Proteomic analysis of plasma in healthy adults receiving recombinant vaccinia virus provides novel insights into HIV-1 neutralizing antibodies.

Human immunodeficiency virus (HIV) infection is still a global public health issue, and the development of an effective prophylactic vaccine inducing potent neutralizing antibodies remains a significant challenge. This study aims to explore the inflammation-related proteins associated with the neutralizing antibodies induced by the DNA/rTV vaccine. In this study, we employed the Olink chip to analyze the inflammation-related proteins in plasma in healthy individuals receiving HIV candidate vaccine (DNA priming and recombinant vaccinia virus rTV boosting) and compared the differences between neutralizing antibody-positive (nab + ) and -negative(nab-) groups. We identified 25 differentially expressed factors and conducted enrichment and correlation analysis on them. Our results revealed that significant expression differences in artemin (ARTN) and C-C motif chemokine ligand 23 (CCL23) between nab+ and -nab- groups. Notably, the expression of CCL23 was negatively corelated to the ID50 of neutralizing antibodies and the intensity of the CD4+ T cell responses. This study enriches our understanding of the immune picture induced by the DNA/rTV vaccine, and provides insights for future HIV vaccine development.

Multi-omics analysis reveals the dynamic interplay between Vero host chromatin structure and function during vaccinia virus infection.

The genome folds into complex configurations and structures thought to profoundly impact its function. The intricacies of this dynamic structure-function relationship are not well understood particularly in the context of viral infection. To unravel this interplay, here we provide a comprehensive investigation of simultaneous host chromatin structural (via Hi-C and ATAC-seq) and functional changes (via RNA-seq) in response to vaccinia virus infection. Over time, infection significantly impacts global and local chromatin structure by increasing long-range intra-chromosomal interactions and B compartmentalization and by decreasing chromatin accessibility and inter-chromosomal interactions. Local accessibility changes are independent of broad-scale chromatin compartment exchange (~12% of the genome), underscoring potential independent mechanisms for global and local chromatin reorganization. While infection structurally condenses the host genome, there is nearly equal bidirectional differential gene expression. Despite global weakening of intra-TAD interactions, functional changes including downregulated immunity genes are associated with alterations in local accessibility and loop domain restructuring. Therefore, chromatin accessibility and local structure profiling provide impactful predictions for host responses and may improve development of efficacious anti-viral counter measures including the optimization of vaccine design.

Identifying Protein Interactions with Viral DNA Genomes during Virus Infection.

Viruses exploit the host cell machinery to enable infection and propagation. This review discusses the complex landscape of DNA virus-host interactions, focusing primarily on herpesviruses and adenoviruses, which replicate in the nucleus of infected cells, and vaccinia virus, which replicates in the cytoplasm. We discuss experimental approaches used to discover and validate interactions of host proteins with viral genomes and how these interactions impact processes that occur during infection, including the host DNA damage response and viral genome replication, repair, and transcription. We highlight the current state of knowledge regarding virus-host protein interactions and also outline emerging areas and future directions for research.

Vaccinia Virus: Mechanisms Supporting Immune Evasion and Successful Long-Term Protective Immunity.

Vaccinia virus is the most successful vaccine in human history and functions as a protective vaccine against smallpox and monkeypox, highlighting the importance of ongoing research into vaccinia due to its genetic similarity to other emergent poxviruses. Moreover, vaccinia's ability to accommodate large genetic insertions makes it promising for vaccine development and potential therapeutic applications, such as oncolytic agents. Thus, understanding how superior immunity is generated by vaccinia is crucial for designing other effective and safe vaccine strategies. During vaccinia inoculation by scarification, the skin serves as a primary site for the virus-host interaction, with various cell types playing distinct roles. During this process, hematopoietic cells undergo abortive infections, while non-hematopoietic cells support the full viral life cycle. This differential permissiveness to viral replication influences subsequent innate and adaptive immune responses. Dendritic cells (DCs), key immune sentinels in peripheral tissues such as skin, are pivotal in generating T cell memory during vaccinia immunization. DCs residing in the skin capture viral antigens and migrate to the draining lymph nodes (dLN), where they undergo maturation and present processed antigens to T cells. Notably, CD8+ T cells are particularly significant in viral clearance and the establishment of long-term protective immunity. Here, we will discuss vaccinia virus, its continued relevance to public health, and viral strategies permissive to immune escape. We will also discuss key events and populations leading to long-term protective immunity and remaining key gaps.

Safety and Efficacy of the Modified Vaccinia Ankara-Bavaria Nordic Vaccine Against Mpox in the Real World: Systematic Review and Meta-Analysis.

In May 2022, mpox began to spread worldwide, posing a serious threat to human public health. Modified Vaccinia Ankara-Bavaria Nordic (MVA-BN) is a live attenuated orthopoxvirus vaccine that has been authorized by the U.S. Food and Drug Administration as the vaccine of choice for the prevention of mpox. In this study, we conducted a meta-analysis of all currently published literature on the efficacy and safety of the MVA-BN vaccine in the real world, showing that the MVA-BN vaccine is effective and safe, with efficacy of up to 75% with a single dose and up to 80% with a two-dose vaccine. Meanwhile, we found that subcutaneous injection has lower local and systemic adverse events than intradermal injection, regardless of single- or two-dose vaccination, and subcutaneous injection is better tolerated in children, the elderly, or people with underlying medical conditions. These results have important reference value for clinical practice.

DNA vaccine prime and replicating vaccinia vaccine boost induce robust humoral and cellular immune responses against MERS-CoV in mice.

As of December 2022, 2603 laboratory-identified Middle East respiratory syndrome coronavirus (MERS-CoV) infections and 935 associated deaths, with a mortality rate of 36%, had been reported to the World Health Organization (WHO). However, there are still no vaccines for MERS-CoV, which makes the prevention and control of MERS-CoV difficult. In this study, we generated two DNA vaccine candidates by integrating MERS-CoV Spike (S) gene into a replicating Vaccinia Tian Tan (VTT) vector. Compared to homologous immunization with either vaccine, mice immunized with DNA vaccine prime and VTT vaccine boost exhibited much stronger and durable humoral and cellular immune responses. The immunized mice produced robust binding antibodies and broad neutralizing antibodies against the EMC2012, England1 and KNIH strains of MERS-CoV. Prime-Boost immunization also induced strong MERS-S specific T cells responses, with high memory and poly-functional (CD107a-IFN-γ-TNF-α) effector CD8+ T cells. In conclusion, the research demonstrated that DNA-Prime/VTT-Boost strategy could elicit robust and balanced humoral and cellular immune responses against MERS-CoV-S. This study not only provides a promising set of MERS-CoV vaccine candidates, but also proposes a heterologous sequential immunization strategy worthy of further development.

Polyvalent mpox mRNA vaccines elicit robust immune responses and confer potent protection against vaccinia virus.

The 2022 mpox outbreak led the World Health Organization (WHO) to declare it a public health emergency of international concern (PHEIC). There is a need to develop more effective and safer mpox virus (MPXV)-specific vaccines in response to the mpox epidemic. The mRNA vaccine is a promising platform to protect against MPXV infection. In this study, we construct two bivalent MPXV mRNA vaccines, designated LBA (B6R-A29L) and LAM (A35R-M1R), and a quadrivalent mRNA vaccine, LBAAM (B6R-A35R-A29L-M1R). The immunogenicity and protective efficacy of these vaccines alone or in combination were evaluated in a lethal mouse model. All mRNA vaccine candidates could elicit potential antigen-specific humoral and cellular immune responses and provide protection against vaccinia virus (VACV) infection. The protective effect of the combination of two bivalent mRNA vaccines and the quadrivalent vaccine was superior to that of the individual bivalent mRNA vaccine. Our study provides valuable insights for the development of more efficient and safer mRNA vaccines against mpox.

A two-dose viral-vectored Plasmodium vivax multistage vaccine confers durable protection and transmission-blockade in a pre-clinical study.

Among Plasmodium spp. responsible for human malaria, Plasmodium vivax ranks as the second most prevalent and has the widest geographical range; however, vaccine development has lagged behind that of Plasmodium falciparum, the deadliest Plasmodium species. Recently, we developed a multistage vaccine for P. falciparum based on a heterologous prime-boost immunization regimen utilizing the attenuated vaccinia virus strain LC16m8Δ (m8Δ)-prime and adeno-associated virus type 1 (AAV1)-boost, and demonstrated 100% protection and more than 95% transmission-blocking (TB) activity in the mouse model. In this study, we report the feasibility and versatility of this vaccine platform as a P. vivax multistage vaccine, which can provide 100% sterile protection against sporozoite challenge and >95% TB efficacy in the mouse model. Our vaccine comprises m8Δ and AAV1 viral vectors, both harboring the gene encoding two P. vivax circumsporozoite (PvCSP) protein alleles (VK210; PvCSP-Sal and VK247; -PNG) and P25 (Pvs25) expressed as a Pvs25-PvCSP fusion protein. For protective efficacy, the heterologous m8Δ-prime/AAV1-boost immunization regimen showed 100% (short-term; Day 28) and 60% (long-term; Day 242) protection against PvCSP VK210 transgenic Plasmodium berghei sporozoites. For TB efficacy, mouse sera immunized with the vaccine formulation showed >75% TB activity and >95% transmission reduction activity by a direct membrane feeding assay using P. vivax isolates in blood from an infected patient from the Brazilian Amazon region. These findings provide proof-of-concept that the m8Δ/AAV1 vaccine platform is sufficiently versatile for P. vivax vaccine development. Future studies are needed to evaluate the safety, immunogenicity, vaccine efficacy, and synergistic effects on protection and transmission blockade in a non-human primate model for Phase I trials.

Vaccinia virus subverts xenophagy through phosphorylation and nuclear targeting of p62.

Autophagy is an essential degradation program required for cell homeostasis. Among its functions is the engulfment and destruction of cytosolic pathogens, termed xenophagy. Not surprisingly, many pathogens use various strategies to circumvent or co-opt autophagic degradation. For poxviruses, it is known that infection activates autophagy, which however is not required for successful replication. Even though these complex viruses replicate exclusively in the cytoplasm, autophagy-mediated control of poxvirus infection has not been extensively explored. Using the prototypic poxvirus, vaccinia virus (VACV), we show that overexpression of the xenophagy receptors p62, NDP52, and Tax1Bp1 restricts poxvirus infection. While NDP52 and Tax1Bp1 were degraded, p62 initially targeted cytoplasmic virions before being shunted to the nucleus. Nuclear translocation of p62 was dependent upon p62 NLS2 and correlated with VACV kinase mediated phosphorylation of p62 T269/S272. This suggests that VACV targets p62 during the early stages of infection to avoid destruction and further implies that poxviruses exhibit multi-layered control of autophagy to facilitate cytoplasmic replication.

Application of Oncolytic Poxviruses: An Emerging Paradigm in Cancer Therapy.

Despite the significant advancement of new tools and technology in the field of medical biology and molecular biology, the challenges in the treatment of most cancer types remain constant with the problem of developing resistance toward drugs and no substantial enhancement in the overall survival rate of cancer patients. Immunotherapy has shown the most promising results in different clinical and preclinical trials in the treatment of various cancer due to its higher efficacy and minimum collateral damage in many cancer patients as compared to conventional chemotherapy and radiotherapy. An oncolytic virus is a new class of immunotherapy that can selectively replicate in tumor cells and destroy them by the process of cell lysis while exerting minimum or no effect on a normal cell. Besides this, it can also activate the host's innate immune system, which generates an anti-tumor immune response to eliminate the tumor cells. Several wild types and genetically modified viruses have been investigated to show oncolytic behavior. Vaccinia virus has been studied extensively and tested for its promising oncolytic nature on various model systems and clinical trials. Recently, several engineered vaccinia viruses have been developed that express the desired genes encoded for selective penetration in tumor cells and enhanced activation of the immune system for generating anti-tumor immunity. However, further investigation is required to prove their potential and enhance their therapeutic efficacy.

Expression analysis and mapping of Viral-Host Protein interactions of Poxviridae suggests a lead candidate molecule targeting Mpox.

BACKGROUND: Monkeypox (Mpox) is an important human pathogen without etiological treatment. A viral-host interactome study may advance our understanding of molecular pathogenesis and lead to the discovery of suitable therapeutic targets. METHODS: GEO Expression datasets characterizing mRNA profile changes in different host responses to poxviruses were analyzed for shared pathway identification, and then, the Protein-protein interaction (PPI) maps were built. The viral gene expression datasets of Monkeypox virus (MPXV) and Vaccinia virus (VACV) were used to identify the significant viral genes and further investigated for their binding to the library of targeting molecules. RESULTS: Infection with MPXV interferes with various cellular pathways, including interleukin and MAPK signaling. While most host differentially expressed genes (DEGs) are predominantly downregulated upon infection, marked enrichments in histone modifiers and immune-related genes were observed. PPI analysis revealed a set of novel virus-specific protein interactions for the genes in the above functional clusters. The viral DEGs exhibited variable expression patterns in three studied cell types: primary human monocytes, primary human fibroblast, and HeLa, resulting in 118 commonly deregulated proteins. Poxvirus proteins C6R derived protein K7 and K7R of MPXV and VACV were prioritized as targets for potential therapeutic interventions based on their histone-regulating and immunosuppressive properties. In the computational docking and Molecular Dynamics (MD) experiments, these proteins were shown to bind the candidate small molecule S3I-201, which was further prioritized for lead development. RESULTS: MPXV circumvents cellular antiviral defenses by engaging histone modification and immune evasion strategies. C6R-derived protein K7 binding candidate molecule S3I-201 is a priority promising candidate for treating Mpox.

A new MVA ancestor-derived oncolytic vaccinia virus induces immunogenic tumor cell death and robust antitumor immune responses.

Vaccinia viruses (VACVs) are versatile therapeutic agents and different features of various VACV strains allow for a broad range of therapeutic applications. Modified VACV Ankara (MVA) is a particularly altered VACV strain that is highly immunogenic, incapable of replicating in mammalian hosts, and broadly used as a safe vector for vaccination. Alternatively, Western Reserve (WR) or Copenhagen (Cop) are VACV strains that efficiently replicate in cancer cells and, therefore, are used to develop oncolytic viruses. However, the immune evasion capacity of WR or Cop hinders their ability to elicit antitumor immune responses, which is crucial for efficacy in the clinic. Here, we describe a new VACV strain named Immune-Oncolytic VACV Ankara (IOVA), which combines efficient replication in cancer cells with induction of immunogenic tumor cell death (ICD). IOVA was engineered from an MVA ancestor and shows superior cytotoxicity in tumor cells. In addition, the IOVA genome incorporates mutations that lead to massive fusogenesis of tumor cells, which contributes to improved antitumor effects. In syngeneic mouse tumor models, the induction of ICD results in robust antitumor immunity directed against tumor neo-epitopes and eradication of large established tumors. These data present IOVA as an improved immunotherapeutic oncolytic vector.

Circular RNA vaccines against monkeypox virus provide potent protection against vaccinia virus infection in mice.

Since the outbreak of monkeypox (mpox) in 2022, widespread concern has been placed on imposing an urgent demand for specific vaccines that offer safer and more effective protection. Using an efficient and scalable circular RNA (circRNA) platform, we constructed four circRNA vaccines that could induce robust neutralizing antibodies as well as T cell responses by expressing different surface proteins of mpox virus (MPXV), resulting in potent protection against vaccinia virus (VACV) in mice. Strikingly, the combination of the four circular RNA vaccines demonstrated the best protection against VACV challenge among all the tested vaccines. Our study provides a favorable approach for developing MPXV-specific vaccines by using a circular mRNA platform and opens up novel avenues for future vaccine research.

The Recombinant Oncolytic Virus VV-GMCSF-Lact and Chemotherapy Drugs against Human Glioma.

Virotherapy is one of the perspective technologies in the treatment of malignant neoplasms. Previously, we have developed oncolytic vaccinia virus VV-GMCSF-Lact and its high cytotoxic activity and antitumor efficacy against glioma was shown. In this work, using immortalized and patient-derived cells with different sensitivity to VV-GMCSF-Lact, we evaluated the cytotoxic effect of chemotherapy agents. Additionally, we studied the combination of VV-GMCSF-Lact with temozolomide which is the most preferred drug for glioma treatment. Experimental results indicate that first adding temozolomide and then the virus to the cells is inherently more efficient than dosing it in the reverse order. Testing these regimens in the U87 MG xenograft glioblastoma model confirmed this effect, as assessed by tumor growth inhibition index and histological analysis. Moreover, VV-GMCSF-Lact as monotherapy is more effective against U87 MG glioblastoma xenografts comparing temozolomide.

Recent progress in combination therapy of oncolytic vaccinia virus.

In recent years, oncolytic viruses have emerged as promising agents for treating various cancers. An oncolytic virus is a non-pathogenic virus that, due to genetic manipulation, tends to replicate in and cause lysis of cancerous cells while leaving healthy cells unaffected. Among these viruses, vaccinia virus is an attractive platform for use as an oncolytic platform due to its 190 Kb genome with a high capacity for encoding therapeutic payloads. Combining oncolytic VV therapy with other conventional cancer treatments has been shown to be synergistic and more effective than monotherapies. Additionally, OVV can be used as a vector to deliver therapeutic payloads, alone or in combination with other treatments, to increase overall efficacy. Here, we present a comprehensive analysis of preclinical and clinical studies that have evaluated the efficacy of oncolytic vaccinia viruses in cancer immunotherapy. We discuss the outcomes of these studies, including tumor regression rates, overall survival benefits, and long-term responses. Moreover, we provide insights into the challenges and limitations associated with oncolytic vaccinia virus- based therapies, including immune evasion mechanisms, potential toxicities, and the development of resistance.

Engineered Luminescent Oncolytic Vaccinia Virus Activation of Photodynamic-Immune Combination Therapy for Colorectal Cancer.

Oncolytic virus therapy is currently regarded as a promising approach in cancer immunotherapy. It has greater therapeutic advantages for colorectal cancer that is prone to distant metastasis. However, the therapeutic efficacy and clinical application of viral agents alone for colorectal cancer remain suboptimal. In this study, an engineered oncolytic vaccinia virus (OVV-Luc) that expresses the firefly luciferase gene is developed and loaded Chlorin e6 (Ce6) onto the virus surface through covalent coupling, resulting in OVV-Luc@Ce6 (OV@C). The OV@C infiltrates tumor tissue and induces endogenous luminescence through substrate catalysis, resulting in the production of reactive oxygen species. This unique system eliminates the need for an external light source, making it suitable for photodynamic therapy (PDT) in deep tissues. Moreover, this synergistic effect between PDT and viral immunotherapy enhances dendritic cell maturation, macrophage polarization, and reversal of the immunosuppressive microenvironment. This synergistic effect has the potential to convert a "cold" into a "hot" tumor, it offers valuable insights for clinical translation and application.

Cardiac glycosides inhibit early and late vaccinia virus protein expression.

Cardiac glycosides (CGs) are natural steroid glycosides, which act as inhibitors of the cellular sodium-potassium ATPase pump. Although traditionally considered toxic to human cells, CGs are widely used as drugs for the treatment of cardiovascular-related medical conditions. More recently, CGs have been explored as potential anti-viral drugs and inhibit replication of a range of RNA and DNA viruses. Previously, a compound screen identified CGs that inhibited vaccinia virus (VACV) infection. However, no further investigation of the inhibitory potential of these compounds was performed, nor was there investigation of the stage(s) of the poxvirus lifecycle they impacted. Here, we investigated the anti-poxvirus activity of a broad panel of CGs. We found that all CGs tested were potent inhibitors of VACV replication. Our virological experiments showed that CGs did not impact virus infectivity, binding, or entry. Rather, experiments using recombinant viruses expressing reporter proteins controlled by VACV promoters and arabinoside release assays demonstrated that CGs inhibited early and late VACV protein expression at different concentrations. Lack of virus assembly in the presence of CGs was confirmed using electron microscopy. Thus, we expand our understanding of compounds with anti-poxvirus activity and highlight a yet unrecognized mechanism by which poxvirus replication can be inhibited.

An IRF2-Expressing Oncolytic Virus Changes the Susceptibility of Tumor Cells to Antitumor T Cells and Promotes Tumor Clearance.

IFN regulatory factor 1 (IRF1) can promote antitumor immunity. However, we have shown previously that in the tumor cell, IRF1 can promote tumor growth, and IRF1-deficient tumor cells exhibit severely restricted tumor growth in several syngeneic mouse tumor models. Here, we investigate the potential of functionally modulating IRF1 to reduce tumor progression and prolong survival. Using inducible IRF1 expression, we established that it is possible to regulate IRF1 expression to modulate tumor progression in established B16-F10 tumors. Expression of IRF2, which is a functional antagonist of IRF1, downregulated IFNγ-induced expression of inhibitory ligands, upregulated MHC-related molecules, and slowed tumor growth and extended survival. We characterized the functional domain(s) of IRF2 needed for this antitumor activity, showing that a full-length IRF2 was required for its antitumor functions. Finally, using an oncolytic vaccinia virus as a delivery platform, we showed that IRF2-expressing vaccinia virus suppressed tumor progression and prolonged survival in multiple tumor models. These results suggest the potency of targeting IRF1 and using IRF2 to modulate immunotherapy.

Oncolytic vaccinia virus immunotherapy antagonizes image-guided radiotherapy in mouse mammary tumor models.

Ionizing radiation (IR) and oncolytic viruses are both used to treat cancer, and the effectiveness of both agents depends upon stimulating an immune response against the tumor. In this study we tested whether combining image guided ionizing radiation (IG-IR) with an oncolytic vaccinia virus (VACV) could yield a better therapeutic response than either treatment alone. ΔF4LΔJ2R VACV grew well on irradiated human and mouse breast cancer cells, and the virus can be combined with 4 or 8 Gy of IR to kill cells in an additive or weakly synergistic manner. To test efficacy in vivo we used immune competent mice bearing orthotopic TUBO mammary tumors. IG-IR worked well with 10 Gy producing 80% complete responses, but this was halved when the tumors were treated with VACV starting 2 days after IG-IR. VACV monotherapy was ineffective in this model. The antagonism was time dependent as waiting for 21 days after IG-IR eliminated the inhibitory effect but without yielding any further benefits over IR alone. In irradiated tumors, VACV replication was also lower, suggesting that irradiation created an environment that did not support infection as well in vivo as in vitro. A study of how four different treatment regimens affected the immune composition of the tumor microenvironment showed that treating irradiated tumors with VACV altered the immunological profiles in tumors exposed to IR or VACV alone. We detected more PD-1 and PD-L1 expression in tumors exposed to IR+VACV but adding an αPD-1 antibody to the protocol did not change the way VACV interferes with IG-IR therapy. VACV encodes many immunosuppressive gene products that may interfere with the ability of radiotherapy to induce an effective anti-tumor immune response through the release of danger-associated molecular patterns. These data suggest that infecting irradiated tumors with VACV, too soon after exposure, may interfere in the innate and linked adaptive immune responses that are triggered by radiotherapy to achieve a beneficial impact.

Deletions initiated by the vaccinia virus TopIB protein in yeast.

The type IB topoisomerase of budding yeast (yTop1) generates small deletions in tandem repeats through a sequential cleavage mechanism and larger deletions with random endpoints through the nonhomologous end-joining (NHEJ) pathway. Vaccinia virus Top1 (vTop1) is a minimized version of the eukaryal TopIB enzymes and uniquely has a strong consensus cleavage sequence: the pentanucleotide (T/C)CCTTp↓. To define the relationship between the position of TopIB cleavage and mutagenic outcomes, we expressed vTop1 in yeast top1Δ strains containing reporter constructs with a single CCCTT site, tandem CCCTT sites, or CCCTT sites separated by 42 bp. vTop1 cleavage at a single CCCTT site was associated with small, NHEJ-dependent deletions. As observed with yTop1, vTop1 generated 5-bp deletions at tandem CCCTT sites. In contrast to yTop1-initiated deletions, however, 5-bp deletions associated with vTop1 expression were not affected by the level of ribonucleotides in genomic DNA. vTop1 expression was associated with a 47-bp deletion when CCCTT sites were separated by 42 bp. Unlike yTop1-initiated large deletions, the vTop1-mediated 47-bp deletion did not require NHEJ, consistent with a model in which re-ligation of enzyme-associated double-strand breaks is catalyzed by vTop1.