env-specific B cells, which further induced proliferation of CD4+ T cells by presentation of liposome-derived peptides on MHC-II molecules. The peptide encapsulation process was then quantitatively improved by an electrostatically driven approach using an overall anionic lipid formulation. We demonstrated that peptides delivered by liposomes were presented by DCs in secondary lymphoid organs after intramuscular immunization of mice. UFO (uncleaved prefusion optimized) Env trimers were covalently coupled to peptide-loaded anionic liposomes by His-tag/NTA(Ni) interactions and EDC/Sulfo-NHS crosslinking. EM imaging revealed a moderately dense array of well-folded Env trimers on the liposomal surface. The conformation was verified by liposomal surface FACS. Furthermore, anionic Env-coupled T helper liposomes effectively induced Env-specific B cell activation and proliferation in a comparable range to T helper VLPs. Taken together, we demonstrated that T helper VLPs can be substituted with customizable and GMP-scalable liposomal nanoparticles as a perspective for future preclinical and clinical HIV vaccine applications. The functional nanoparticle characterization assays shown in this study can be applied to other systems of synthetic nanoparticles delivering antigens derived from various pathogens.

The display of native-like human immunodeficiency virus type 1 envelope (HIV-1 Env) trimers on liposomes has gained wide attention over the last few years. Currently, available methods have enabled the preparation of Env-liposome conjugates of unprecedented quality. However, these protocols require the Env trimer to be tagged and/or to carry a specific functional group. For this reason, we have investigated N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide/N-Hydroxysulfosuccinimide (EDC/Sulfo-NHS) chemistry for its potential to covalently conjugate tag-free, non-functionalized native-like Env trimers onto the surface of carboxyl-functionalized liposomes. The preservation of the liposome’s physical integrity and the immunogen’s conformation required a fine-tuned two-step approach based on the controlled use of β-mercaptoethanol. The display of Env trimers was strictly limited to activated liposomes of positive charge, i.e., liposomes with a positive zeta potential that carry amine-reactive Sulfo-NHS esters on their surface. In agreement with that, conjugation was found to be highly ionic strength- and pH-dependent. Overall, we have identified electrostatic pre-concentration (i.e., close proximity between negatively charged Env trimers and positively charged liposomes established through electrostatic attraction) to be crucial for conjugation reactions to proceed. The present study highlights the requirements and limitations of potentially scalable EDC/Sulfo-NHS-based approaches and represents a solid basis for further research into the controlled conjugation of tag-free, non-functionalized native-like Env trimers on the surface of liposomes, and other nanoparticles.

Due to the low efficacy and the need for seasonal adaptation of currently licensed influenza A vaccines, the importance of alternative vaccination strategies is increasingly recognized. Considering that DNA vaccines can be rapidly manufactured and readily adapted with novel antigen sequences, genetic vaccination is a promising immunization platform. However, the applicability of different genetic adjuvants to this approach still represents a complex challenge. Immune checkpoints are a class of molecules involved in adaptive immune responses and germinal center reactions. In this study, we immunized mice by intramuscular electroporation with a DNA-vaccine encoding hemagglutinin (HA) and nucleoprotein (NP) of the influenza A virus. The DNA-vaccine was applied either alone or in combination with genetic adjuvants encoding the soluble ectodomains of programmed cell death protein-1 (sPD-1) or its ligand (sPD-L1). Co-administration of genetic checkpoint adjuvants did not significantly alter immune responses against NP. In contrast, sPD-1 co-electroporation elevated HA-specific CD4⁺ T cell responses, decreased regulatory CD4⁺ T cell pools, and modulated the IgG2a-biased HA antibody pattern towards an isotype-balanced IgG response with a trend to higher influenza neutralization in vitro. Taken together, our data demonstrate that a genetic DNA-adjuvant encoding soluble ectodomains of sPD-1 was able to modulate immune responses induced by a co-administered influenza DNA vaccine.

The importance of a balanced T_H1/T_H2 humoral immune response against the HIV-1 envelope protein (Env) for antibody-mediated HIV-1 control is increasingly recognized. However, there is no defined vaccination strategy to raise it. Since immune checkpoints are involved in the induction of adoptive immunity and their inhibitors (monoclonal antibodies) are licensed for cancer therapy, we investigated the effect of checkpoint blockade after HIV-1 genetic vaccination on enhancement and modulation of antiviral antibody responses. By intraperitoneal administration of checkpoint antibodies in mice we observed an induction of anti-drug antibodies which may interfere with immunomodulation by checkpoint inhibitors. Therefore, we blocked immune checkpoints locally by co-electroporation of DNA vaccines encoding the active soluble ectodomains of programmed cell death protein-1 (PD-1) or its ligand (PD-L1), respectively. Plasmid-encoded immune checkpoints did not elicit a detectable antibody response, suggesting no interference with their immunomodulatory effects. Co-electroporation of a HIV-1 DNA vaccine formulation with soluble PD-L1 ectodomain increased HIV-1 Env-specific T_H1 CD4 T cell and IgG2a antibody responses. The overall antibody response was hereby shifted towards a more T_H1/T_H2 balanced subtype pattern. These findings indicate that co-electroporation of soluble checkpoint ectodomains together with DNA-based vaccines has modulatory effects on vaccine-induced immune responses that could improve vaccine efficacies.

Since the first use of liposomes as carriers for antigens, much work has been done to elucidate the mechanisms involved in the encapsulation of vaccine-relevant biomolecules. However, only a few studies have specifically investigated the encapsulation of hydrophilic, non-conformational peptide epitopes. We performed comprehensive and systematic screening studies, in order to identify conditions that favor the electrostatic interaction of such peptides with lipid membranes. Moreover, we have explored bi-terminal sequence extension as an approach to modify the isoelectric point of peptides, in order to modulate their membrane binding behavior and eventually shift/expand the working range under which they can be efficiently encapsulated in an electrostatically driven manner. The findings of our membrane interaction studies were then applied to preparing peptide-loaded liposomes. Our results show that the magnitude of membrane binding observed in our exploratory in situ setup translates to corresponding levels of encapsulation efficiency in both of the two most commonly employed methods for the preparation of liposomes, i.e., thin-film hydration and microfluidic mixing. We believe that the methods and findings described in the present studies will be of use to a wide audience and can be applied to address the ongoing relevant issue of the efficient encapsulation of hydrophilic biomolecules.

Incorporation of immunodominant T-helper epitopes of licensed vaccines into virus-like particles (VLP) allows to harness T-helper cells induced by the licensed vaccines to provide intrastructural help (ISH) for B-cell responses against the surface proteins of the VLPs. To explore whether ISH could also improve antibody responses to calcium phosphate (CaP) nanoparticle vaccines we loaded the nanoparticle core with a universal T-helper epitope of Tetanus toxoid (p30) and functionalized the surface of CaP nanoparticles with stabilized trimers of the HIV-1 envelope (Env) resulting in Env-CaP-p30 nanoparticles. In contrast to soluble Env trimers, Env containing CaP nanoparticles induced activation of naïve Env-specific B-cells in vitro. Mice previously vaccinated against Tetanus raised stronger humoral immune responses against Env after immunization with Env-CaP-p30 than mice not vaccinated against Tetanus. The enhancing effect of ISH on anti-Env antibody levels was not attended with increased Env-specific IFN-γ CD4 T-cell responses that otherwise may potentially influence the susceptibility to HIV-1 infection. Thus, CaP nanoparticles functionalized with stabilized HIV-1 Env trimers and heterologous T-helper epitopes are able to recruit heterologous T-helper cells induced by a licensed vaccine and improve anti-Env antibody responses by intrastructural help.

The great advantage of virus-like particle (VLP) nano-vaccines is their structural identity to wild-type viruses, ensuring that antigen-specific B-cells encounter viral proteins in their natural conformation. “Wild-type” viral nanoparticles can be further genetically or biochemically functionalized with biomolecules (antigens and adjuvants). Flagellin is a potent inducer of innate immunity and it has demonstrated adjuvant effectiveness due to its affinity for toll-like receptor 5 (TLR5). In contrast to most TLR ligands, flagellin is a protein and can induce an immune response against itself. To avoid side-effects, we incorporated a less inflammatory and less immunogenic form of flagellin as an adjuvant into HIV-based nanoparticle B-cell-targeting vaccines that display either the HIV-1 envelope protein (Env) or a model antigen, hen egg lysozyme (HEL). While flagellin significantly enhanced HEL-specific IgG responses, anti-Env antibody responses were suppressed. We demonstrated that flagellin did not activate B-cells directly in vitro, but might compete for CD4+ T-cell help in vivo. Therefore, we hypothesize that in the context of VLP-based B-cell nano-vaccines, flagellin serves as an antigen itself and may outcompete a less immunogenic antigen with its antibody response. In contrast, in combination with a strong immunogen, the adjuvant activity of flagellin may dominate over its immunogenicity.