Research Group of K. Überla/ V. Temchura

Virology

Head of Institute:
Prof. Dr. med. Klaus Überla

HIV Vaccine Research

With annual HIV infection rates still exceeding 2 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 persued:

GagPol-specific CD4+ T-cells increase the antibody response to Env by intrastructural help

Immunization of rhesus macaques against Gag of SIV resulted in a more rapid appearance of Env antibodies after infection with SIV or SHIV challenge viruses although the vaccines lacked an Env component. One explanation by which Gag-specific immunity may increase Env-specific antibody responses is based on intrastructural help, a mechanism described three decades ago for influenza virus and hepatitis B virus. Naïve B-cells specific for external viral proteins may take-up the entire viral particle and subsequently present peptides derived from external and internal viral proteins on their MHC-II molecules. Thus, Gag-specific T helper cells induced by vaccination could provide cognate help for Env-specific B cells and thus accelerate the production of Env-specific antibody responses early after infection. We therefore explored whether T helper cells specific for internal HIV proteins could provide intrastructural help for Env-specific B cells and thus increase the Env antibody response. To test this hypothesis, we performed immunization and T cell transfer experiments in mice, and explored intrastructural help by in vitro B and T cell co-culture experiments. Mice were immunized by adenoviral vector or DNA vaccines against GagPol and then boosted with virus-like particles (VLP) containing GagPol and Env. Env-specific antibody levels after the VLP booster immunizations were significantly higher in GagPol-immunized mice than in mock-vaccinated controls. Adoptive transfer of CD4+ T cells from GagPol-immunized mice also enhanced the Env antibody response to VLP immunization in the recipient mice. Depending on the presence of VLPs, co-cultivation of CD4+ T cells from GagPol-primed mice with BCR transgenic B cells specific for a protein presented on the surface of the VLPs also resulted in the activation of the B and T cells. Our study indicates that GagPol-specific T helper cells may provide intrastructural help for Env antibody responses. This cross-talk between immune responses directed against different components of the retroviral particle may be relevant for the immunopathogenesis of retroviral infections and allow to improve virus like particle vaccine approaches against HIV.

Enhancing the quality of antibodies to HIV-1 Env by GagPol-specific T helper cells

The importance of Fcγ receptor (FcγR)-dependent effector functions of antibodies induced by vaccination is increasingly recognized. However, HIV-1 Env immunogens seem to preferentially induce antibodies with little Fc-dependent effector functionality. A bias in the Env antibody response was indeed detected after DNA or virus-like particle (VLP) immunization of mice and could be traced back to skewed Env-specific T helper cells. To modulate the Env-specific antibody response, GagPol-specific CD4+ T cells were induced prior to immunization with VLPs containing GagPol and Env. The GagPol-specific CD4+ T cells provided intrastructural help (ISH) to Env-specific B cells during the VLP booster immunization, leading to a balanced humoral immune response against Env. The glycosylation of Env-specific antibodies was also affected by ISH, indicating the impact of CD4+ T cells on the glycosylation of immunoglobulins in vivo. The increased quality in the antibody response against Env was confirmed by a surrogate assay for ADCC activity. We also observed a bias in the Env-specific T helper cell responses in participants of the RV138 HIV vaccine trial. Optimizing the antibody response by ISH from T-helper cells induced by previous viral infections or vaccination is therefore an attractive novel approach to increase the efficacy of prophylactic HIV Env-based vaccines and may also be applicable to other particulate vaccines such as nanoparticles and liposomes.

Avoiding HIV-specific T helper cells to improve vaccine efficacy

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 explors in relevant animal models the possibility to substitute HIV-specific T cell help by T helper cells with other specificities.

Direct effects of HIV-derived lentiviral particles on T-cell independent activation and differentiation of naïve cognate conventional B2-cells in vitro

Since little is known about the direct impact of VLP on antigen-specific B cells, we incorporated Hen Egg Lysozyme (HEL) into VLP (HEL-VLP) derived from HIV to study their effect on HEL-specific, B cell receptor-transgenic B-cells (HEL+ B-cells) in vitro. We observed preferential binding of HEL-VLP to HEL+ B-cells and their efficient internalization. HEL-VLP were able to effectively cross-link B-cell receptors as indicated by the loss of surface CD62L. In the absence of CD4+ T-cells, other activation events induced by VLP in cognate naïve B-cells included increased expression of activation and co-stimulatory molecules as well as an enhanced proliferative response. Additionally, the B-cell phenotype shifted toward a germinal center pattern with further differentiation into memory and IgG3- and IgA-producing cells. The observed CD4+ T-cell independent activation and differentiation may be due to VLP-induced expression of CD40L by a subset of cognate B-cells. Thus, even in the absence of CD4+ T-cells VLP provide strong direct activation signals to cognate naïve B-cells, which may contribute to the strong humoral immune responses observed after VLP immunization.

Generation of T-follicular helper like cells in vitro

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 virus-like particles, 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. Dendritic cells supported the generation of functional TFH-like cells, but were unable to induce them without cognate B-cells. 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.

Testing of calcium phosphate nanoparticle-based vaccines

Different kinds of synthetic nanoparticles have been suggested for vaccination. However, there are some advantages to use calcium phosphate nanoparticles (CaP) for targeted delivery of antigens: CaP occur in the body as mineral component of mammalian bone and are therefore not harmful to the body, they 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 and, finally, their surface can be covalently functionalized with biomolecules to an effective cell targeting. In this collaborative project with Prof. Epple’s group (Essen), we have performed a side-by-side comparison of CaP nanoparticles functionalized with HEL protein (CaP-HEL) on targeting, binding and internalisation by cognate B-cells in vitro. The nanoparticles were characterized by scanning electron microscopy and dynamic light scattering. The functionalized calcium phosphate nanoparticles were preferentially bound and internalized by HEL-specific B-cells. Co-cultivation of HEL-specific B-cells with the functionalized nanoparticles also increased surface expression of B-cell activation markers. Functionalized nanoparticles were able to effectively cross-link B-cell receptors at the surface of antigen-matched B-cells and were 100-fold more efficient in the activation of B-cells than soluble HEL. Thus, calcium phosphate nanoparticles coated with protein antigens are promising vaccine candidates for induction of humoral immunity. Different TLR ligands can be further included into the calcium phosphate nanoparticles to modulate antigen-specific immune response in different B-cell subpopulations. The calcium phosphate nanoparticle platform is thus a versatile carrier system that can be adapted to the particular needs of vaccines targeting various diseases.

Sterilizing immunity after passive immunization

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 100-fold reduction of HIV-1 Env-mediated infection events. Inhibition of infection was also detected under non-neutralizing conditions. Thus, antiviral antibodies can reach sufficient levels at mucosal entry sites to provide sterilizing immunity in the strictest sense.

 
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Summary