Rapid, sensitive and cost-effective determination of immune checkpoint inhibitor activity using a magnetic bead-based binding assay.

Immune checkpoint Inhibitors (ICIs) are effective immunno-therapeutic agents for cancer. Rapid and sensitive determination of the blocking activity of ICIs is important for ICIs development and immunological research. Among various immune checkpoint (IC) binding assays, cell-based binding assays are widely regarded, and the functional ELISA is a convenient alternative. However, these methodologies are limited by time-consuming preparation of cell lines stably expressing IC molecules, or long turnaround time with high cost. In this study, two magnetic bead based binding assays were developed to evaluate activity of ICIs, which was determined by a soluble ligand/bead immobilized receptor based binding assay (sL/bR binding assay) that assessed efficacy to block binding of one soluble IC ligand on its cognate receptor immobilized beads, or by a soluble receptor/bead immobilized ligand based binding assay (sR/bL binding assay) that assessed efficacy to block binding of soluble IC receptor on its cognate ligand immobilized beads. Half maximal inhibitory concentration (IC50) values of ICIs were calculated to determine ICIs activity. The sL/bR binding assay accurately determined the activity of two TIGIT blocking antibodies, since the relative blocking activity of two TIGIT antibodies determined by the sL/bR binding assay established in this study and that by the cell based binding assay were almost identical. In contrast, the sR/bL binding assay showed significantly improved sensitivity to determine activity of two PD-1 blocking antibodies than the sL/bR binding assay that was tested in this study and previous reports. Moreover, both amount of the used recombinant protein of ICI receptor/ligand and turnaround time of the two binding assays were more than 10 times less than those of the functional ELISA. These data indicate that the two magnetic bead based binding assays are sensitive, rapid and cost-effective methods to determine blocking activity of ICIs.

[The construction of attenuated Tiantan recombinant vaccinia virus vector with IFN-gamma receptor gene deletion].

OBJECTIVE: B8R gene encodes a secreted protein with homology to IFN-gamma receptor, which neutralizes the antiviral and immunological regulation activities of IFN-gamma. To improve the safety of vaccinia virus vector, an attenuated recombinant vaccinia virus with the B8R gene deletion from Tiantan vaccine strain (VTT) was constructed. METHODS: The transfer vectors were generated by joining B8R left flank, B8R right flank, vv promoter, LacZ, multicloning site and pBRSK fragments. The recombinant viruses VTTdeltaB8RLacZ (VTT with B8R deletion and LacZ insertion) were constructed by homologous recombination. RESULTS: The B8R deletion mutants were confirmed by dot blot with B8R gene probe and PCR amplification. The replication ability of VTTdeltaB8RLacZ strain in vitro was similar to that of the VTT. The skin lesions formed by VTTdeltaB8RLacZ (10(6) pfu) were significantly smaller and healed faster than those formed by VTT when injected intradermally to the rabbits,and no visible ulceration occurred. Meanwhile LacZ in VTKgpedeltaB8RLacZ was expressed stably. CONCLUSIONS: The attenuated vector with B8R gene deletion improves the safety of recombinant vaccinia virus vaccine B8R locus may be used as a new site for insertion of foreign genes in vaccinia virus vector.

[Immunogenicity analysis of HIV vaccine based on replicating vaccinia virus Tiantan vector].

OBJECTIVE: To investigate the immunogenicity of HIV vaccine vTKgpe based on Vaccinia Virus Tiantan vector in mice and rabbits. METHODS: Mice were inoculated with HIV vaccine vTKgpe by intramuscular (i.m.), intradermal (i.d.) or subcutaneous (s.c.) injections. Blood sample were collected every week, and then antibodies against HIV and vaccinia virus vector were detected. At week 4, some mice were killed and cellular immune responses were examined by flow cytometer. Additionally two rabbits were vaccinated subcutaneously, blood sample were tested as done with mice. RESULTS: In mice i.m. and s.c. groups, HIV specific antibodies emerged at week 2 and declined at week 4. Antibodies against vector elevated rapidly at week 4, and potentially affected HIV specific antibody detection. Cellular immune responses were only detected in s.c. group. Serum of rabbit showed that anti-HIV antibody appeared at week 2 and maintained for several weeks. CONCLUSION: Vaccine vTKgpe innoculated by i.m. and s.c routes inclined to induce humoral immune responses in mice, but in i.d. group, inclined to induce cellular immune responses. Response to the recombinant vaccinia virus was more sensitive in rabbit than in mice.