A chimeric vesicular stomatitis virus with the glycoprotein of the lymphocytic choriomeningitis virus, VSV-GP, is a potent viral vaccine vector that overcomes several of the limitations of wild-type VSV. Here, we evaluated the potential of VSV-GP as an HIV vaccine vector. We introduced genes for different variants of the HIV-1 envelope protein Env, i.e., secreted or membrane-anchored, intact or mutated furin cleavage site or different C-termini, into the genome of VSV-GP. We found that the addition of the Env antigen did not attenuate VSV-GP replication. All HIV-1 Env variants were expressed in VSV-GP infected cells and some were incorporated very efficiently into VSV-GP particles. Crucial epitopes for binding of broadly neutralizing antibodies against HIV-1 such as MPER (membrane-proximal external region), CD4 binding site, V1V2 and V3 loop were present on the surface of VSV-GP-Env particles. Binding of quaternary antibodies indicated a trimeric structure of VSV-GP incorporated Env. We detected high HIV-1 antibody titers in mice and showed that vectors expressing membrane-anchored Env elicited higher antibody titers than vectors that secreted Envs. In rabbits, Tier 1A HIV-1 neutralizing antibodies were detectable after prime immunization and titers further increased after boosting with a second immunization. Taken together, VSV-GP-Env is a promising vector vaccine against HIV-1 infection since this vector permits incorporation of native monomeric and/or trimeric HIV-1 Env into a viral membrane.

Heterologous prime-boost settings with a protein vaccine and the viral vector vesicular stomatitis virus, both expressing tumor-associated antigens (KISIMA-TAA and VSV-GP-TAA), have been previously shown to generate potent antitumor immunity. In the cold TC-1 model (HPV antigen) and the immune-infiltrate MC-38 model (Adpgk, Reps1 and Rpl18 neo-antigens), we further investigated pivotal immune cells that educate CD8+ T cells. Heterologous prime-boost vaccination induced a superior antitumor response characterized by the increase in number and functionality of antigen-specific CD8+ T cells, recruitment of cross-presenting dendritic cells, and polarization of CD4+ T cells towards an antitumor Th1 phenotype within the tumor and tumor-draining lymph nodes, turning the cold TC-1 tumor into a hot, inflamed tumor. In the inflamed MC-38 tumor model, treatment combination markedly prolonged the overall survival of mice. Treatment with multi-epitope vaccines also induced high frequencies of multiple antigen specificities in the periphery and in the tumor. Prime-boost treatment reduced tumor-infiltrating regulatory CD4+ T cells whilst increasing cross-presenting dendritic cells in tumor-draining lymph nodes. In conclusion, heterologous prime-boost vaccination possesses the ability to induce a potent anti-tumor response in both immune-excluded and immune-infiltrated mouse tumor models. Additionally, this study highlights the design of a multi-epitope vaccine for cancer immunotherapy.

One important prerequisite for developing a therapeutic monoclonal antibody is to evaluate its in vivo efficacy. We tested the therapeutic potential of an anti-CD96 antibody alone or in combination with an anti-PD-1 antibody in a mouse colon cancer model. Early anti-PD-1 treatment significantly decreased tumor growth and the combination with anti-CD96 further increased the therapeutic benefit, while anti-CD96 treatment alone had no effect. In late therapeutic settings, the treatment combination resulted in enhanced CD8⁺ T cell infiltration of tumors and an increased CD8/Treg ratio. Measured anti-PD-1 concentrations were as expected in animals treated with anti-PD-1 alone, but lower at later time points in animals receiving combination treatment. Moreover, anti-CD96 concentrations dropped dramatically after 10 days and were undetectable thereafter in most animals due to the occurrence of anti-drug antibodies that were increasing antibody clearance. Comparison of the anti-PD-1 concentrations with tumor growth showed that higher antibody concentrations in plasma correlated with better therapeutic efficacy. The therapeutic effect of anti-CD96 treatment could not be evaluated, because plasma concentrations were too low. Our findings strongly support the notion of measuring both plasma concentration and anti-drug antibody formation throughout in vivo studies, in order to interpret pharmacodynamic data correctly.

Numerous factors influence the magnitude and effector phenotype of vaccine-induced CD8⁺ T cells, thereby potentially impacting treatment efficacy. Here, we investigate the effect of vaccination dose, route of immunization, presence of a target antigen-expressing tumor, and heterologous prime-boost with peptide vaccine partner following vaccination with antigen-armed VSV-GP. Our results indicate that a higher vaccine dose increases antigen-specific CD8⁺ T cell proportions while altering the phenotype. The intravenous route induces the highest proportion of antigen-specific CD8⁺ T cells together with the lowest anti-viral response followed by the intraperitoneal, intramuscular, and subcutaneous routes. Moreover, the presence of a B16-OVA tumor serves as pre-prime, thereby increasing OVA-specific CD8⁺ T cells upon vaccination and thus altering the ratio of anti-tumor versus anti-viral CD8⁺ T cells. Interestingly, tumor-specific CD8⁺ T cells exhibit a different phenotype compared to bystander anti-viral CD8⁺ T cells. Finally, the heterologous combination of peptide and viral vaccine elicits the highest proportion of antigen-specific CD8⁺ T cells in the tumor and tumor-draining lymph nodes. In summary, we provide a basic immune characterization of various factors that affect anti-viral and vaccine target-specific CD8⁺ T cell proportions and phenotypes, thereby enhancing our vaccinology knowledge for future vaccine regimen designs.