Antibiotic-dependent expression of early transcription factor subunits leads to stringent control of vaccinia virus replication

AbstractThe use of vaccinia virus (VACV) as the vaccine against variola virus resulted in the eradication of smallpox. VACV has since been used in the development of recombinant vaccine and therapeutic vectors, but complications associated with uncontrolled viral replication have constrained its use as a live viral vector. We propose to improve the safety of VACV as a live-replicating vector by using elements of the tet operon to control the transcription of genes that are essential for viral growth. Poxviruses encode all enzymes and factors necessary for their replication within the host cell cytoplasm. One essential VACV factor is the vaccinia early transcription factor (VETF) packaged into the viral core. This heterodimeric protein is required for expression of early VACV genes. VETF is composed of a large subunit encoded by the A7L gene and a small subunit encoded by the D6R gene. Two recombinant VACVs were generated in which either the A7L or D6R gene was placed under the control of tet operon elements to allow their transcription, and therefore viral replication, to be dependent on tetracycline antibiotics such as doxycycline. In the absence of inducers, no plaques were produced but abortively infected cells could be identified by expression of a reporter gene. In the presence of doxycycline, both recombinant viruses replicated indistinguishably from the wild-type strain. This stringent control of VACV replication can be used for the development of safer, next-generation VACV vaccines and therapeutic vectors. Such replication-inducible VACVs would only replicate when administered with tetracycline antibiotics, and if adverse events were to occur, treatment would be as simple as antibiotic cessation

Development of Repressible Systems to Control Gene Expression in Vaccinia Virus

Two vaccinia virus (VACV) expression systems that contain elements from the lactose (lac) and the tetracycline (tet) operons of E. coli were developed to repress the expression of a reporter gene, enhanced green fluorescent protein (EGFP), in the presence of tet operon inducers. In the first system, lac and tet operon elements were arranged in a gene circuit, and in the presence of increasing concentrations of a lac operon inducer (isopropyl-β-D-thiogalactoside, IPTG), EGFP expression increased in a dose dependent manner and at high IPTG concentrations, expression reached the same levels as a positive control virus. Importantly, in the presence of increasing concentrations of the tet operon inducer (doxycycline, DOX), EGFP expression decreased in a dose dependent manner and at high concentrations of DOX, expression was repressed to the degree observed in a negative control virus that does not express EGFP. In the second system, recombinant VACVs constitutively expressing six mutant versions of the tet repressor gene (tetR) shown to bind tet operators in the presence, but not absence of inducers (reverse tetR genes), were developed. In the presence of tetracyclines (TCs), the recombinant VACVs exhibited various degrees of repression of EGFP expression, with increasing concentrations of TCs leading to EGFP repression in a dose dependent manner, and in some instances, down to the degree observed in a negative control virus. In light of the renewed interest for the use of VACV as vaccine and therapeutic cancer vectors, the repressible VACV expression system developed here can be used to tightly regulate genes essential for VACV replication, thus functioning as a built-in safety mechanism to conditionally control viral replication

Development of Replication-Repressible Vaccinia Virus Vectors for Vaccines and Therapeutics

Archival abstract submitte

Development of Repressible Systems to Control Gene Expression in Vaccinia Virus

Two vaccinia virus (VACV) expression systems that contain elements from the lactose (lac) and the tetracycline (tet) operons of E. coli were developed to repress the expression of a reporter gene, enhanced green fluorescent protein (EGFP), in the presence of tet operon inducers. In the first system, lac and tet operon elements were arranged in a gene circuit, and in the presence of increasing concentrations of a lac operon inducer (isopropyl-β-D-thiogalactoside, IPTG), EGFP expression increased in a dose dependent manner and at high IPTG concentrations, expression reached the same levels as a positive control virus. Importantly, in the presence of increasing concentrations of the tet operon inducer (doxycycline, DOX), EGFP expression decreased in a dose dependent manner and at high concentrations of DOX, expression was repressed to the degree observed in a negative control virus that does not express EGFP. In the second system, recombinant VACVs constitutively expressing six mutant versions of the tet repressor gene (tetR) shown to bind tet operators in the presence, but not absence of inducers (reverse tetR genes), were developed. In the presence of tetracyclines (TCs), the recombinant VACVs exhibited various degrees of repression of EGFP expression, with increasing concentrations of TCs leading to EGFP repression in a dose dependent manner, and in some instances, down to the degree observed in a negative control virus. In light of the renewed interest for the use of VACV as vaccine and therapeutic cancer vectors, the repressible VACV expression system developed here can be used to tightly regulate genes essential for VACV replication, thus functioning as a built-in safety mechanism to conditionally control viral replication

Replication-inducible vaccinia virus vectors with enhanced safety in vivo.

Vaccinia virus (VACV) has been used extensively as the vaccine against smallpox and as a viral vector for the development of recombinant vaccines and cancer therapies. Replication-competent, non-attenuated VACVs induce strong, long-lived humoral and cell-mediated immune responses and can be effective oncolytic vectors. However, complications from uncontrolled VACV replication in vaccinees and their close contacts can be severe, particularly in individuals with predisposing conditions. In an effort to develop replication-competent VACV vectors with improved safety, we placed VACV late genes encoding core or virion morphogenesis proteins under the control of tet operon elements to regulate their expression with tetracycline antibiotics. These replication-inducible VACVs would only express the selected genes in the presence of tetracyclines. VACVs inducibly expressing the A3L or A6L genes replicated indistinguishably from wild-type VACV in the presence of tetracyclines, whereas there was no evidence of replication in the absence of antibiotics. These outcomes were reflected in mice, where the VACV inducibly expressing the A6L gene caused weight loss and mortality equivalent to wild-type VACV in the presence of tetracyclines. In the absence of tetracyclines, mice were protected from weight loss and mortality, and viral replication was not detected. These findings indicate that replication-inducible VACVs based on the conditional expression of the A3L or A6L genes can be used for the development of safer, next-generation live VACV vectors and vaccines. The design allows for administration of replication-inducible VACV in the absence of tetracyclines (as a replication-defective vector) or in the presence of tetracyclines (as a replication-competent vector) with enhanced safety

Development of multi-specific humanized llama antibodies blocking SARS-CoV-2/ACE2 interaction with high affinity and avidity

Coronaviruses cause severe human viral diseases including SARS, MERS and COVID-19. Most recently SARS-CoV-2 virus (causing COVID-19) has led to a pandemic with no successful therapeutics. The SARS-CoV-2 infection relies on trimeric spike (S) proteins to facilitate virus entry into host cells by binding to ACE2 receptor on host cell membranes. Therefore, blocking this interaction with antibodies are promising agents against SARS-CoV-2. Here we describe using humanized llama antibody VHHs against SARS-CoV-2 that would overcome the limitations associated with polyclonal and monoclonal combination therapies. From two llama VHH libraries, unique humanized VHHs that bind to S protein and block the S/ACE2 interaction were identified. Furthermore, pairwise combination of VHHs showed synergistic blocking. Multi-specific antibodies with enhanced affinity and avidity, and improved S/ACE2 blocking are currently being developed using an in-silico approach that also fuses VHHs to Fc domains. Importantly, our current bi-specific antibody shows potent S/ACE2 blocking (KD – 0.25 nM, IC100 ∼ 36.7 nM, IC95 ∼ 12.2 nM, IC50 ∼ 1 nM) which is significantly better than individual monoclonal VHH-Fcs. Overall, this design would equip the VHH-Fcs multiple mechanisms of actions against SARS-CoV-2. Thus, we aim to contribute to the battle against COVID-19 by developing therapeutic antibodies as well as diagnostics

Development of humanized tri-specific nanobodies with potent neutralization for SARS-CoV-2

SARS-CoV-2 is a newly emergent coronavirus, which has adversely impacted human health and has led to the COVID-19 pandemic. There is an unmet need to develop therapies against SARS-CoV-2 due to its severity and lack of treatment options. A promising approach to combat COVID-19 is through the neutralization of SARS-CoV-2 by therapeutic antibodies. Previously, we described a strategy to rapidly identify and generate llama nanobodies (VHH) from naïve and synthetic humanized VHH phage libraries that specifically bind the S1 SARS-CoV-2 spike protein, and block the interaction with the human ACE2 receptor. In this study we used computer-aided design to construct multi-specific VHH antibodies fused to human IgG1 Fc domains based on the epitope predictions for leading VHHs. The resulting tri-specific VHH-Fc antibodies show more potent S1 binding, S1/ACE2 blocking, and SARS-CoV-2 pseudovirus neutralization than the bi-specific VHH-Fcs or combination of individual monoclonal VHH-Fcs. Furthermore, protein stability analysis of the VHH-Fcs shows favorable developability features, which enable them to be quickly and successfully developed into therapeutics against COVID-19