Oncolytic virotherapy is a promising therapeutic approach treating tumors, where oncolytic viruses (OVs) can selectively infect and lyse tumor cells through replication, while also triggering long-lasting anti-tumor immune responses. Vaccinia virus (VV) has emerged as a leading candidate for use as an OV due to its broad cytophilicity and robust capacity to express exogenous genes. Consequently, oncolytic vaccinia virus (OVV) has entered clinical trials. This review provides an overview of the key strategies used in the development of OVV, summarizes the findings from clinical trials, and addresses the challenges that must be overcome in the advancement of OVV-based therapies. Furthermore, it explores potential future strategies for enhancing the development and clinical application of OVV, intending to improve tumor treatment outcomes. The review aims to facilitate the further development and clinical adoption of OVV, thereby advancing tumor therapies.
Vaccinia virus/physiology* ; Humans ; Oncolytic Virotherapy/methods* ; Oncolytic Viruses/physiology* ; Neoplasms/therapy* ; Animals

For rapid and accurate screening of recombinant modified vaccinia virus Ankara (rMVA) that satisfied the quality standards of clinical trials, a novel shuttle vector that can delete the marker gene automatically during virus propagation was construted: pZL-EGFP. To construct the pZL-EGFP, the original shuttle vector pSC11 was modified by replacing the LacZ marker gene with enhanced green fluorescent protein (EGFP) and then inserting homologous sequences of TKL into the flank regions of EGFP. Baby hamster kidney (BHK)-21 cells were cotransfected with pZL-EGFP and MVA, and underwent ten passages and one plaque screening to obtain the EGFP-free rMVA carrying the exogenous gene. Resulting rMVA was tested by polymerase chain reaction and western blotting to verify pZL-EGFP function. A novel shuttle vector pZL-EGFP containing an EGFP marker gene which could be deleted automatically was constructed. This gene deletion had no effect on the activities of rMVA, and the exogenous gene could be expressed stably. These results suggest that rMVA can be packaged efficiently by homologous recombination between pZL-EGFP and MVA in BHK-21 cells, and that the carried EGFP gene can be removed automatically by intramolecular homologous recombination during virus passage. Meanwhile, the gene deletion had no influence on the activities of rMVA and the expression of exogenous target gene. This study lays a solid foundation for the future research.
Animals ; Biomarkers ; Cricetinae ; Epithelial Cells ; virology ; Gene Deletion ; Genetic Engineering ; methods ; Genetic Vectors ; genetics ; metabolism ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Recombination, Genetic ; Vaccinia ; virology ; Vaccinia virus ; genetics ; physiology ; Virus Replication

Orthopoxvirus vector has a broad prospect in recombinant vaccine research, but the rarely severe side-effect impedes its development. Vaccinia virus and Cowpox virus of Orthopoxvirus have broad host range, and they have typical host range genes as K1L, CP77 and C7L. These three genes affect host range of Vaccinia virus, disturb the cell signaling pathways, suppress immune response and are related to virulence.
Cell Line ; Cowpox virus ; genetics ; immunology ; pathogenicity ; physiology ; Genetic Vectors ; Host Specificity ; genetics ; Orthopoxvirus ; genetics ; immunology ; pathogenicity ; physiology ; Signal Transduction ; Vaccines, Synthetic ; immunology ; Vaccinia virus ; genetics ; immunology ; pathogenicity ; physiology ; Viral Proteins ; genetics ; metabolism ; Viral Vaccines ; immunology ; Virulence

BACKGROUND/AIMS: JX-594 is an oncolytic virus derived from the Wyeth vaccinia strain that causes replication-dependent cytolysis and antitumor immunity. Starting with a cross-examination of clinical-trial samples from advanced hepatocellular carcinoma patients having high levels of aldosterone and virus amplification in JX-594 treatment, we investigated the association between virus amplification and aldosterone in human cancer cell lines. METHODS: Cell proliferation was determined by a cell-counting-kit-based colorimetric assay, and vaccinia virus quantitation was performed by quantitative polymerase chain reaction (qPCR) and a viral plaque assay. Also, the intracellular pH was measured using a pH-sensitive dye. RESULTS: Simultaneous treatment with JX-594 and aldosterone significantly increased viral replication in A2780, PC-3, and HepG2 cell lines, but not in U2OS cell lines. Furthermore, the aldosterone treatment time altered the JX-594 replication according to the cell line. The JX-594 replication peaked after 48 and 24 hours of treatment in PC-3 and HepG2 cells, respectively. qPCR showed that JX-594 entry across the plasma membrane was increased, however, the changes are not significant by the treatment. This was inhibited by treatment with spironolactone (an aldosterone-receptor inhibitor). JX-594 entry was significantly decreased by treatment with EIPA [5-(N-ethyl-N-isopropyl)amiloride; a Na+/H+-exchange inhibitor], but aldosterone significantly restored JX-594 entry even in the presence of EIPA. Intracellular alkalization was observed after aldosterone treatment but was acidified by EIPA treatment. CONCLUSIONS: Aldosterone stimulates JX-594 amplification via increased virus entry by affecting the H+ gradient.
Aldosterone/*pharmacology ; Aldosterone Antagonists/pharmacology ; Amiloride/analogs & derivatives/pharmacology ; Animals ; Carcinoma, Hepatocellular/blood/virology ; Cell Line, Tumor ; Humans ; Hydrocortisone/blood ; Hydrogen-Ion Concentration ; Liver Neoplasms/blood/virology ; Neuroprotective Agents/pharmacology ; Oncolytic Virotherapy ; Rabbits ; Spironolactone/pharmacology ; Vaccinia virus/*drug effects/genetics/metabolism/*physiology ; Virus Replication/*drug effects

OBJECTIVEB8R 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.

METHODSThe 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.

RESULTSThe 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.

CONCLUSIONSThe 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.

Animals ; Cell Line ; Chick Embryo ; Gene Deletion ; Genetic Vectors ; Humans ; Rabbits ; Receptors, Interferon ; genetics ; physiology ; Recombination, Genetic ; Vaccines, Attenuated ; immunology ; Vaccinia virus ; genetics ; immunology ; pathogenicity ; Virulence ; Virus Replication

BACKGROUNDTo investigate expression of IL-6 in non?replicating vaccinia virus and its immune effects on recombinant virus.

METHODSThe recombinant non replicating vaccinia virus RVJ123 delta CK11 beta 75IL6 was constructed with non?replicating vaccinia virus vector pNEOCK11beta75IL6 and replicating vaccinia virus RVJ123. In animal model, immunization with the recombinant virus was carried out and its immune response was studied.

RESULTSThe recombinant virus could express IL-nd HBsAg simultaneously. Southern blot analysis demonstrated that the genes between vaccinia virus Hind? C and K fragments were deleted and IL-6 gene was integrated stably. Given intranasal inocula of the virus to immunize BALB/c mouse and New Zealand Rabbit, the elevated anti-HBsAg IgA and IgG antibody secreting cells in mouse lung lymphoid to vectors expressing IL-6 was at about two?fold higher level than those elicited by control virus at day 14 after immunization. Authors also could detect elevated anti-HBsAg IgA and IgG antibody conversion in mouse serum and lung fluid, rabbits serum, lung fluid, saliva, vagina and nasal washing samples.

CONCLUSIONSIL-6 expressed by non-replicating recombinant vaccinia virus could enhance the induced?immune effects, it could serve as the effective adjuvant for recombinant vector vaccine.

Adjuvants, Immunologic ; Animals ; Cells, Cultured ; Chick Embryo ; Female ; Genetic Vectors ; Humans ; Immunoglobulin A ; analysis ; Immunoglobulin G ; analysis ; Interleukin-6 ; biosynthesis ; genetics ; immunology ; Lung ; immunology ; Mice ; Mice, Inbred BALB C ; Rabbits ; Recombination, Genetic ; Vaccinia virus ; immunology ; metabolism ; physiology ; Virus Replication

OBJECTIVETo generate an HPV16 prophylactic vaccine candidate for cervical cancer.

METHODSHPV16 major capsid protein L1 gene and minor capsid protein L2 gene were amplified using PCR. These genes were mutated by PCR site-directed mutagenesis for removal of sequence motifs (TTTTTNT) which would cause transcription termination when expressed from a vaccinia virus early promoter, then inserted into a vaccinia virus expression vector. A strain replication-deficient recombinant vaccinia virus containing the mutant sequences was obtained through a homologous recombination and identified.

RESULTSThe nucleotide sequence remained the correct amino acid sequence of the L1 and L2 proteins after mutated. Full-length L1 and L2 proteins were generated in cells infected with the recombinant virus. The virus strain propagated at very low titer or could not reproduce in some kinds of cell derived from different human tissues.

CONCLUSIONSThe authors have generated a strain replication-deficient recombinant vaccinia virus expressing HPV16 L1 plus L2 proteins as an HPV16 prophylactic vaccine candidate for cervical cancer.

Capsid ; Capsid Proteins ; genetics ; Cell Line ; Cloning, Molecular ; Female ; Gene Expression ; Genetic Vectors ; Humans ; Oncogene Proteins, Viral ; genetics ; Papillomaviridae ; genetics ; physiology ; Papillomavirus Infections ; prevention & control ; Transfection ; Tumor Virus Infections ; prevention & control ; Uterine Cervical Neoplasms ; virology ; Vaccinia virus ; genetics ; physiology ; Virus Replication

Over 60 different types of human papillomavirus (HPV) have been identified, and they are classified into high and low risk groups based on the risk for malignant progression of HPV associated lesions. HPVs belonging to a high risk group have been shown to express two major transforming proteins, E6 and E7. With respect to the transforming activity of these proteins, many investigators have reported the location of these proteins in the cell, but their results are still controversial. In the present study, HPV type 16 E6 or E7 open reading frame (ORF) proteins were expressed and localized in human epidermal keratinocytes (RHEK-1) using the vaccinia virus as an expression vector. Immunofluorescence detection using monoclonal antibodies against E6 or E7 ORF proteins revealed that E6 or E7 proteins of HPV type 16 were located in the cytoplasm of RHEK-1 cells. These results suggest that E6 and E7 proteins bind to the tumor suppressor counterparts, thereby preventing transport of these proteins into the nucleus. These antioncogene products that fail to be rapidly transported out of the cytosol may be degraded by certain proteases such as the ubiquitin dependent system. In this way, the precise function of antioncogene products in the regulation of cell growth could be destroyed, and abnormal cell growth could occur.
Animal ; Base Sequence ; Cell Line ; Fluorescent Antibody Technique ; Haplorhini ; Human ; Keratinocytes/metabolism ; Molecular Sequence Data ; Oncogene Proteins, Viral/*biosynthesis ; Open Reading Frames/*physiology ; Papillomavirus, Human/*chemistry ; Plaque Assay ; Polymerase Chain Reaction ; Recombinant Proteins/biosynthesis ; Support, Non-U.S. Gov't ; Vaccinia virus/genetics