With annual HIV infection rates still exceeding two million people a year, development of an HIV vaccine remains a world-wide urgency. The HIV vaccine research group is therefore exploring novel vaccination strategies and aims at a better understanding of immune responses that are induced by HIV or are required for protection. Currently, the following projects are pursued:
Antigen-specific T helper cells provide critical signals for cognate B-cells to differentiate into memory B-cells and plasma cells. In the context of particulate vaccines composed of different proteins, T helper cell epitopes and B-cell epitopes do not need be covalently linked. HIV-Env specific B-cells for example can internalize virus-like particles (VLPs) containing HIV Env and Gag. This leads to presentation of Env- and Gag-derived peptides on their MHC-II molecules explaining our observation that Gag-specific T helper cells can provide help for Env-specific antibody responses. In mouse experiments, Gag-specific T helper cells not only enhanced anti-Env IgG levels up to 100-fold but also modulated the IgG subtype response. A major aim of our research is therefore to develop novel vaccine platforms that exploit T helper cells induced by common licensed vaccines to improve the antibody response to heterologous viral antigens.
As a proof-of-concept, we introduced T helper cell epitopes of tetanus toxoid (TT) into HIV VLPs and immunized naïve mice or mice that had been immunized by Tetanol, a commercially available Tetanus vaccine, with HIV VLPs. Much higher HIV Env IgG1 antibody responses were observed in Tetanol-immunized mice that received VLPs containing the TT T helper cell epitopes than in naïve mice receiving the same TT-VLPs or Tetanol-primed mice receiving VLPs lacking the TT epitopes. Since the IgG1 subtype response induced in Tetanol-primed mice has less efficient Fc-effector functions we also identified T helper cell epitopes induced by HBVAXPRO, a licensed hepatitis B vaccine, and incorporated them into HIV VLPs. We currently exploring intrastructural help effects induced by different hepatitis B vaccines. To prepare for clinical studies, we have also identified HB-virus S-antigen-specific T helper cell epitopes recognized by most human vaccines.
In addition to VLP vaccines, liposomes may also be an attractive vaccine platform to harness T helper cells induced by licensed vaccine. In collaboration with Polymun Scientific Immunbiologische Forschung GmbH the incorporation of T helper cell epitopes into liposomes was optimized and we are currently exploring the coupling of HIV Env trimers to the surface of liposomes.
This project follows the innovative strategy to improve the efficacy of HIV vaccines by induction of affinity-matured HIV Env antibodies in the absence of HIV-specific T helper cells. The rational behind this strategy is based on observations that passive immunization with neutralizing antibodies can prevent infection and evidence that HIV-specific cellular immune responses may increase the susceptibility of acquisition of HIV infection. Since induction of the neutralizing antibodies is expected to depend on T-cell help, the project explores in relevant animal models the possibility to substitute HIV-specific T-cell help by T helper cells with other specificities.
Antibodies targeting the HIV-1 Env protein have been shown to prevent systemic infection in non-human primate models of AIDS. To explore whether these antibodies can block infection of the “first cell” at the viral portal of entry, challenge viruses based on simian immunodeficiency virus (SIV) were developed that use HIV-1 Env for entry into target cells during the first replication cycle, but then switch to SIV Env for all subsequent rounds of infection. Passive immunization of monkeys with a broadly neutralizing antibody binding to Env of HIV-1, but not SIV, prior to rectal exposure to the switching challenge virus led to a >50-fold reduction of HIV-1 Env-mediated infection events. Inhibition of infection was also detected under non-neutralizing conditions. Based on the Env switching challenge virus a simultaneous challenge model is currently developed to address the breadth of protection in a non-human primate model.
Since little was known about the direct impact of VLPs on antigen-specific B-cells, we incorporated hen egg lysozyme (HEL) into VLPs derived from HIV (HEL-VLPs) to study their effect on HEL-specific, B-cell receptor (BCR) transgenic B-cells. We observed that B-cells preferentially bind and internalized cognate VLPs in vitro. HEL-VLPs were able to effectively cross-link BCRs, increase expression of activation and co-stimulatory molecules, and enhance proliferative responses. Additionally, the
B-cell phenotype was shifted toward a germinal center pattern with further differentiation into memory and IgG3- and IgA-producing cells. Upon activation by HEL-VLPs, some cognate B-cells were able to produce CD40L, which might provide the required co-stimulatory signals in the absence of CD4+ T-cells in vitro. The patterns of cognate B-cell activation obtained open up the path for the development of VLP-based vaccines inducing rapid humoral responses in patients with AIDS or after organ transplantation when CD4+ T-cell function is impaired.
In vivo, we compared the efficacy of HEL-VLPs, delivered by subcutaneous (s.c.) or intravenous (i.v.) immunization to simultaneously stimulate primary cognate B-cell proliferative responses in different lymphoid organs. The observed differences argue for further studies on the quality of immune responses after intravenous administration of VLPs, an approach rarely pursued in the past.
T follicular helper (TFH) cells provide help for B-cells and are important for the formation and maintenance of germinal centers. Using co-cultivation of TCR-transgenic CD4+ T-cells together with dendritic and BCR-transgenic B-cells in the presence of cognate VLPs, we induced co-expression of the TFH-master regulator transcription factor BCL-6 together with CXCR5 in up to 40% of the CD4+ T-cells. Other phenotypic markers of for TFH cells could additionally be detected in these cells. Production of IL-21 and isotype switching by B-cells to IgG1 in the presence of induced TFH-like cells indicate a helper function of these cells in vitro. Thus, our study presents a robust experimental system for efficient generation of TFH-like cells in vitro and confirms the importance of cognate B- and T-cell cross-talk for the TFH-differentiation process.
Different kinds of synthetic nanoparticles have been suggested for vaccination. However, there are some advantages to use calcium phosphate nanoparticles (CaP-NPs) for targeted delivery of antigens: CaP is a mineral component of mammalian bone and therefore not harmful to the body, CaP-NPs can be prepared in a multi-shell way in order to protect biomolecules inside and after cellular uptake release their cargo inside the cell upon lysosomal dissolution at lower pH. Their surfaces can be covalently functionalized with biomolecules to an effective cell targeting. In this collaborative project with Prof. Epple (Essen), we performed a side-by-side comparison of CaP-NPs functionalized with HEL protein (CaP-HEL-NPs) on targeting, binding and internalization by cognate B-cells in vitro. CaP-HEL-NPs were preferentially bound and internalized by HEL-specific B-cells. They effectively cross-linked B-cell receptors and were 100-fold more efficient in the activation of B-cells than soluble HEL. In vivo, we explored different administration routes for the CaP-HEL-NPs and confirmed their ability to reach draining lymph nodes and deliver the protein in its native conformation to the B-cell areas. By using well-defined immunological adjuvants we increased the immunogenicity of CaP-HEL-NPs and were able to modulate the strength and quality of the systemic and the mucosal humoral immune responses. The results indicate that CaP-NPs represent a flexible platform to modulate humoral immune responses on demand that can be adapted to the particular needs of vaccines targeting various diseases.
HIV infection causes profound and often irreversible changes to the innate and adaptive immune system. For example, a permanent state of immune activation as well as depletion and dysfunction of CD4+ T-cells that are important regulators of humoral and cellular responses, all contribute to an overall impaired immunity even in ART-treated HIV infection. The main aim of this project is to study the effect of ART-treated HIV infection on the immunity gained from vaccines administered prior to HIV infection. For this purpose, an initial focus is set on immune responses of adults to the measles virus and tetanus toxin (both primed in childhood). The various objectives currently being evaluated include measurements of (i) cellular and soluble markers of inflammation, (ii) antigen-specific T-cell responses, (iii) antigen-specific antibody levels and (iv) transcriptome analysis of sorted antigen-specific T-cells. Gaining a deeper understanding of how HIV infection alters antigen-specific responses may provide clues for how these responses could be improved to the benefit of an enhanced vaccine-induced protective immunity. Samples from HIV-infected subjects are obtained in collaboration with Prof. Dr. med. Thomas Harrer (Medizinische Klinik 3 – UK Erlangen).