Receptor-targeted viral vectors: Tracking of stem cells and side by side comparison of AAV and lentiviral vectors

In recent years, substantial progress in gene therapy has been made as proofed by several successful clinical trials providing substantial benefit to patients and the first marketing authorization of an adeno-associated virus (AAV) vector-based medical product. Especially lentiviral and AAV vectors represent promising tools for gene transfer. They have been further improved to ensure safety and efficiency. One strategy to customize these viral vectors is the generation of receptor-targeted vectors that restrict gene delivery to cells expressing the targeted receptor. The first part of this thesis compares lentiviral and AAV vectors targeted to the receptor Her2/neu which is overexpressed in various tumor cells. This is for the first time a true side by side comparison of this totally different vector types, since here, both use the same receptor for cell entry. The second part investigates the potential of receptor-targeted lentiviral gene transfer into human hematopoietic stem cells (HSCs) via the cell surface protein CD105 and evaluates if CD105 is a marker for human long-term repopulating HSCs. The Her2-targeted lentiviral and AAV vector had been generated and characterized before (Münch et al., 2011; Münch et al., 2013). Both particles display a Her2/neu specific targeting ligand, the designed ankyrin repeat protein 9.29. First, functional, genomic and physical titers of Her2-LV and Her2-AAV vector stocks were determined side by side to allow precise normalization of both vector types. While the Her2-LV vector stocks showed higher genomic titers, Her2-AAV vectors comprised more functional particles per genome containing particles. Accordingly, about 10-fold more genome copies of Her2-LV than Her2-AAV had to be administered systemically in a subcutaneous tumor mouse model for detectable transgene expression. Analysis of the vector distribution short time after systemic administration in vivo revealed that the non-enveloped Her2-AAV vector circulated stably in the blood of mice for a prolonged time compared to Her2-LV. Accumulation of Her2-AAV within the target tissue occurred only after 24 hours. Lentiviral vectors are currently the preferred vector type for the modification of HSCs due to their capability of integrating the transgene into the host cell’s genome. Thereby the entire hematopoietic system can be reconstituted with cells carrying the corrected gene. True HSCs which are capable of self-renewal and differentiate into all hematopoietic lineages can be identified by the expression of specific cell surface markers. In mice, CD105 was previously shown to be present on most immature, long-term repopulating HSCs. After confirming that human CD105 is expressed on 30-80% of human CD34+ cells, CD34+ cells were transduced with a lentiviral vector targeted to human CD105 (CD105-LV) and transplanted into NOD-scid IL2Rγ-/- mice. Stable reporter gene expression in engrafted cells was detected long-term in all human hematopoietic lineages in bone marrow, spleen and blood. In addition, competitive repopulation experiments in mice showed a superior engraftment of CD105-LV transduced CD34+ cells in bone marrow and spleen compared to cells transduced with a conventional non-targeted lentiviral vector confirming CD105 as a marker for early HSCs with high repopulating capacity. The data shown in this thesis highlight the potential of receptor-targeted vectors to trace cell subsets and identify new markers for specific cell populations. In addition, it demonstrates the potential of comparing vectors derived from different virus families once they have been targeted to the same entry receptor

Neuraminidase-Inhibiting Antibody Titers Correlate with Protection from Heterologous Influenza Virus Strains of the Same Neuraminidase Subtype.

Immune responses induced by currently licensed inactivated influenza vaccines are mainly directed against the hemagglutinin (HA) glycoprotein, the immunodominant antigen of influenza viruses. The resulting antigenic drift of HA requires frequent updating of the vaccine composition and annual revaccination. On the other hand, the levels of antibodies directed against the neuraminidase (NA) glycoprotein, the second major influenza virus antigen, vary greatly. To investigate the potential of the more conserved NA protein for the induction of subtype-specific protection, vesicular stomatitis virus-based replicons expressing a panel of N1 proteins from prototypic seasonal and pandemic H1N1 strains and human H5N1 and H7N9 isolates were generated. Immunization of mice and ferrets with the replicon carrying the matched N1 protein resulted in robust humoral and cellular immune responses and protected against challenge with the homologous influenza virus with an efficacy similar to that of the matched HA protein, illustrating the potential of the NA protein as a vaccine antigen. The extent of protection after immunization with mismatched N1 proteins correlated with the level of cross-reactive neuraminidase-inhibiting antibody titers. Passive serum transfer experiments in mice confirmed that these functional antibodies determine subtype-specific cross-protection. Our findings illustrate the potential of NA-specific immunity for achieving broader protection against antigenic drift variants or newly emerging viruses carrying the same NA but a different HA subtype. Despite the availability of vaccines, annual influenza virus epidemics cause 250,000 to 500,000 deaths worldwide. Currently licensed inactivated vaccines, which are standardized for the amount of the hemagglutinin (HA) antigen, primarily induce strain-specific antibodies, whereas the immune response to the neuraminidase (NA) antigen, which is also present on the viral surface, is usually low. Using NA-expressing single-cycle vesicular stomatitis virus replicons, we show that the NA antigen conferred protection of mice and ferrets against not only the matched influenza virus strains but also viruses carrying NA proteins from other strains of the same subtype. The extent of protection correlated with the level of cross-reactive NA-inhibiting antibodies. This highlights the potential of the NA antigen for the development of more broadly protective influenza vaccines. Such vaccines may also provide partial protection against newly emerging strains with the same NA but a different HA subtype

A Third Dose of the COVID-19 Vaccine, CVnCoV, Increased the Neutralizing Activity against the SARS-CoV-2 Wild-Type and Delta Variant

A third dose of CVnCoV, a former candidate mRNA vaccine against SARS-CoV-2, was previously shown to boost neutralizing antibody responses against SARS-CoV-2 wild-type in adults aged 18–60 and >60 years in a phase 2a clinical study. In the present study, we report the neutralizing antibody responses to a wild-type and a variant of concern, Delta, after a third dose of the vaccine on day (D)57 and D180. Neutralization activity was assessed using a microneutralization assay. Comparable levels of neutralizing antibodies against the wild-type and Delta were induced. These were higher than those observed after the first two doses, irrespective of age or pre-SARS-CoV-2-exposure status, indicating that the first two doses induced immune memory. Four weeks after the third dose on D180, the neutralizing titers for wild-type and Delta were two-fold higher in younger participants than in older participants; seroconversion rates were 100% for wild-type and Delta in the younger group and for Delta in the older group. A third CVnCoV dose induced similar levels of neutralizing responses against wild-type virus and the Delta variant in both naïve and pre-exposed participants, aligning with current knowledge from licensed COVID-19 vaccines that a third dose is beneficial against SARS-CoV-2 variants

Enhancing the Oncolytic Activity of CD133-Targeted Measles Virus: Receptor Extension or Chimerism with Vesicular Stomatitis Virus Are Most Effective

Therapy resistance and tumor recurrence are often linked to a small refractory and highly tumorigenic subpopulation of neoplastic cells, known as cancer stem cells (CSCs). A putative marker of CSCs is CD133 (prominin-1). We have previously described a CD133-targeted oncolytic measles virus (MV-CD133) as a promising approach to specifically eliminate CD133-positive tumor cells. Selectivity was introduced at the level of cell entry by an engineered MV hemagglutinin (H). The H protein was blinded for its native receptors and displayed a CD133-specific single-chain antibody fragment (scFv) as targeting domain. Interestingly, MV-CD133 was more active in killing CD133-positive tumors than the unmodified MV-NSe despite being highly selective for its target cells. To further enhance the antitumoral activity of MV-CD133, we here pursued arming technologies, receptor extension, and chimeras between MV-CD133 and vesicular stomatitis virus (VSV). All newly generated viruses including VSV-CD133 were highly selective in eliminating CD133-positive cells. MV-CD46/CD133 killed in addition CD133-negative cells being positive for the MV receptors. In an orthotopic glioma model, MV-CD46/CD133 and MVSCD-CD133, which encodes the super cytosine deaminase, were most effective. Notably, VSV-CD133 caused fatal neurotoxicity in this tumor model. Use of CD133 as receptor could be excluded as being causative. In a subcutaneous tumor model of hepatocellular cancer, VSV-CD133 revealed the most potent oncolytic activity and also significantly prolonged survival of the mice when injected intravenously. Compared to MV-CD133, VSV-CD133 infected a more than 104-fold larger area of the tumor within the same time period. Our data not only suggest new concepts and approaches toward enhancing the oncolytic activity of CD133-targeted oncolytic viruses but also raise awareness about careful toxicity testing of novel virus types